Gyula Mózsik, András Dömötör, Tibor Past, Viktória Vas, Pál Perjési, Mónika Kuzma, Gyula Blazics, János Szolcsányi Capsaicinoids, From The Plant Cultivation To The Production of The Human Medical Drug

168x238_paprika_cimnegyed_vegleges.qxd 2009.04.21.
16:32 Page 1
CAPSAICINOIDS
From the Plant Cultivation to the Production
of the Human Medical Drug
168x238_paprika_cimnegyed_vegleges.qxd 2009.04.21. 16:32 Page 3
CAPSAICINOIDS
From the Plant Cultivation to the Production
of the Human Medical Drug
By
Gyula Mzsik, Andrs Dmtr, Tibor Past, Viktria Vas,
Pl Perjsi, Mnika Kuzma, Gyula Blazics, Jnos Szolcsnyi
AKADMIAI KIAD
168x238_paprika_cimnegyed_vegleges.qxd 2009.04.21. 16:32 Page 4
The publication of this book was supported by the grant of the National Office for Research
and Technology Pzmny Pter Program Ret-II, 08/2005 MEDIPOLISZ
ISBN 978 963 05 8694 8
Gyula Mzsik, 2009
Published by Akadmiai Kiad

Member of Wolters Kluwer Group
P.O. Box 245, H1519 Budapest, Hungary
www.akkrt.hu
All rights reserved. No part of this book may be reproduced by any means or transmitted
or translated into machine language without the written permission of the publisher.
Printed in Hungary
paprika2.qxd 4/15/09 12:17 PM Page 5
Contents
Foreword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
1. General introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
1.1. General introduction to the interactions of foods
(or food components) and drugs to be used in the prevention
and treatment of different diseases in patients. . . . . . . . . . . . . . . . . . . . . . . . . . 15
1.1.1. Foods and food components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
1.1.2. Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
1.1.3. Levels of the drugfood interactions in healthy human
beings and in patients with different diseases . . . . . . . . . . . . . . . . . . . . . 17
1.1.4. A short historic background of the interaction between
the effects of capsaicin (capsaicinoids) and NSAIDs in animal
experiments and healthy human subjects . . . . . . . . . . . . . . . . . . . . . . . . 18
1.1.5. Goals of the drug (drug-combination) production. . . . . . . . . . . . . . . . . . 20
2. Capsaicin (capsaicinoids) is (are) a famous family of the spices . . . . . . . . . . . . 21

2.1. Cultural background of spices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.2. History of spices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.2.1. Terminology and modern history of Capsicum . . . . . . . . . . . . . . . . . . . . 23
2.2.2. History of Capsicum in Hungary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3. Botanical taxonomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
4. Cultivation of Capsicum or paprika . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

4.1. Cultivation of Capsicums in India . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
4.2. Cultivation of Capsicums in other Asian countries . . . . . . . . . . . . . . . . . . . . . . 33
4.3. Cultivation of Capsicums in Africa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
4.4. Cultivation of Capsicums in America . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
4.5. Cultivation of Capsicums in Europe and Hungary . . . . . . . . . . . . . . . . . . . . . . 34
4.5.1. Cultivar types in Hungary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
4.5.1.1. Sweet Capsicum cultivar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
4.5.1.2. Paprika varieties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
5
5. General chemical structure and composition of Capsicums . . . . . . . . . . . . . . . 39

5.1. Macroscopic characteristics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
5.2. Microscopic characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
5.3. General chemical composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
6. Chemical taxonomy of the functional parts of the Capsicums. . . . . . . . . . . . . . 43
6.1. Capsicum: Botanical aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
6.2. Capsicum: Chemical constituents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
6.2.1. Volatiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
6.2.2. Coloring pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
6.2.2.1. Methods for determination of red and total carotenoids . . . . . . 53
6.2.3. Capsaicinoids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
6.2.3.1. Chemistry of capsaicinoids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
6.3. Capsicum: Quality control. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
6.3.1. Capsicum fruits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
6.3.1.1. The European Pharmacopeia (Ph. Eur. 5.0) . . . . . . . . . . . . . . . . 61
6.3.2. Capsicum extracts Oleoresin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
6.3.2.1. Capsicum extracts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
6.3.2.2. Capsicum Oleoresin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
6.3.3. Quantitation of capsicum pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
6.3.3.1. The Color Matching Method . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
6.3.3.2. Spectrophotometric methods . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
6.3.3.2.1. The EOA Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
6.3.3.2.2. The American Spice Trade Association
(ASTA) Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
6.3.3.2.3. The Hungarian Standard Method . . . . . . . . . . . . . . . . 68
6.3.4. Quantitation of pungent principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
6.3.4.1. Official methods for organoleptic determination of pungency . . . . 69
6.3.4.1.1. The Scoville Method. . . . . . . . . . . . . . . . . . . . . . . . . . 69
6.3.4.1.2. The EOA Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
6.3.4.1.3. The British Standard Method . . . . . . . . . . . . . . . . . . . 71
6.3.4.1.4. The International Standard Organization
(ISO) Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
6.3.4.1.5. The ASTA Method . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
6.3.4.2. Quantitation of capsaicinoids . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
6.3.4.2.1. Early direct methods . . . . . . . . . . . . . . . . . . . . . . . . . . 72
6.3.4.2.2. Methods based on separation of capsaicinoids. . . . . . 72
6.3.4.2.3. Newer chromatographic micromethods
Thin Layer Chromatography . . . . . . . . . . . . . . . . . . . 73
Gas Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . 73
High Performance Liquid Chromatography . . . . . . . . 75
6.3.4.2.6. The ASTA method for determination
of capsaicinoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
6.3.4.2.7. The United States Pharmacopeia (USP) . . . . . . . . . . . 77
6.3.4.3. Correlation of pungency and capsaicinoid content . . . . . . . . . . 79
6.3.4.4. Stability of capsaicinoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
6
7. General physiology of retinoids and carotenoids. . . . . . . . . . . . . . . . . . . . . . . . . 81

7.1. Biochemistry of retinoids and carotenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
7.1.1. General physiology of retinoids and carotenoids . . . . . . . . . . . . . . . . . . 82
7.1.2. Retinoids and chemical carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . 83
7.1.3. Effect of antioxidants on colorectal epithelial cell proliferation,
polyp recurrence and carcinogenesis: Clinical trials in patients . . . . . . . 84
7.2. Prevalence and importance of the checked human GI diseases. . . . . . . . . . . . . 85
7.3. Gastric cytoprotection, as a special form of defensive mechanisms
to gastrointestinal (GI) mucosal injury produced by retinoids . . . . . . . . . . . . . 90
7.4. New results in the biological actions of retinoids and carotenoids in animals,
healthy human subjects and patients with different gastrointestinal disorders . . . 91
7.4.1. Gastrointestinal mucosal protective effects produced
by retinoids in animal experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
7.4.2. Effects of vitamin A on the gastric secretory responses
and indomethacin-induced gastric microbleedings in healthy
human subjects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
7.4.3. Ulcer healing effect of vitamin A in patients with chronic gastric
ulcer (multiclinical randomized, prospective study) . . . . . . . . . . . . . . . . 93
7.4.4. Changes in serum levels of retinoids in patients with different
gastrointestinal inflammatory diseases . . . . . . . . . . . . . . . . . . . . . . . . . . 93
7.4.5. Changes in serum levels of retinoids in patients with different
gastrointestinal cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
7.4.5.1. Leiden mutation in patients with different
gastrointestinal tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
7.4.5.2. Correlation between the prevalence of Leiden mutation
and decrease of serum levels of vitamin A and zeaxanthin
in patients with different gastrointestinal tumors. . . . . . . . . . . . 97
8. Animal and human observations with capsaicinoids . . . . . . . . . . . . . . . . . . . . 100
8.1. Physiological and pharmacological research tool by capsaicin
(Short overview) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
8.1.1. The chemistry of capsaicinoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
8.1.2. Source of capsaicin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
8.1.3. Selective sensory effects of capsaicin . . . . . . . . . . . . . . . . . . . . . . . . . . 101
8.1.4. Mechanism of action of capsaicin on sensory receptors. . . . . . . . . . . . 102
8.1.5. Capsaicin actions in the gastrointestinal tract of animals . . . . . . . . . . . 103
8.1.6. Capsaicin-sensitive sensory nerves and gastric acid secretion . . . . . . . 105
8.1.7. Molecular-pharmacological studies . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
8.1.8. Capsaicin actions in healthy human subjects and in patients
with different gastrointestinal disorders . . . . . . . . . . . . . . . . . . . . . . . . 113
8.1.8.1. Results of the comparative molecular-pharmacological
studies of capsaicin, atropine, omeprazole, famotidine,
ranitidine and cimetidine on the gastric basal acid output
(BAO) in human subjects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
8.1.9. Side effects of aspirin and other nonsteroidal anti-inflammatory
drugs (NSAIDs) in the gastrointestinal tract of patients . . . . . . . . . . . . 118
7
9. Toxicological studies with capsaicin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

9.1. Animal observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
9.1.1. Acute toxicology studies of capsaicin in animal experiments . . . . . . . 120
9.1.2. Acute toxicity studies with pure trans-capsaicin derivates
in dogs after intravenous administration . . . . . . . . . . . . . . . . . . . . . . . . 121
9.1.2.1. Acute effects on cardiovascular and respiratory parameters . . . 123
9.1.2.2. Plasma levels of capsaicin . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
9.1.3. Results of subacute toxicology of capsaicin in dogs. . . . . . . . . . . . . . . 124
9.1.3.1. Two weeks treatment with trans-capsaicin . . . . . . . . . . . . . . . 124
9.1.3.2. Clinical chemistry and hematology . . . . . . . . . . . . . . . . . . . . . 124
9.1.3.3. Organ weights, macroscopic and microscopic observations . . . 125
9.1.3.4. Pharmacokinetic data after 14-days treatment with
trans-capsaicin in dogs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
9.1.3.5. Absorption and metabolism of oral application
of capsaicinoids in animal experiments. . . . . . . . . . . . . . . . . . 125
9.1.3.6. Summary and conclusions of the administration
of different doses of trans-capsaicin in acute and subacute
experiments in dogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
9.1.3.7. Chronic toxicity studies in animals . . . . . . . . . . . . . . . . . . . . . 128
9.1.4. Metabolism of capsaicin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
9.1.4.1. The potential routes of metabolism of capsaicin . . . . . . . . . . . 129
9.1.4.1.1. Enzymatic oxidative metabolism of capsaicin . . . . 129
9.1.4.1.2. Non-oxidative metabolism of capsaicin . . . . . . . . . 130
9.1.4.2. Role of metabolic activation in capsaicin-induced toxicity . . . . 131
9.1.5. Effects of capsaicin on xenobiotic metabolism and chemically
induced mutagenesis and carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . 133
9.1.6. Hepatoprotection of capsaicin in rats . . . . . . . . . . . . . . . . . . . . . . . . . . 134
9.1.7. Genotoxicity studies with capsaicin or trans-capsaicin . . . . . . . . . . . . 135
9.1.7.1. Ames assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
9.1.7.2. Mouse lymphoma cell mutation assay . . . . . . . . . . . . . . . . . . 137
9.1.7.3. Mouse in vivo micronucleus assay . . . . . . . . . . . . . . . . . . . . . 137
9.1.7.4. Chromosomal aberration in human peripheral blood
lymphocytes (HPBL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
9.1.7.5. Brief summary of the main results of observations
with capsaicin in animals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
9.2. Human observations with capsaicin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
9.2.1. Observations with capsaicin in healthy human subjects . . . . . . . . . . . . 141
9.2.1.1. Dose-response curves of capsaicin in the human
stomach acute observation. . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
9.2.1.2. Changes in laboratory parameters and complaints
of healthy human subjects during the study with capsaicin . . 142
9.2.2. Subchronic observations with capsaicin in healthy human subjects . . . . 142
9.2.2.1. Two weeks treatment with capsaicin . . . . . . . . . . . . . . . . . . . . 142
9.2.2.2. Biochemical measurements and complaints in healthy
human subjects during two weeks capsaicin treatment . . . . . . 142
9.2.3. Human chronic observations with capsaicinoids . . . . . . . . . . . . . . . . . 143
9.2.4. Preventive effects of capsaicin against the selective
and non-selective inhibitory actions produced by nonsteroidal
anti-inflammatory drugs on COX-1 and COX-2 enzymes . . . . . . . . . . 143
8
9.2.5. Summary of the observations with capsaicin alone or in combination

with selective and non-selective inhibition of COX-1 and COX-2
enzymes by nonsteroidal anti-inflammatory compounds (drugs)
in animal experiments and in human observations . . . . . . . . . . . . . . . . 145
10. Nature and characteristics of the innovative drug research. . . . . . . . . . . . . . 147
10.1. Characterization of the innovative drug research . . . . . . . . . . . . . . . . . . . . 147
11. Pharmaceutical industrial research and development . . . . . . . . . . . . . . . . . . 157
11.1. Product design and development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
11.1.1. General goals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
11.2. Chemical-pharmaceutical aspects of the product development . . . . . . . . . 158
11.2.1 Pharmaceutical form . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
11.2.2 Composition and quality of starting materials. . . . . . . . . . . . . . . . . 159
11.2.2.1. Active Pharmaceutical Ingredient (API) of the product . . . . 160
11.2.2.2. Excipients quality of PH EUR requirements. . . . . . . . . . 161
11.2.2.3. Coating powder mixture quality . . . . . . . . . . . . . . . . . . . . 161
11.2.3. Packaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
11.3. Formulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
11.3.1. Formulation steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
11.3.2. FP Specification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
11.3.3. Samples for clinical trial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
11.3.3.1. Preparation of samples for clinical trial . . . . . . . . . . . . . . 163
11.3.3.2. Exposing, stability and packaging of planned final
product . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
11.4. Plant batch manufacturing and process validation . . . . . . . . . . . . . . . . . . . 163
11.5. Summary of the Chemical-Pharmaceutical development . . . . . . . . . . . . . 164
12. Clinical pharmacological studies with capsaicinoids alone and with
combination of capsaicinoids with nonsteroidal anti-inflammatory drugs. . . . 165
12.1. Main aims of clinical pharmacology and its relation
to the evidence-based medicine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
12.2. Our special scientific problems in the human clinical pharmacology
of capsaicinoids alone and capsaicinoids with together application
of aspirin, diclofenac and Naproxen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
12.3. Principal schedules for the human Phase III studies with capsaicinoids
alone and together with aspririn, diclofenac and Naproxen . . . . . . . . . . . . 168
12.3.1. Preparation of protocols for the human clinical pharmacological
studies (including Phase I to IV). . . . . . . . . . . . . . . . . . . . . . . . . . . 168
12.3.1.1. Medical points of the preparation of the study
protocols. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
12.3.2. Control of the protocols by the National or Regional Clinical
Pharmacological and Ethical Commitees.. . . . . . . . . . . . . . . . . . . . 169
12.3.3. Pharmacokinetic and pharmacodynamic effects
of capsaicinoids only. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
12.3.3.1. Human Phase I clinical pharmacological study. . . . . . . . 169
12.3.3.2. Human clinical pharmacological Phase I study
with capsaicinoids plus nonsteroidal anti-inflammatory
drugs in healthy human subjects . . . . . . . . . . . . . . . . . . . 170
9
12.3.3.2.1. Human clinical pharmacological phase

I study with capsaicinoids plus aspirin . . . . . 170
12.3.3.2.2. Human clinical pharmacological Phase
I study with capsaicinoids plus diclofenac. . . 170
I study with capsaicinoids plus Naproxen . . . 171
12.3.3.3. Human clinical pharmacological Phase II studies
in patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
II study in patients with thromboembolic
diseases (myocardial infarction, stroke,
thromboembolic events). . . . . . . . . . . . . . . . . 171
II studies in patients with different
degenerative locomotor diseases . . . . . . . . . . 171
Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
1. European Commission. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
1.1 Opinion of the scientific committee on food on capsaicin . . . . . . . . . . . . . . . . 207
1.2. European Herbal Practitioners Association . . . . . . . . . . . . . . . . . . . . . . . . . . 213
1.3. Commission Decision of 18 May 2005 amending Decision
1999/217/EC as regards the register of flavouring substances
used in or on foodstuffs (notified under document number C(2005) 1437)
(Text with EEA relevance) (2005/389/EC) . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
2. Medical products for human use: Common Technical Document (CTD) . . . 219
2.1. Foreword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
2.2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
2.3. Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
2.4. Presentation of European Marketing Authorisation Applications . . . . . . . . . 221
2.5. Presentation of Applications in the Mutual Recognition Procedure
or Decentralised Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
2.6. Presentation of Follow-up Measures, Specific Obligations and PSURs . . . . 222
2.7. Reformatting of dossiers of already authorised products . . . . . . . . . . . . . . . . 222
2.8. Presentation of the application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
2.9. Administrative, regional or national information is provided
in different Modules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
2.10. Preparing and Organizing the CTD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
2.11. Pagination and Segregation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
2.12. Information about national administrative requirements . . . . . . . . . . . . . . . 226
2.13. Special guidance for different kinds of applications . . . . . . . . . . . . . . . . . . 226
2.14. Special guidance on herbal medicinal products . . . . . . . . . . . . . . . . . . . . . . 229
2.15. Variation of an ASMF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
2.16. European Certificate of Suitability of monographs of the European
Pharmacopoeia (CEP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
2.17. European Community Guidelines on Quality, Safety and Efficacy . . . . . . . 231
About the authors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
Foreword
The potential role(s) of the scientific research works is (are) extremely well accepted
in our days, when the Establishment of European Union celebrated its 50th years
anniversary (2007). Consequently the countries of the European Union have chosen
a new and common pathway for the economical, social and scientific development
in Europe for the forthcoming time period (hopefully for all of the countries and for
many centuries).
Seven countries (France, West Germany, Italy, Spain, The Netherlands, Luxem-
burg, Belgium) established the European Economical Committee in Rome, Italy in
1957, when Europe demonstrated clearly the signs of survival over the Second World
War to other parts of the World. Later on, this European Economical Committee has
incorporated other countries including the countries liberated from the Soviet power.
The final aim of these European political and economical changes is to establish an
eminent, economically, politically and scientifically stable European Union.
Hungary was accepted and entered the European Union by January 1, 2004. There-
after many assets of our life have changed significantly (including economy, policy,
social life, science). We have to try to find the most prominent lines still going on
from the life in Hungary, which help us to reach the streams of the European Union.
The pharmacological research and industry in Hungary were internationally well
accepted by the different countries of Europe, North America (USA, Canada), Asia
(Japan, China, India, and Pakistan).
The essential and key role of so-called innovative pharmacological research has
been especially accepted by the Hungarian Research Departments (including the
Hungarian Academy of Sciences) and by the Hungarian Government.
The different Regional University Science Centers (Debrecen, Budapest, Szeged
and Pcs) have been established in 2004 by the Hungarian Government.
Our (Pcs) University has received a grant for carrying out the innovative pharma-
cological research in the time period of 20052008 (National Office for Research
and Technology Pzmny Pter Program, RET-II, 08/2005).
The members (altogether 21) of the research are working in the University of
Pcs, First Department of Medicine and Pharmaceutical Chemical Institute, Medical
Faculty, University of Pcs, Hungary and PannonPharma Pharmaceutical Ltd. (Pcs-
vrad, Hungary).
These researchers never worked together in a common project. So we found our-
selves before an extremely new challenge in our common innovative pharmacologi-
11
cal research, considering the very short time period (from 2005 to 2008) of our grant
and our common success to produce new drug combinations of orally applicable
capsaicin (capsaicinoids*) alone, capsaicinoids plus nonsteroidal anti-inflammatory
compounds (aspirin, diclofenac, Naproxen) for patients suffering from myocardial
infarction, pain, degenerative joint diseases, topically applied itraconazole (as one of
the antifungal drugs) and etacrinic acid alone or in some combination to be used as
an eyedrop preparation for patients suffering from glaucoma.
The scale of this innovative pharmacological research is very wide and multidis-
ciplinary. Furthermore, we had a very short time to realize the development of these
scientific and industrial aims.
We had several different scientific, industrial, marketing problems in the realiza-
tion of our innovative pharmacological research.
We met firstly with the problems of the use of capsaicin as chemical compound
used as drug material. Capsaicin can be obtained from the plants, and this compound
does not represent a uniform chemical entity (chemically capsaicin contains at least
5 to 6 chemical components (which are similar chemically and act in the same man-
ner). This compound was never used as an orally applicable drug. Furthermore, we
had very limited knowledge on the chemical compounds used in the cultivation of
plants for extraction of capsaicin (capsaicinoids).
We have learned a great deal of scientific matters from the lawyers, authorities,
medical, industrial and patent work experts. The main point was focused, in these
problems, to obtain the preparate of capsaicin (capsaicinoids) from the different
plants. The environmental factors (application of different chemicals) played an
essential role in the cultivation of capsaicin-containing plants. Briefly, the capsaicin
(capsaicinoids) is (are) of plant origin (but not chemically produced) chemical com-
pound (compounds).
Consequently, the acceptance of a chemical compound of plant origin cannot be
separated from the applied chemicals to produce larger quantities of the chemical
compound (compounds), which may be environmentally harmful. Furthermore, we
planned to produce capsaicin-containing drug combinations for oral application.
During our innovative pharmacological research, we met a lot of different (medi-
cal, industrial, technological, botanical, agriculture, chemical, pharmacological,
national and international juridical and patent) problems.
All of us worked hard. Our aim was to give a short (but complete) summary of our
common works in the years between 2005 and 2008. We hope very much that the
results of our scientific endeavour can help the works of those participating in inno-
vative research.
Pcs, Hungary, September 2008

The Authors
* Important note: The term of capsaicin is used in the text, however, capsaicinoids of natu-
ral origin are used during different studies. Sometimes the term capsaicinoids is used to
emphasize their plant origin in industrial research; suitable methods are used in the human
clinical pharmacology (phase III).
12
Preface
Generally, books and monographs are published to summarize the actually most
considerable results of the relevant research topics. We have also published a series
of different monographs, important books and congress proceedings up to now.
Our studies with capsaicin (capsaicinoids) have been started from the 1980s in
animal experiments and from 1997 in healthy human subjects and patients with dif-
ferent gastrointestinal (GI) disorders. The main point of the capsaicin research can
be summarized by mentioning that capsaicin modifies significantly the function of
the capsaicin-sensitive afferent nerves (the effects depending on the doses of
applied capsaicin).
The application of capsaicin is able to produce gastrointestinal mucosal defense.
The gastrointestinal mucosal damage can be produced by different noxious agents,
including nonsteroidal antiinflammatory drugs, NSAIDs in animal experiments and
humans. The gastrointestinal mucosal defense induced by small doses of capsaicin
(against Indomethacin, ethanol) has been proven in healthy human subjects, under
conditions of Helsinki Declaration and respecting the rules of the Good Clinical
Practice (GCP).
The nonsteroidal antiinflammatory drugs (NSAIDs) are widely used in the treatment
of patients with acute coronary syndrome (ACS), myocardial infarction, thrombophilia,
stroke (aspirin) and with different degenerative diseases of the locomotor system [selec-
tive cyclooxygenase (COX) I inhibitors and non-selective COX-1 and COX-2 inhi-
bitors]. These drugs produced different gastrointestinal diseases (mucosal damage,
ulceration, bleedings, and gastropathia). The results of animal experiments and human
observations have proved that capsaicin (given in small doses) is able to prevent the
NSAIDs-induced GI complaints (mucosal damage, ulceration, bleedings, etc.).
Different drugs (anticholinergic and antigastrinic drugs, histamine 2-receptor
antagonists, proton-pump inhibitors, and scavengers) are widely used in the every-
day medical practice to prevent the NSAIDs-induced GI side effects.
The stimulation of capsaicin-sensitive nerves, by the application of capsaicin in small
doses, represents a new pharmacological approach to prevent NSAIDs induced GI
mucosal damage in healthy human subjects and in patients with different GI disorders.
The Hungarian National Office for Research and Technology has offered us a
grant for doing innovative drug research in humans. We submitted our application
to produce a new drug (or drug combinations) to be applied in the patients treated
with NSAIDs.
13
The participants of this multidisciplinary group receiving the grant (National

Office for Research and Technology Pzmny Pter Program RET-II, 08/2005)
came from the Department of the Pharmaceutical Chemistry, First Department of
Medicine, Department of Ophthalmology, Medical Faculty, Pcs University, Hun-
gary and from the PannonPharma Pharmaceutical Ltd, Pcsvrad, Hungary (including
21 researchers altogether, among them internist, ophthalmologist, chemists, chemical
engineers, biologist, bioenergineers, pharmacologist, horticultural engineer).
We have met considerably different problems of medical, chemical, technologi-
cal, industrial research, national and international laws, and marketing during our
research work. We all together started with the innovative drug research and we
had to learn many new things (results) from the different medical, chemical, phar-
maceutical, industrial research during a very short time in 2005, in the pathways of
mutually induced group education (including the research, legal, ethical, drug indus-
trial fields; the necessity of the contact to the National Institute of Pharmacy (Orsz-
gos Gygyszerszeti Intzet, OGYI); to evaluate successfully the problems given by
the National Pharmaceutical Institute in the field of our innovative drug research;
marketing fields, etc.
In our days, the role of innovative drug research has been widely accepted not
only in Hungary but all over the World.
Consequently we had a very short time to find the successful possibilities in the
pathways of this research field.
We had to learn the possibilities and basic requirements for the international regis-
tration of three patents of the produced new drug (or drug combinations) in the year
of 2007:
1. Production of a new drug (or drug combinations): small doses of capsaicin (as
orally given drug) applied alone or in combination with aspirin, diclofenac and
Naproxen;
2. Production of a topically applicable antifungal drug in form of solution and in nail
polish;
3. Production of a new drug combination of etachrinic acid with other chemical com-
pounds to be applied in the patients with glaucoma.
This book indicates and details the cultural history of spices (capsaicin) in the
World; species, taxonomy, chemistry, medical research (in animal experiments and
human observations), pharmacokinetic and pharmacodynamic determinations of
drug (or drug combinations), preparation of suitable clinical pharmacological proto-
cols, which are used in human studies (phase III); the actuality of the related and
valid national (Hungarian) and international (European Union, United States, Japan,
India, China) laws which give the principal bases of new patents and marketing.
This book represents the very effective application of human power of the resear-
chers to the enormous spiritual and physical challenges of our country, the European
Union and other parts of the World in the field of introduction of a new drug (or of
drug combinations). The research in this field is an extremely hard and interdiscipli-
nary work.
We believe that the experiments of our innovative drug research can help others,
who are participating in these types of the innovative and multidisciplinary research.
Pcs, Hungary, September 2008 The Authors

1. General introduction
1.1. General introduction to the interactions of foods (or food

components) and drugs to be used in the prevention
and treatment of different diseases in patients
1.1.1. Foods and food components
The prevention and treatment of the different human diseases is the final form of our
medical activities to keep the health conditions in the human beings. Nutrition and
pharmacological treatment are also incorporated in this field.
Nutrition alone is very important (including the consume of different macro- and
micronutrients, trace elements, etc. in terms of their quantities and preparations)
representing the basis of sustainance of our life, by supplying the necessary energeti-
cal background.
The production of foods, establishment of the new foods, their biological and
nutritional qualifications, cultivation of plants to be used for food production, indus-
trial background cover a very wide range of natural sciences (physiology, pharma-
cology, nutrition, agriculture, food industry, medicine) - in terms of prevention and
treatment - and of the different social sciences, marketing, etc. (Mozsik, Figler,
2005). The correct evaluation of these questions represents very urgent and impor-
tant scientific aspects in our personal life, as well as in the World.
The spices (chemical compounds of plant origin) have been used to modify the
use of the different foods since 9500 years ago in the human culture. These spices
are different chemically; they produce different changes in the process of nutritional
habits, and on the other hand, are able to produce different physiological processes
in healthy human beings and in patients with different diseases.
Capsaicin (functionally capsaicinoids) is (are) one famous chemical compound
among the spices.
The historical and scientific history of capsaicin (capsaicinoids) is especially inte-
resting:
1. capsaicin (capsaicinoids) does (do) not represent an energy supply for the man;
2. capsaicin (capsaicinoids) is (are) able to modify the carbohydrate metabolism
(Domotor et al., 2006);
3. the spreading of capsaicin (capsaicinoids) has changed significantly over the
World during the last 9500 years (Govindajaran, 1985, 1986a-f; Govindajaran et
al., 1977, 1987; Govindajaran, Sathyanarayana 1991);
4. the cultivation of plants containing capsaicin changed significantly from time to
time, especially in the last two centuries (Govindajaran, 1985, 1986a-f; Govinda-
jaran et al., 1977, 1987; Govindajaran, Sathyanarayana 1991);
5. the discovery of a very wide scale of capsaicin-induced physiological regulatory
mechanisms (Roosterman et al., 2006);
6. the discovery of the specific stimulatory effect of capsaicin on the afferent sensory
nerves ("capsaicin sensitive afferent nerves") (Jancso-Gabor, 1959; Jancso et al.,
1968, 1985; 1987; Jancso-Gabor, Szolcsanyi 1972; Szolcsanyi, 1977, 1982,
1983a,b, 1984a,b, 1985, 1990a,b, 1993, 1996, 2004; Buck et al., 1982; Monsee-
renusorn et al., 1982; Fitzgerald, 1983; Chahl et al., 1984; Hori, 1984; Russel,
Burchiel, 1984; Holzer, Sametz, 1986; Buck, Burks, 1986; Maggi, Meli, 1988;
Holzer, 1988, 1990, 1991a,b, 1992a,b, 1998; Maggi et al., 1989; Holzer, Lippe,
1988; Holzer et al., 1989; Maggi 1995; Jancso-Gabor et al., 1997);
7. The application of capsaicin produces different physiological (pharmacological)
regulatory steps (Szolcsanyi, 1990a,b, 1993, 1996, 2004);
8. The application of capsaicin offered a new research tool to approach the different
biological regulatory mechanisms in animals and human beings (Mozsik et al.,
2007b) and in patients with different diseases (Domotor et al., 2005, 2006, 2007).
1.1.2. Drugs
The research and production of different drugs are the keystones both in the drug
industry and in the human medical treatment.
The progress of medical treatments was based on two main research lines:
1. The evaluation of the results of the "classical and historical" medical practice
dominantly started from Asia (from the Chinese medicine). The application of
these medicines was based on the efficacy of the medical treatment carried out;
2. The planning of chemically, pharmacologically, biologically active compound(s)
for production of the new drugs to be used in the treatment of patients with diffe-
rent diseases (that has been dominating in the last decades).
Our attention is focused on the nonsteroidal anti-inflammatory drugs (NSAIDs).

The most famous compound of this drug family was produced by the Pharmaceutical
Company of Bayer (Germany) in the 1880s (Aspirin). This compound is very widely
used in the everyday medical treatment (as analgetic, antipyretic agent, and platelet
aggregation inhibitor), however, its most important indication is in the prevention of
myocardial infarction, stroke, thromboembolic events and prevention of different
colorectal malignancies.
The main actions of NSAIDs are the following:
1. inhibitory action on the platelet aggregation;
2. production of gastric mucosal damage chemically (in the pathway of inhibition of
dissociation of weak acid in the presence of a strong acid);
3. inhibition of the bicarbonate secretion produced by the pancreas;
4. inhibition of the prostaglandin (PG) synthesis and
5. modification of the blood flow in the gastrointestinal tract by the inhibition of the
PG synthesis.
The number of patients suffering from myocardial infarction or from different
degenerative locomotor diseases is very large. The application of aspirin is gene-
rally and internationally accepted in the prevention of the myocardial reinfarction
(given in doses of 100 mg/day or in large doses of 300 mg/day in so-called Aspirin
16
insensitive patients). The administration of aspirin means a long-lasting treatment in
all patients with cardiological and thromboembolic events.
Patients suffering from different degenerative diseases of the locomotor system
are treated with one of the compounds of NSAIDs.
The actions and pharmacokinetic behaviors of the NSAIDs can be modified by the
application of different foods (fats, aperitive compounds, aldehyde and keto-sugars,
dietary fibres, etc.).
1.1.3. Levels of the drug-food interactions in healthy human

beings and in patients with different diseases
There is a specific field of the human nutrition (including that of both healthy sub-
jects and patients) considering that we use generally several macronutrients (pro-
teins, carbohydrates, fats, etc.) together. There is no question that the absorption,
metabolism, excretion and utilization of the macronutrients taken alone differ signi-
ficantly from those when they are consumed together. Furthermore, the complemen-
tation of macronutrients with each other is a basic nutritional question (e.g. comple-
mentation of cereals with beans). Clinicians were taught that these problems (related
to the application form of macronutrients) significantly differ in healthy subjects and
in patients with different diseases. The different diseases are able to modify the
digestion (all of the physiological processes which are able to prepare the foods in a
suitable form for absorption), absorption and post-absorptive phases (transfer of the
drugs from the small intestine to the target organ, excretion by the different path-
ways, utilization, including their anabolism and catabolism). The analysis of these
medical questions requires a thorough medical knowledge.
The detailed analysis of digestion, absorption and utilization of the different foods
covers the main topics of the medical sciences (such as gastroenterology, nutrition,
medical pathophysiology, organspecific topics of medicine as hepatology, nephro-
logy, hematology, as well as different other subjects).
Different drugs are used in the medical practice to modify the different physiolo-
gical and pathological processes in healthy human subjects and in patients. Drug
application to healthy subjects causes the transitory modification of the different
functions in healthy human subjects (pain killers, coffee, laxative agents, many other
drugs). Drug application in the patients with different diseases originally aims at
decreasing the complaints of the patients. We have to know that these drugs modify
the consumed foods (their absorption, digestion, utilization, transportation in the
human body) (Table 1).
A few examples will be given to explain these questions:
1. The prokinetic drugs decrease the transit time of the different foods in the GI tract
of healthy humans, just like diarrhea (as disease). Consequently the time for the
absorption of different foods is short;
2. Vitamin B is absorbed together with its intrinsic factor from the distal part of the
l 2
small intestine. If the patients have no intrinsic factor, then the B absorption
) 2
becomes insufficient, however, in patients with ileitis terminalis B absorption

1 2
17
T a b l e I . T h e scientific a p p r o a c h of t h e m a i n q u e s t i o n s o f c l i n i c a l nutrition a n d dietetics a p p e a r s
as f o l l o w s ( M o z s i k , Figler, 2 0 0 5 ) *
1. F o o d a n d f o o d i n t e r a c t i o n s at t h e l e v e l s of
1.1. pre-absorptive p h a s e (digestion),
1.2. absorptive phase,
1.3. post-absorptive p h a s e .
2. T h e d r u g a n d f o o d a c t i o n s w i l l m a n i f e s t at t h e l e v e l s of
2.1. pre-absorptive l e v e l (digestion),
2.2. absorptive level,
2.3. post-absorptive l e v e l .
* Mozsik, Gy., Figler, M. (2005): Metabolic Ward in Human Clinical Nutrition and Dietetics. Research Signpost,
Kerala (with permission)
becomes insufficient (partly due to the extremely decreased transit time) without
any absence of vitamin B and its intrinsic factor; 1 2
3. Most of the macronutrients (carbohydrates, proteins) are absorbed by an active

transport process from the small intestine. Strophantin g (and other digitalis com-
pounds) specifically inhibits the active transport processes in the human gastroin-
testinal tract. In non-tropical sprue (gluten sensitive enteropathy) the patients have
no brush border in the small intestine and consequently the mentioned food com-
ponents will not be absorbed from the GI tract (without or with application of any
kind of digitalis compounds);
4. Finally, the application of different drugs (nonsteroidal anti-inflammatory com-
pounds) can modify the transport of food by the modification of the linkage of
nutritional compounds and hormones to albumin, the excretion (e.g. diuretics)
and the function of the target organ (positive and negative enzyme inductors).
Moreover, in patients with hypalbuminemia (when the drugs are linked in smaller
quantities to albumin) we can produce easily drug intoxication in the patients during
different drug treatments.
These examples from the everyday medical practice clearly indicate the extreme
complexity of the correlation between nutrition and drug therapy in healthy human
subjects and in patients with different diseases.
1.1.4. A short historic background of the interaction between the

effects of capsaicin (capsaicinoids) and NSAIDs in animal
experiments and healthy human subjects
The mechanism of action produced by capsaicin (capsaicinoids) is very wide both in

animal experiments and human beings (including the studies with isolated and culti-
vated cells, in vitro and in vivo studies, and whole living individuals) (Roosterman
et al., 2006).
18
Our attention is focused on the mechanisms of action of NSAIDs and capsaicin
both in animals and human beings.
There is a vast literature dealing with the gastrointestinal mucosal damage pro-
duced by NSAIDs.
The mechanisms of the NSAIDs-induced gastrointestinal mucosal damage can be
detected at the levels of:
1. physico-chemical laws (Davenport, 1970, 1973; Mozsik et al., 1981);
2. prostaglandin synthesis (many authors worked in this field);
3. cellular energy systems (Mozsik et al., 1981; Mozsik, 2006);
4. oxygen free radicals (Mozsik, 2006).
Szolcsanyi and Bartho (1981) were the first who proved that capsaicin prevents
the gastric mucosal damage, when it is given in a small dose. In contrast, the gastric
mucosal damage is enhanced by the application of higher doses of capsaicin. These
results clearly demonstrated that the effect of capsaicin on the gastrointestinal tract
depends on its doses applied in the experiments.
After this original observation many researchers demonstrated the gastrointestinal
preventive effect of capsaicin in animal experiments (Holzer, 1988, 1990, 1991a,b,
1992a,b, 1998; Holzer etal., 1989).
A very systematic research work has been carried out by our team since 1980. Its
main results and critical overview were summarized in a book (Mozsik et al., 1997a).
The final conclusion of this book was that the application of capsaicin in the dif-
ferent animal experiments offers a new research tool for understanding the different
mechanisms existing in the gastrointestinal tract.
Because one of us (Gy.M.) is internist, nutritional expert and clinical pharmacolo-
gist, our attention turned toward the human physiological, pharmacological aspects
of gastroenterology, the gastrointestinal tract and the mechanisms of actions of cap-
saicin related to other fields of medicine.
The human clinical nutritional studies with capsaicin have been carried out from
1997, according to the Good Clinical Practice (GCP), respecting the Helsinski Dec-
laration, and by the permission of the Regional Ethical Committee of Pecs Uni-
versity, Hungary.
One of the main results of these studies was:
Capsaicin (capsaicinoids) prevents (prevent) the Indomethacin (which is a non-
selective cyclooxygenase enzyme inhibitor)-induced gastrointestinal side effect
(microbleeding), however, it prevents the topically applied ethanol-induced decrease
of gastric transmucosal potential difference (GTPD), and enhances the gastric trans-
mucosal potential difference (GTPD) in healthy human subjects in association with
gastric inhibitory actions on the gastric basal acid output (BAO) (Mozsik et al., 1999,
2004, 2005a, 2007b).
These results offered a new possibility to plan and to create a new drug (or drug
combinations), in which the gastrointestinal mucosal damaging NSAIDs are com-
bined with the gastroprotective capsaicin in a suitable concentration and formula-
tion.
19
1.1.5. Goals of the drug (drug-combination) production
The NSAIDs are widely used in the everyday medical practice (prevention of throm-
boembolic episodes, myocardial reinfarction, stroke and colorectal cancers). These
compounds act at the levels of enzymes of COX-1 and COX-2 by the modification of
functions of these enzymes. The functions of COX-1 and COX-2 are involved in
many therapeutic steps, new pathological events in the development of different di-
seases (myocardial infarct, damage in the gastrointestinal tract, development of dif-
ferent gastrointestinal malignancies, drugs-induced gastrointestinal injuries, etc.).
The further (present) drug production research was based on our earlier obtained
research works in the everyday clinical (cardiological, gastrointestinal, oncological)
practice.
The production of a new drug (or new drug combination) is a very hard and com-
plicated work. This research is extremely well regulated internationally, covering the
chemistry, animal experiments, stability studies, genotoxicology, metabolism, phar-
macokinetic observations in animals and in human beings.
The authors of this book participated in the establishments of clinical pharmaco-
logy (in the 1960s), clinical nutrition (in 1977) in the production of many drugs.
These investigators work in the basic pharmaceutical research as well as in clinical
nutrition, clinical pharmacology and medicine.
This research represented a new challenge for all of us. Because this so-called
innovative drug research does not represent a "typical research" pathway to us (inde-
pendently all of us participated actively in the classical medical and chemical
research), we decided that we try to give the necessary information of this "innova-
tive drug research" for those, who want to participate in this process.
In the course of this process we have gained knowledge considering national and
international new scientific information, organization of the "innovative research
team" to estimate our national and international possibilities including our limits,
preparation of patents, preparation of the preclinical dossier, organization of human
phase III trials, medical problems, marketing, etc.).
20
2. Capsaicin (capsaicinoids) is (are)
a famous family of the spices
2 . 1 . Cultural background of spices
Since the time man started gathering food, the addition of small amounts of some
plant materials may have been used for the powerful impact they had on the appear-
ance or eating quality. Initially, these additions were valued for masking the off-fla-
vor of stored, decomposing foods; later on, in medieval Europe, they were imported
as rural spices for their capacity to slow down deterioration during storage.
Gradually, when definitive ethnic cuisines developed, the individual spices came to
be valued in the modern sense for their real contribution, which is to flavor general-
ly insipid meat and cereal foods and to make the food more acceptable and preferred.
Spices have become indispensable in the modern kitchens of individual homes, insti-
tutions, and the food manufacturing industries. They provide individually to the
dishes and distinguishing gourmet foods.
In the countries of their origin and growth, the Hindustan, the Spice Island, and
China, the highly systemized use of spices and other aromatic plants in foods, for
increased acceptance and physiological effects of the extracts, has been known
through ancient Vedic texts and the Ayurvedic text of Susrata and Caraca. For seve-
ral centuries Before Christ (B.C.) spices, along with silk, gold, and precious stones,
were principal articles of trade between the East and West. As early as 1600 B.C.,
the Chinese brought spices along the Chinese coast and overland along Tibet to
Kashgar. The Arabs then took the spices west to Baghdad, Alexandria, and Persian
Gulf ports. These were hazardous journeys, and great mystery surrounded the ori-
gin, collection and transport such that the values of the spices were very great. Later,
the spice route shifted to sea routes (and included products besides those of Gulf
ports) and then to the merchants in Alexandria and Baghdad. Knowledge of spices
and their uses reached Egypt and spread to the Mediterranean region. Hippocrates,
the father of modern medicine, and Theophrastus, the Greek philosopher and bo-
tanist, wrote treatises on medicinal plants that included many spices.
During the first few centuries A.D., many cities (Alexandria, Constantinople,
Basra and Venice) underwent violent changes as they became focal points of the
spice trade.
The values of trade in pepper and a few other spices were so great to the economy
of the kingdoms in Europe that kings sent costly expeditions to spice-growing coun-
tries. In the late 15th century, Marco Polo of Venice, Vasco da Gama and Cabral of
Portugal, and later Columbus and Cortes from Spain, undertook hazardous voyages
21
to establish sea routes to trading ports in the spice-producing countries of Asia. The
trading became so intense that there were wars between Portugal, The Netherlands
and England, while the local populations in Asia were forcibly enslaved and crip-
pling taxes were levied. The entry of U.S.A. into the world of spices broke the
monopoly of the European nations and the important trading centers shifted to
Singapore and Calcutta in the East, and New York, London and Hamburg in West.
The colonization of distant lands and the internationalization of the spice trade
also had desirable changes, world trade in spices was increased and spices were
introduced from one region to another. Black pepper, cardamom, ginger and turmer-
ic from Asia were introduced into South America and Africa, and Capsicum (red
pepper), vanilla and other spices were introduced from the New World of Central
and South America to the Old World of Europe, Asia and Africa.
2.2. History of spices
Among the spices, capsicums are the most colorful in appearance, and are important
in terms of their history, antiquity and influence on many cuisines of the world.
During his first expedition, Columbus (1492 to 1493) observed that the natives of
the New World used a colorful red fruit calls aji or axi with most of their foods.
This additive was found to be much stronger than the black pepper of Asia (Piper
nigrum L.) in search of which he had undertaken this expedition. He took samples of
this fruit back to Spain and named it "red pepper". De Cuneo, who accompanied
Columbus on the second voyage to the New World in 1495, made the more defini-
tive observations that "rose-like bushes have fruits as long as cinnamon, full of small
grains, as biting as pepper; those Caribes and Indians eat that fruit like we eat
apples".
Chanca, the physician with the expedition, observed that the natives used this food
additive both as a condiment and in medicine. Other travelers, who extensively tra-
veled in Spanish America in the 16th and 17th centuries, described the popular use
of Uchu, a colorful fiery pod of a plant, in the food of Peruvian Indians. Since early
times, the internal use of a similar fruit as a cure for cramps and diarrhea by Mayan
Indians of northern Guatemala has been recorded. The highly irritating smoke from
the burning dried fruit is reported to have been used by the native Indians against
Spanish invaders.
For many years (centuries) prior to the Spanish occupation, the native Indians had
been cultivating this piquant food additive. There was a considerable variation in
color, shapes and sizes in the different regions. The great variety of capsicums used
by the native tribes was thus known by different names, as mentioned earlier.
Chilli or Chili, a name now commonly used in Asia and Africa, is said to have
come from the Nahuatl dialect of Mexico and Central America. A wealth of original
material thus became available and is still available in these areas for taxonomic and
developmental work.
22
2.2.1. Terminology and modern history of Capsicum
The term "chilli" is a rather confusing terminology: "chilli", "aji", "paprika",

"chile", "chile" and Capsicum are all used frequently and interchangeably for "chilli
pepper" under the genus of Capsicum, which belongs to a dicotyledonous group of
flowering plants. A particular species of Capsicum is called "chili pepper" in parts
of Mexico, southwestern United States and parts of Central America. To make mat-
ters still more confusing, a "sweet bell pepper" is often referred to as "Capsicum
pepper", whereas to refer to the term "chilli", "hot pepper" is used. The term "bell
pepper" is used to refer to a non-pungent, chunky, sweet chilli type, whereas "chilli
pepper" generally refers to a pungent chilli variety.
Red peppers have been familiar to all Spanish South Americans by the Arawakan
name "aji" and by the Nahuatlan name "chilli" in Mexico and Central America. The
genus Capsicum, which is commonly known as "red chile", "chilli pepper", "hot
red pepper", "tabasco", "paprika" and "cayenne", belongs to the family Solanaceae
(Night shade family) that includes tomato, potato, petunias and tobacco (Hawkes et
al., 1979; Macrae, 1993). According to some references, the popular name "chili" or
"chilli" originates from the hot pepper specially cultivated in the South American
country, Chile. However, the name "chilli" seems to have nothing to do with the
country name, on the contrary, it is believed to have originated from a district of
Central America (De, 1992, 1993, 1994, 2000).
One of the very first sources of life among the Inca, the "Commentaries Reales"
by Garcilaso de la Vega, "El Inca", in 1609, mentions the common or even daily use
of "chillies". According to de la Vega, there are three different kinds of chilli, two of
which can be identified as "aji" and "rocoto", while the third is only insufficiently
described.
Even though the chilli is referred to by different names within the same country,
and even in different states or provinces, the botanical name of chilli is the Latin
name Capsicum. The word comes from a Greek based derivate of Latin "Kapto"
meaning "to bite". The word "chili" is a variation of "cil" derivated from the
Nahuatl (Aztec) dialect, which referred to plants now known as Capsicum, whereas
"aji" is a variation of "axi" from the Arawak dialect of the Caribbean. This brings
us up to the point of the correct way to spell "chile" (Domencini, 1983). The "e"
ending in chile is the authentic Hispanic spelling of the word, whereas English lin-
guists have changed the "e" to an "i". From the Nahuatl dialect of the Aztec lan-
guage, the name "Chilrepin" has been derived. This was the name given to the ear-
liest known word chile, the combination resulting in "Flea Chile", which is
believed to allude to the sharp biting taste of the chilli pepper. Down the ages the
original name has been slowly reformulated as "chile + reprintl" to "chilecping" to
"chilrepin" to "chilepiquin", the latter two names being frequently interchan-
geable. The version used depends upon the source of information. However,
Capsicum annuum var. aviculare is the modern scientific name of this earliest
known variety (Macrae, 1993).
However, a multilinguistic nation (like India) represents a unique case, where the
same specimen Capsicum annuum L. is referred to by different names in different
parts of the nation. In original Sanskrit the plant is known as "Mairichi phalam
23
and "Bruchi". However, in modern Indian languages it is known by different names,
and even by more than one within the same language as given in Table 2.
T a b l e 2. S u m m a r y of C a p s i c u m s in t h e l a n g u a g e s of different n a t i o n s *
Language Common name
Pharm Fructus C a p s i c i a c e r
Arabic Felfel, B i s b a s , F u l f u l h a l u
Amharic Mit'mita, Berbere
Assam i Joloka
Bulgarian C h e r v e n piper
Bengali Lanka, M o r i c h
Burmese N g a yut thee, Nil thee
Chinese Lup-Chew
Danish Chili
Dutch S p a a n s e peper, C a y e n n e p e p e r
English C a y e n n e pepper, R e d pepper, C h i l l i , Chili
Estonian Kibe paprika
Finnish Chilipippuri
French P o i v r e r o u g e , P i m e n t e n r a g e , P i m e n t fort, P i m e n t - a i s e a u , P o i v r e d e C a y e n n e
German R o t e r Pfeffer, C a y e n n e - P f e f f e r , Chili-Pfeffer, B e i f t b e e r e
Gujarati Lai m a r c h a (red), Lila m a r c h a (green)
Hebrew Pilpel adorn
Hindi Lai m i r c h (red), H a r i m i r c h (green)
Hungarian C s i l i p a p r i k a , Igen eros a p r o , C a y e n n e bors, C a y e n n i b o r s ,

M a c s k a p o c s paprika, Aranybors, Ordogbors, Chilipaprika
Icelandic Chilipipar, Cayennepipar
Indonesian Lombok, Cabe, Cabai
Italian P e p e r o n e , D i a v o l e t t o , P e p e r o n c i n o , P e p e di C a i e n n e , P e p e rosso p i c a n t e
Japanese Togarashi
Kannada Menashinakay
Laotian M a k p h e t kunsi
Malay Lada merah
Malayalam Mulagu
Marathi Lai m i r c h y a (red), H i r v y a m i r c h y a (green)
Oriya Lankamaricha
Pashto Murgh
Portuguese P i m e n t a o , Piri-piri, P i m e n t a d e c a i e n a
Punjabi Lal-mircha
Russian Perets krasni
Sanskrit Marichiphala, Ujjvala
Singhalese Rathu miris, Gasmiris
Spanish C h i l e , G u i n d i l l a , C a y e n a inglesa, P i m i e n t a d e C a y e n a , P i m i e n t a p i c a n t e , Ajf
24
Table 2. Cont'd
Language Common name
Swahili Pilipili h o h o
Swedish Chilipeppar
Tagalog S i l i n g l a b y o , Sili
Tamil Mulagu
Telegu Mirapakya
Thai Pisi h u i , Prik k h e e , Prik
Tibetan S i p e n m a r p o , Si p a n d m a r p o
Turkish A c i kirmizi biber
Urdu Lalmarach
Vietnamese Ot
* After the paper of Basu, S. K., De, A. K. (2003): Capsicum: Historical and Botanical Perspectives.
Taylor and Francis Ltd., London (with permission)
The chilli pepper is one of the very old domesticated plants of Middle America. In
the Valle de Tehuacan (Puebla) which is one of the best documented examples of an
early settlement in Mesoamerica, archeological evidence for the consumption of chilli
peppers dates back to the seventh millennium B.C., long before the cultivation of
maize and beans. The early findings of peppers - in coprolites and charred remains -
were probably harvested in the wild. However, domesticated chillies similar to their
modern varieties in both size and shape can be found from the fifth millennium B.C.
onwards. In pre-Columbian Mexico, chilli was one of the preferred tributes which
dependent city-states had to deliver to their hegemonial powers. The paying of tribute
also in the form of chilli peppers was later continued by the new Castilian rules.
Capsicum has been known since the beginning of civilization in the Western
Hemisphere. It has been a part of the human diet since about 7500 B.C. (MacNeish,
1964). It was the ancient ancestors of the native people, who took the wild chilli
piquin and selected the values types known today. Heiser (1967) stated that between
5200 and 3400 B.C., the native Americans were growing chilli plants. This places
chilli among the oldest cultivated crops of the Americas. Seeds found in early
dwellings indicate that the natives were enjoying the peppers in 7000 B.C., along
with potatoes in the Andes. In Mexico dry pepper fruits and seeds were received
from 9500-year-old burials in Tamaulipas and Tehuacan. Domestication might have
taken place 10,000 to 12,000 years ago.
Christopher Columbus is believed to be the first European to discover chilli du-
ring one of his legendary travels to America around 1493. He was looking for an
alternative source of black pepper which at that time was the favorite spice of
Europe. What he discovered was a small fiery pod that for centuries provided sea-
soning for native Americans, the hot chilli pepper. It has to be emphasized that chilli
or capsicum is not related to the Piper genus, which contains Piper nigrum L. of the
family Piperaceae, the source of the black and white pepper. Within a century after
its discovery hot chilli pepper attained a worldwide distribution. According to some
25
other sources, the American origin of Capsicum was first reported in 1494 by
Chanca, a physician who accompanied Columbus in his second voyage to the West
Indies (Macrae, 1993).
Chilli peppers grow as a perennial shrub in suitable climatic conditions. The ge-
nus usually represents glabrous, perennial, woody subshrubs, some tending to be
vines, rarely herbs, which are native to Central and South America, live for a decade
or more in the tropical conditions of their natural habitat, but are mostly cultivated
as annuals elsewhere. Chilli is native to the Western Hemisphere and probably
evolved from an ancestor from the area of Bolivia and Peru.
The first chillies consumed were probably collected from wild plants. Prehistoric
Americans took the wild chilli "Piquin" and selected it for the various pod-types
known today. However, domesticated chilli was apparently not grown prehistorical-
ly in New Mexico (Macrae, 1993).
In fact, it is not known exactly when chillies were introduced into New Mexico.
Chillies may have been used by the indigenous peoples as a medicine, a practice
common among the Mayans. By the time the Spanish arrived in Mexico, Aztec plant
breeders had already dozens of varieties. Undoubtedly, these chillies were the pre-
cursors to the large number of varieties found in Mexico today. Whether chillies
were traded and used in New Mexican pueblos is still not clear (Macrae, 1993).
Capsicum species have been thought to be of Central American origin, but one spe-
cies has been reported to be introduced in Europe in the fifteenth century. By the middle
of the seventeenth century, Capsicum was cultivated throughout Southern and Middle
Europe as a spice and/or medicinal drug. One species was introduced into Japan and
about five species were introduced into India, of which Capsicum annuum L. and C.
frutescens L. were cultivated on a large scale (The Wealth of India, 1992).
In commerce the description given applies to various African commercial varie-
ties and these and the Japanese variety are sold in the United Kingdom as chillies,
while the larger but less pungent Bombay and Natal fruits are sold as Capsicums.
Very large Capsicum fruits that resemble tomatoes in texture and are practically non-
pungent are widely grown in Southern Europe as vegetables (Evans, 1996).
Records of the prehistoric Capsicum species around burial sites in Peru indicate
that the original home of the chillies may be tropical South America. These seem to
have been diffused from there to Mexico, or an independent origin in the latter coun-
try, where a great diversity of the genus is found. While Capsicum annuum has not
been recorded in the wild state and Capsicum frutescens doubtfully so, they have
now naturalized in the tropics of many countries and are easily disseminated by birds
(The Wealth of India, 1992).
The plant was introduced into Spain by Columbus, from where it spread widely.
Subsequently, the prolonged viability, easy germination, and easy transportation
assisted its spread all across the globe. The original distributions of this species
appear to have been extending from the South of Mexico into Columbia (The Wealth
of India, 1992). "Ginnie Pepper" was well known in England in 1597 and was grown
by Gerarde (Evans, 1996).
The Portuguese introduced chilli into India. Chilli is used as a condiment in large
quantities in India, Africa and tropical America, where the fruit develops greater
pungency than in colder regions. It has now, however, become a popular condiment
26
all over the World. The long, thin fruits constitute the source of dry chillies used for
commerce. The wide popularity of chilli and its extensive cultivation are due to its
being a short duration crop and its ease of cultivation under a wide range of climatic
and adaptive conditions, particularly in comparison with black pepper {Pepper
nigrum L.). The cultivation and utility of both Capsicum spp. are similar, except for
local peculiarities (The Wealth of India, 1992).
Capsicums are mentioned in a classic text of the Tibetan medical tradition, the
"Blue Beryll": "Capsicum (tsi-tra-ka) increases digestive warmth of the stomach, and
is the supreme medication for the alleviation of edema, hemorrhoids, animalcules,
leprosy and wind". Another passage tells us "bad-kan-nad-sel tsje-'phel tsi-tra-ka
mar sbrang sbyar - to alleviate diseases of phlegm and prolong the lifespan: [use]
Capsicum mixed with butter and honey". Interestingly, the spice translated as cayenne
pepper has another name, Yer-ma, and is generally used to treat wind disorders.
In pre-Hispanic medicine, chilli peppers were used to treat a host of conditions,
often in combination with other plant and mineral substances. For example, chilli
was used to treat diseases of the gastrointestinal tract (infections, diarrhoea), in addi-
tion to tooth pain, cough and lack of appetite. It was popular as an aphrodisiac, as
well. For that reason, the Spanish Padre Acosta, traveling through New Spain in the
sixteenth century warned that high consumption of the chilli peppers would be detri-
mental to the "soul's health" because it "promoted sensuality" (Acosta, 1985). Chilli
peppers had other uses as draconic punishment in children's education, and even in
warfare - the enemy was driven out of his fortification by the employing the acrid
smoke of smoldering chillies. This tactic was employed in pre-Columbian times, but
also during the Mexican revolution at the beginning of the twentieth century.
2.2.2. History of Capsicum in Hungary
Spain cultivated the pimientos, valued for the brilliant red color, characteristic aro-
ma, and mild pungency. These are more and more being produced as an industry
with close state control, integrating the steps of cultivation, processing, quality con-
trol and marketing. In Europe, Hungary and the Balkan countries have been produ-
cing the carefully selected cultivars of paprika since the 18th century and possibly
still earlier.
Hungary and the Balkan countries have been the traditional producers of highly
valued burgundy red, elongated conical, fleshy smooth erect fruit of paprika for its
color and flower. The main areas of cultivation in Hungary are Szeged and Kalocsa
alongside the Danube (Govindarajan, 1985).
The first written records of the appearance of paprika in Hungary are from the
middle of the sixteenth century. Paprika was grown as a rarity in the garden of Mar-
git Szechy, the step-mother of the distinguished general Miklos Zrinyi. It can be
assumed from the information of the next two to three centuries that Capsicum was
cultivated only to be used as a condiment. The cultivation of the sweet Capsicum
known today began at the end of nineteenth century. Bulgarians were the first grow-
ers who cultivated Capsicum in the southern part of the country (Szentes and sur-
roundings) (Somogyi et al., 2003).
27
At the beginning of the twentieth century, production of sweet Capsicum spread
to other parts of Hungary. It is interesting to note that the sweet Capsicum growing
regions are not the same as the traditional paprika producing areas. Important sweet
Capsicum areas were in the middle of the country (Boldog, Nagykoros, Cegled), and
in the south-southeastern part (Gyula, Baja, Bogyiszlo). But today this typical vege-
table is cultivated all over the country.
The production of condiment paprika in Hungary is highly labor-intensive. It
became a significant crop at the end of the nineteenth century, although reports of its
cultivation exist from the sixteenth century in Szeged, where the crop was mentioned
as one of the crops grown. It was introduced from Turkey by monks, who excelled in
healing, and was used as an effective medicine against malaria.
Looking at the history of Hungarian paprika production we can distinguish seve-
ral classical periods:
- Until the middle of the nineteenth century feudalistic family self-sufficiency and
the beginning of the production for the market;
- Until the beginning of the First World War paprika production is market-oriented
based on free competition;
- From the end of the First World War until the end of Second World War a state-
regulated production order was characteristic;
- Paprika milling was under state monopoly for the period of 1940-1990;
- In the last decade of the twentieth century again free market: production and pro-
cessing are based on competition.
Spice made of paprika is known as "Hungaricum" worldwide, and is an essential
element of the Hungarian cuisine. Until the turn of the nineteenth century it was
known in public life mainly as medicine.
Paprika (Capsicum annuum L.) originated from South America and came to
Europe - probably first to Spain in 1493, after the discovery of the American conti-
nent (Pickersgill, 1986, 1989). From there it came to Hungary across the Balkan
through Turkish growers. The first paprika plants were planted at the end of the
1500s. At first paprika was considered as an ornamental plant and was grown for
culinary usage at the beginning of the seventeenth century. The plant later enjoyed
tremendous popularity around the time of Napoleon (Somos, 1981). The book con-
taining its first detailed description was written by Csapo (1775). According to this
book it is grown in vegetable gardens and the long red fruit is dried and crushed to a
powder.
Veszelszky (1798) mentioned around that time that the farmers of Fot, Palota and
Dunakeszi grew paprika.
The first cultivation trials were conducted at the botanical garden of the University
of Pest in 1788. Since that time different Capsicum varieties were found in the "In-
dex seminum" of the botanical garden (Augustin, 1907). In letters that Count
Hoffmansegg sent his wife about his journey in Hungary, he mentioned "here I real-
ly liked a Hungarian dish, meat with paprika. I must be very healthy" (Balint, 1962).
Augustin, a German traveler did not talk so nicely about the Hungarian paprika in
his book, "Die Ungarn wie sie sind" (1831). He called paprika "Diablische Paprika
Briihe". He wrote that for people who are not used to it, the effect on the palate is
like embers or even worse (see for details Somogyi et al., 2003).
28
3. Botanical taxonomy
Capsicum is a genus of the family Solanaceae and is closely related to another ge-
nus, Solanum, which covers many economically important plants such as the potato
{Solarium tuberosum L.) eggplant {S. melongene L.) tomato (Lycopersieon esculen-
tum Mill.), and tobacco (Nicotiana tobacum L.).
Some 20 to 30 species of Capsicum are reported to have their origin in the New
World covering the tropical area of Northern South America, Central America,
Mexico, and the islands in that region. Many of these occur in the wild as natural
growth in areas of undisturbed vegetation, though their fruits were collected and dis-
tributed throughout the marketplace.
Exploration and plant collection throughout the New World had given us a gener-
al but false impression of specification in the genus. Humans selected several taxa
and in moving them toward domestication selected the same morphological shapes,
size and colors in at least three distinct species. The early explorations in Latin
America were designed to sample the flora of a particular region. Thus, any collec-
tion of Capsicum was a matter of chance and usually yielded a very limited sample
of pepper from that area. Only with the advent of collecting trips designed to inves-
tigate a particular taxon did the range of variation with species begin to be under-
stood. One needs only to borrow specimens from the international network of
herbaria to appreciate what a limited sample exists for most taxa, particularly for
collections made prior to 1950. The domesticated Capsicum pubescent, e.g., that is
wide-spread in the mid-elevation Andes from Columbia to Bolivia, is barely repre-
sented in the herbaticum collection of the world.
The taxonomy of Capsicum represents a very complex approach to different spe-
cies. Many classifications were published earlier (see Govindarajan, 1985; Basu,
De, 2003).
In 1957 Smith and Heiser recognized five species only, which gives the "Big Five"
species of Capsicum. Further recent works by Eshbaugh, Pickersgill and Hunziker
identified 22 other wild species (Macrae, 1993).
The wild gene pool, tightly linked to the domesticated, is designated Capsicum
baccatum var. baccatum and is most common in Bolivia, Brazil, Chile and Argentina.
Its flowers are solitary at each node. The pedicels are erected or declining at anthe-
sis. Corollas are white or greenish-white, with diffuse yellow spots at the base of
corolla lobes or either side of middle-vein (the flower is white with yellowish spots,
anthers are white but turn brownish-yellow with age): the corolla lobes usually are
29
slightly revolving. The calyx of mature fruit is without annular constriction at its
junction with the pedicel (though sometimes irregularly wrinkled), veins prolonged
into prominent teeth. The fruit flesh is firm. Seeds are straw-colored. Chromosome
number is 2n=24, with one pair of acrocentric chromosomes (Escabeche, Peru).
The following five major species are morphologically definable:
Capsicum annuum var. annuum L., Capsicum frutescences L., Capsicum chi-
nense Jacq, Capsicum baccatum var. pendulum wild, Capsicum pubescent Riuz and
Pav (Table 3)
T a b l e 3 . T h e m o r p h o l o g i c a l i d e n t i f i c a t i o n of t h e f i v e m a j o r s p e c i e s *
Species Flower color Number flw/node Seed color Calyx constriction
C. annuum white 1 tan absent

C. frutescens greenish 2-5 tan absent
C. Chinese white/greenish 2-5 tan present
C. baccatum white with 1-2 tan absent
y e l l o w spot
C. pubescens purple 1-2 black absent
* After the paper Basu S.K., De, A.K. (2003): Capsicum: Historical and Botanical Perspectives Taylor and Francis
Ltd., London (with permission)
The haploid chromosomal count of the cultivated and wild species is 12. There
are wide variations among and within the species, whether wild or cultivated, as no
karyotype is characteristic for any single species and certain characteristics are
observed among the majority of the members. Natural polyploidy is reported in the
case of Capsicum, although a spontaneous tetraploidy has also been reported in an
intravarietal cross. Induced polyploidy with colchicine has also been reported where
the induced polyploid exhibits a high vitamin C content profile. Diploids showing
mitotic abnormalities and irregulaties have also been reported (The Wealth of India,
1992).
Generally, there appears to be a well-developed sterility barrier between cultiva-
ted species. It is impossible to cross Capsicum pubescens with other species. Several
crosses between Capsicum annuum, Capsicum frutescens and Capsicum baccatum
var. pendulum have produced a few Fl hybrids, but are mostly highly sterile. In the
case of favorable crosses, the success or failure depends on the direction of the cross.
Reciprocal differences were observed in the case of fertility. The results of hybridiza-
tion also differed according to the parental cultivars. Viable seeds have easily been
produced from Capsicum annuum X Capsicum chinense and Capsicum frutescens X
Capsicum pendulum. The crosses Capsicum annuum X Capsicum frutescens and
Capsicum frutescens X Capsicum chinense have yielded a few viable F l , F2 and
bud cross seeds (The Wealth of India, 1992)
Pronounced differences have been found in the capsaicinoid composition of the
individual species, and the chemotaxonomic basis for identification is in line with
the earlier classification based on floral morphology and can therefore be used as an
30
additional method. The key for the identification of the individual Capsicum species
is given based on the limits of the total and the three individual capsaicinoids and
their properties in Table 4.
T a b l e 4 . A c h e m o t a x o n o m i c k e y t o t h e i d e n t i f i c a t i o n of c u l t i v a t e d C a p s i c u m s *
N D H C fraction o v e r 9 . 5 %
1.1 C fraction o v e r 5 6 % Capsicum baccatum var. pendulum
1.2 C fraction u n d e r 5 6 % Capsicum annuum var. annuum
2 N D H C C Fraction 9 . 5 %
2.1 C f r a c t i o n , 4 2 t o 5 7 % Capsicum annuum var. annuum
2.2 C f r a c t i o n u n d e r 4 2 % Capsicum baccatum var. pendulum
2.3 C f r a c t i o n , 5 7 t o 7 3 %
D H C fraction 2 6 t o 3 4 %
Total c a p s a i c i n o i d s , u n d e r 0 . 3 5 % Capsicum baccatum var. pendulum
2.4 C fraction o v e r 6 3 %
D H C Fraction u n d e r 3 2 %
Total c a p s a i c i n o i d s , o v e r 0 . 3 5 % Capsicum frutescens -
Capsicum chinense complex
* After Jurenitsch J . , Kubelka, W., Jentzsch, K. (1979c): Identification of Cultivated Taxa of Capsicum -
Taxonomy, Anatomy and Composition of Pungent Principles. Plant. Med. 35: 174-180 (with permission)
Abbreviations: C-Capsaicin; NDHC-Nordihydrocapsaicin; DHC-Dihydrocapsaicin
Thus, on the basis of the above studies, one may come to the conclusion that the
genus Capsicum represents a very wide and divergent taxonomic group consisting
of both wild and cultivated species. Some workers consider Capsicum to consist of
three principal species, C. annuum, C. frutescens and C. chinense; meanwhile others
have divided the genus into a divergent spectrum of species. Probably, molecular
biology and molecular genetics, phytochemistry and cell biology can contribute to a
deeper recognition of the taxonomy status of the genus Capsicum.
31
4. Cultivation of Capsicum or paprika
Capsicums, indigenous to South and Central America, Mexico and West India, con-
tinue to be cultivated there and have been introduced and widely cultivated through
temperate, subtropical Europe, the southern of United States of America, tropical
Africa, India, East Asia and China.
The cultivation - through the last 400 years in the different soils, cultivation con-
ditions, natural hybridization and selections - has given us a variety of Capsicums.
Valued solely for their color and secondarily for aroma and mild pungency are the
Spanish and Hungarian paprikas. For color, medium to high pungency and aroma
are the chillies grown in India, Southeast Asia, China and America, and essentially
for pungency the chillies in tropical Africa. Typical practices of the cultivation of
Capsicums are written briefly below.
4 . 1 . Cultivation of Capsicums in India
Chillies have been cultivated in India for over 200 years and have spread to most of
countries, from sea level to 1600 m above sea level with annual rainfall of 600 to
1250 mm. Throughout India, 14 states cultivate more than 10,000 ha each, and 3 of
them over 150,000 ha. The major chilli-producing states are Andhra Pradesh, Ma-
harasthra, Orissa, Karnataka and Tamil Nadu.
Chilli is a warm season crop, but low humidity with high temperature results in
the shedding of buds, flowers and young fruits. Very low temperatures also result in
poor growth. Chilli is started in three seasons, in different parts of the country. The
main crop on the southern plains in India - which produces 70% of the crop (total of
nearly 500,000 tons annually - is started in May to June, transplanted in 5 to 6 weeks,
and harvested from October. In the Punjab area the cultivation of crop starts at the
end of the cold season (in March to April), to avoid damage from frost, and harvest-
ed in August. In the Gangetic plains the crops are started in September and harvested
in February.
Chilli is cultivated in many areas in the north as a rain-fed crop, but in the south it
is increasingly cultivated as an irrigated crop, especially in Andhra Pradesh and Ta-
mil Nadu. The crop is grown in many types of soils but well drained loamy soils rich
in organic matter are considered the most suitable. Clayey loams which can retain
moisture are those in which chilli is cultivated as a rain-fed crop.
32
A variety of chillies, combining different sizes and degrees of pungency, are
grown in India, each area having their characteristic cultivar of Capsicum annuum
var. annuum. Generally, the medium to long and medium to high pungency red varie-
ties are grown in the plains and the long, deep red, thick-fleshed, low pungency types
are grown at higher elevations. Some amounts of vegetable capsicums and red short
blunt conical types are also grown. Minor amounts of the highly pungent, small coni-
cal variety of Capsicum frutescens are grown or collected from semi-wild growth.
A typical cultivation in Tamil Nadu, India is as follows; seedlings are started and
raised in carefully prepared beds with natural shade and near a water source.
4.2. Cultivation of Capsicums in other Asian countries
In the Indian subcontinent, other notable producers are Pakistan and Bangladesh,
where the cultivation practices are similar to that in India, in amounts of internal
consumption, with only a small percentage of export of a short conical variety
("dandicut cherry"). The other consumers of a number of Capsicum annuum, with
moderate to high pungency and good color, are Thailand, Indonesia and Malaysia.
In Southern Asia, South Korea is a substantial producer of highly colored and rather
high pungency chilli varieties, but with a very high consumption, it is not an exporter.
Since the 1950s, China has emerged as a major producer. A Federal Agricultural
Organization (FAO) estimate of the annual production of fresh chillies is put at 1
million ton. China has become a top exporter, equalling, and even exceeding India.
4.3. Cultivation of Capsicums in Africa
In the African continent, the crop was introduced early by the European conquests,
but largely remained as semi-wild plants and exists as an organized cultivation in
small holdings in only the North and West African countries. Both medium-sized
mild and high pungency small chillies have been produced. The mild form is pre-
ferred for local consumption, the export being dominated by the crop collected from
the wild growth in East Africa, the cultivated Zanzibar type from Sierra Leone, and
the mild to medium type from Kenya and Nigeria. Ethiopia is notable for the large-
scale cultivation of a variety of Capsicums, the mild to medium pungency red chillies
and more recently, paprika for oleoresin production.
4.4. Cultivation of Capsicums in America
In the Americas, the source of Capsicum, the Central and South American countries
continue to produce a great variety of Capsicums by collection of wild growths and
by rudimentary cultivation of specific local varieties mostly for internal consump-
tion. The wild Capsicums are reportedly used fresh, and medium pungency types are
dried product. The highly pungent small varieties from all the five species cultivated
are used essentially for therapeutic use. Mexico alone in this group appears to pro-
33
duce mild and high pungency chillies in amounts to have a substantial surplus for
export to the United States. It has been difficult to obtain information on organized
cultivation from these countries.
In the United States, Capsicums have been cultivated since the 1920s in California,
South Carolina, Louisiana and New Mexico. Considerable developmental work on
the selection of suitable cultivars adapted to this region optimal cultivation practices.
Different cultivars which combine the qualities of color and pungency in the required
proportions are grown and account for nearly half of the total requirement of chillies
and paprika, both of which had been earlier wholly imported. Moderately pungent
("Louisiana sport", "Carolinas" or "Anaheim" in California), the high pungency
("Bahanian" in South Carolina and "Tabasco" in Louisiana), the green bell Capsicum
("California Wonder" and "Florida Giant" for the fresh market in Florida and New
Jersey), paprika ("Ruby King" in California) and pimientos ("Perfection") in Geor-
gia exist as flourishing crop, raised through modern cultivation and processing
methods.
Large-scale cultivation and processing practiced in the United States are based on
the basic scientific information collected on the growth of capsicum plant, and the
formation of functionally important components for optimized economic produc-
tion. Considered as a warm season crop, Capsicums are cultivated in spring, summer
and autumn in the upper south of the United States and even in the winter in the
extreme south. Boswell recorded that the optimum temperature for growth is around
24C, and night temperatures lower than 15C or day temperature higher than 32C
are detrimental to growth and fruit set. Additionally, low humidity causes abscission
of flowers, buds and small fruits. At least 3 months of warm weather are necessary
for the quick maturing sweet vegetable Capsicums and 4 to 5 months for most other
cultivars. The small-fruited forms, those of Capsicum frutescent, are much more tol-
erant of higher temperatures and also are late maturing, requiring a longer warm
season. The Capsicums can be grown on well-drained soils, but a fertile loamy soil
rich in lime is considered most suitable. They are generally grown in rain-fed areas
having 600 to 1250 mm of rain annually and redden better under controlled irriga-
tion. Heavy rain and water logging lead to poor fruit set or fruit rot.
4.5. Cultivation of Capsicums in Europe and Hungary
In Europe, since the 18th century, Hungary and the Balkan countries have been pro-
ducing the carefully selected cultivars of paprika and possibly earlier still, Spain has
cultivated the pimientos, valued for their brilliant red color, characteristic aroma,
and mild pungency. These are more and more being produced as an industry with
close control, integrating the steps of cultivation, processing, quality control and
marketing.
Sweet Capsicum can be grown in any region of Hungary with the exception of the
western border of the country where the precipitation is higher and the temperature
is lower than average. Only 8 to 10% of the country's soil and climate conditions
are suitable for growing sweet Capsicum. Nevertheless, as traditional growing
regions evolved, immigrant Bulgarian market gardeners settled at the southern part
34
of the country and started vegetable production. However, in a significant part of the
region we find higher, sandy loam. These soils have inadequate water management
with less humus, the cultivation is easier and the soil warms up faster, at the same
time the quality of the condiment paprika is lower than that obtained from the heavier
soils. These lighter soils are suitable for intensive sweet Capsicum production under
controlled conditions. In the region of Kalocsa the soil is mostly heavy, with rela-
tively low humus content on the former flood plains of the Danube River.
4.5.1. Cultivar types in Hungary
4.5.1.1. Sweet Capsicum cultivar
The sweet Capsicum cultivars in Hungary have changed a lot during the last cen-
turies, especially during the last 50 to 60 years.
In the previous centuries the seed was handed down from father to son and the
growers passed the seed on to each other. The seeds of the best and earliest fruits
were always kept for further sowing. It can be assumed that on many occasions spon-
taneous pollination or mutation created new types or varieties. Breeding techniques
applying genetics go back only to the last 50 to 60 years in Hungary.
The first sweet cultivar which received State registration was "Cecei sweet 3"
bred by Angeli (see Somogyi et al., 2003). He selected this from a pungent white
type and created a non-pungent cultivar. With his work, an active and efficient
Capsicum improvement started and consequently the Hungarian sweet Capsicum
types and an assortment of cultivars have been continuously increased and spread all
over the World. The production of sweet Capsicum hybrid seed cultivars was initia-
ted in Hungary in the middle of the 1960s (Moor, 1969). Cultivars were made by the
breeding work of the state-owned Vegetable Crop Research Institute and its proces-
sors, and the Horticulture Departments of Universities until the middle of 1980s.
Since then, several private breeders' cultivars received Plant Variety Protection. All
the cultivars grown belong to the species of Capsicum annuum L.
The sweet Capsicum cultivars (varieties) are categorized into different groups by
the National Institute for Agricultural Quality Control (Table 5).
Approximately 100 cultivars were registered in the National List of Varieties in
2000.
One of the most important trains of sweet Capsicum cultivation is the sensitivity
to the lack of light (Zatyko, 1979). This characteristic is important for determining
whether the cultivar can be force-grown or not. This means establishing whether the
cultivar may be grown only in the field or whether its production is economical under
green-house conditions out-of-season:
1. Ciklon Fl - indeterminate, white fruit ripening to red, sweet, conical, upright
fruit, for all production systems. Fruits are 12 to 15 cm long and 5 to 6 cm in dia-
2
meter. Yield is 8 to 15 kg/m depending on the production technology. It contains
Tm2 resistance. Under local night conditions, sowing is done at the end of
September and the first 2 cm long fruits appear after a vegetation period of 125 to
130 days. This can decrease by 50% under more intense light conditions;
35
T a b l e 5. C a t e g o r i e s of t h e H u n g a r i a n s w e e t C a p s i c u m c a t e g o r i z e d b y t h e H u n g a r i a n N a t i o n a l
Institute for A g r i c u l t u r a l Q u a l i t y C o n t r o l *
W h i t e fruit, i n d e t e r m i n a t e p u n g e n t or w i t h o u t p u n g e n c y
W h i t e fruit, d e t e r m i n a t e without pungency
Pale g r e e n fruit, i n d e t e r m i n a t e without pungency
Hornshaped, indeterminate pungent, without pungency
P o i n t e d , hot, i n d e t e r m i n a t e without pungency
P o i n t e d , hot, i n d e t e r m i n a t e without pungency
Tomato-shaped without pungency
California W o n d e r type without pungency
Others pungent, without pungency
* After Somogyi, N., Moor, A., Pek, M. (2003): The preservation and production of Capsicum in Hungary.
In: De, A. K. (ed.) Capsicum. The genus Capsicum. Taylor&Francis Ltd, New York. pp. 144-162 (with
permission)
2. Taltos - white fruit turning to red, indeterminate, sweet, conical, blunt, pendulous
fruits, grown in the field. Fruits are 10 to 15 cm long and 5 to 6 cm in diameter.
Potential yield is 30 to 35 tons/ha;
3. "Pungent apple" - white fruit turning to red, indeterminate, pungent, apple-shaped
upright fruit, grown in the field. It is mainly used by the canning industry (to
pickle). Fruits are 6 to 7 cm, in diameter and 4 to 5 cm long. Potential yield is 18
to 25 tons/ha;
4. Feherozon - white fruit turning red, determinate, without pungency, upright fruits,
grown in the field and also under green-house conditions. Fruits are 12 to 15 cm
long and 5 to 6 cm in diameter. Yield in green-house conditions is 25 to 35 tons/ha;
5. Rapires Fl - pale green turning red, indeterminate, pungent, long, conical, pendu-
lous fruits. It can be grown in any type of controlled facilities. Fruits are 15 to 20 cm
long and 3 to 4 cm in diameter. It contains Tm2 resistance. Yield depends on the
2
technology, 7 to 8 kg/m ;
6. Tomato-shaped - green-dark green ripening to red, indeterminate, sweet, flat,
round, seamed, pendulous fruits. Fruits are 8 to 12 cm in diameter and 3 to 5 cm
long. The potential yield of fully ripe fruits is 18 to 20 tons/ha.
4.5.1.2. Paprika varieties
The Hungarian condiment paprika's cultivation period is short, there are only 5 to 5
and half months available for the vegetation period. In spite of the short vegetation
period, the quality of the harvested crop is excellent in most years. The high pigment
and the high dry matter contents guarantee a very good base material for milling.
The Hungarian varieties' yield can be up to 50% more, with improved attributes, by
cultivating them in areas where the vegetation period is longer. That is based on the
Hungarian-Spanish (Somogyi et al., 2003), the Hungarian-Portuguese and the Hun-
garian-Austrian (Derera, 2000) cooperative experiments. The full genetic potential
36
of the Hungarian cultivars is limited by the climatic conditions. Between 1993 and
2000 condiment paprika was grown on an area between 3,000 and 6,500 ha and
between 26,000 and 65,000 tons of raw paprika were produced. Consequently, the
quantity of the milled product varied between 3,000 and 9,400 tons.
All of the condiment paprika cultivars grown in Hungary were bred in Hungary.
They belong to Capsicum annuum L. covar longum by botanical classification. There
are two exceptions (cv. Kalocsai A. cherry type, cv. Kalocsai M. cherry type), regard-
ing the growth habit. They are continuous, semi-determinate and determinate. There
are two types of orientation of the fruits: erect and pendulous.
The categories are indicated below and the pungency is given in brackets:
1. Varieties of continuous growth habit, pendulous fruit: Szegedi 20 Szegedi 80,
Szegedi 57 13, Remeny, Kalmin, Szegedi 178 (pungent), Szegedi 179 (pungent),
Szegedi F-03 (pungent), Kalocsai 50, Kalocsai 90, Kalocsai V-2 (pungent), Kalo-
csai 15 (pungent), Csardas, Folklor;
2. Varieties of continuous growth habit, erect, Kalocsai 37 to 231;
3. Varieties of semi-determinate growth habit, pendulous fruits: Kalocsai 601, Kalo-
csai 702, Zuhatag;
4. Varieties of semi-determinate growth, erect fruit: Kalocsai 622, Rubin;
5. Varieties of determinate growth habit, erect fruits: Kalocsai D 601, Kalocsai D
621 (pungent);
6. The cherry-type paprika is classified as paprika, but it differs from the rest. It is
Capsicum annuum covar. cerasiforme and not longum. It is pungent, its impor-
tance is found in gastronomy. If green fruit is harvested, it can be pickled or made
into salad. When the ripe fruits are harvested they can be used for hot sauces or
dried spice (not milled) flakes. The two cherry cultivars that differ in fruit size and
growth habit are Kalocsai M and Kalocsai A.
A detailed description of the most typical cultivars of each category is given
below:
1. Cultivar of continuous growth habit and erect: Kalocsai 37 to 231, a sweet variety.
The bush is 45 to 55 cm high, its fruits are scattered and 10 to 14 cm long, slight-
ly bent. They are of flaming red color or dark red after post-ripening treatment. Its
main value lies in its good pigment (8 to 9 g/kg) and solids content. It is a middle-
early maturing variety. Its yield potential is 25-16 tons/ ha. It can be transplanted
or directly seeded. It has a good tolerance to diseases;
2. Cultivar of continuous growth habit and pendulous fruit: Szegedi 80, a sweet cul-
tivar. The fruits are 12 to 14 cm long, dark red when ripe, pigment content is 8 to
10 g/kg after post-ripening treatment. The solids content at picking is 20%. Its
yield potential under intensive conditions is 20 to 25 tons/ha. It has a reasonable
tolerance to diseases and can be transplanted or directly seeded. Due to an early
ripeness a reasonable yield can be relied upon before the first frosts. The Szegedi
F-03 is the same type but with pungency;
3. Cultivar of semi-determinate growth habit, with pendulous fruits: Kalocsai 801, a
sweet variety. It has loose spreading foliage. The plant is about 40 to 45 cm long,
the fruits weigh 22 to 28 g. They are straight, gradually tapering toward a pointed
tip, and their color is dark red when ripe. Their pigment content at picking is 6.0
to 7.0 g/kg, going up to 8.0 to 9.0 g/kg after post-harvesting. The dry matter con-
37
tent is 18% when ripe. It is an early, intensive variety bringing a high yield as an
exchange for watering and good nutriment supply. Its potential harvest is 20 to 22
tons/ha and it has a high tolerance to viral diseases;
4. Cultivar of semi-determinate growth habit with erect fruits: Kalocsai M 622, a
sweet cultivar. The bush is 35 to 45 cm high with sparse foliage, and has a rigid
stem with short internodes. Leaves are leathery and thick, so it has a good field
resistance to fungal infections. The fruit is 10 to 15 cm long, gradually tapering
towards a pointed tip, and is dark red when ripe. The pigment content at picking is
6.0 to 8.0 g/kg, increasing to 9.0 to 12.0 g/kg after post-harvest ripening. If trans-
planted, the entire crop can be harvested at the same time due to its short growing
season and early, uniform ripening. It is primarily directly seeded. It is the most
widely spread cultivar in the Kalocsa region. It requires intensive agronomic con-
ditions, but the compensation is a high yield. Its yield potential is 20 to 25 tons/ha.
It also has a high tolerance to diseases, which is the basis of secure production;
5. Cultivar of determinate growth habit and erect fruits: Kalocsai D 601, a sweet
variety with erect fruits appearing in bunches on the stem. The bush is 30 to 35 cm
high and the fruit bunches rise above the foliage and ripen uniformly. Fruits are
10 to 12 cm long, slightly bent, pointed and ripen to a deep red color. It contains
6.0 to 7.0 g/kg pigment at picking. The dry matter content is above average. It has
a short growing season and early, uniform ripening. It is primarily recommended
for direct seeding. On a large scale its can be harvested by machinery in one opera-
tion. The yield potential is 15 to 16 tons/ha;
6. Cherry-type paprika: Kalocsai M cherry type. It is the cultivar of pendulous fruits
and loose foliage. The bush is 40-60 cm high and of continuous growth. The fruit
is 3 to 3.5 cm in diameter, slightly flat and globe-shaped with a closed style point.
Its surface is smooth and its pigment content is 7 to 9 g/kg. The dry matter content
is 20 to 22% when ripe and the capsaicin content at picking is 120 to 140 g/100 g.
It has a good tolerance against viral infections. It has a middle-early, continuous
ripening and a good yield. It is primarily recommended for transplanting and
requires intensive growing conditions. It needs a soil rich in organic matter that
can be easily warmed up.
38
5. General chemical structure
and composition of Capsicums
5.1. Macroscopic characteristics
The dried fruits of the chilli variety are of varying sizes: 6 to 300 mm in length and 5
to 140 mm in width near the calyx. They present a variety of shapes, including the
long thin conical pointed tips, short thin conical blunt tips, short broad conical to
rounded tips, long broad with blunt tips and small round to oval in a few cultivars.
They are laterally compressed except in the small seed-filled varieties and some
paprikas. The vegetable Capsicum varieties are generally big, broad, blunt and
blocky with slightly sloping sides, while the paprikas are of medium length, broad,
blunt and round (Fig. I). The calyx and peduncle are usually attached, except in a
few small-sized fruits and in longer varieties bred for easy destalking during harvest.
Fig. 7. C a p s i c u m fruits v a r y i n g in s h a p e s a n d s i z e . 1 . tiny g l o b u l a r ; 2 . C h i l l i p i q u i n ( M e x i c o ) ;

3. M u n d u ; 4 . S i n d h o o r ; 5. L o n g R e d C a y e n n e ( U S A ) ; 6. M e x i c a n A n c h o ; 7. H u n g a r i a n
p a p r i k a ; 8. B e l l C a p s i c u m (all of t h e s p e c i e s Capsicum annuum var. annuum, L);
9. t a b a s c o ( U S A ) ; 1 0 . bird's e y e ( A f r i c a n ) , ( 9 - 1 0 ) b o t h of t h e s p e c i e s Capsicum frutescens
L; 1 1 . o b l o n g ; 1 2 . m e d i u m b r o a d ; 1 3 . l o n g p o i n t e d fruits of Capsicum baccatum var.
pendulum ( W i l d ) (after G o v i n d a r a j a n , V . S . , 1 9 8 5 : C a p s i c u m - P r o d u c t i o n , t e c h n o l o g y ,
chemistry, a n d q u a l i t y - Part I: History, b o t a n y , c u l t i v a t i o n , a n d p r i m a r y p r o c e s s i n g .
C R C , C r i t i c a l Rev. F o o d S c i . Nutr. 2 2 : 1 0 9 - 1 7 6 ) ( w i t h p e r m i s s i o n )
39
The pericarp is lustrous, smooth and its color generally ranges from deep red to
brownish red or orange. A few cultivars with the ripe mature color such as deep pur-
ple or white are known as also partially bleached orange color due to poor dyeing.
Paprika and pimientos are generally of intense burgundy red, while vegetable
Capsicums are known with both green and deep red colors at fully maturity. The
fruit has two to four loci marked by longitudinal ridges with a central placenta
extending from the base under the calyx to the apex end, except in vegetable
Capsicums where the placenta is confined to the base (Fig. 2). Seeds are numerous,
attached to the central placenta, are yellow (black in one species), smooth, 2.1 to 5
mm, discoid with thickened edge, and prominent pointed micropyle.
Fig. 2. Cross-sections of C a p s i c u m a n d c h i l l i fruits. P e : p e r i c a r p ; D : d i s s i p i g m e n t s ; S: s e e d s ;

P I : p l a c e n t a (after C o v i n d a r a j a n , V . S . , 1 9 8 5 : C a p s i c u m - P r o d u c t i o n , t e c h n o l o g y ,
c h e m i s t r y , a n d q u a l i t y - Part I: History, botany, c u l t i v a t i o n , a n d p r i m a r y p r o c e s s i n g .
C R C , C r i t i c a l Rev. F o o d S c i . Nutr. 2 2 : 1 0 9 - 1 7 6 ) ( w i t h p e r m i s s i o n )
5.2. Microscopic characteristics
The microscopic characteristics of Capsicums are seen on the cross-section.

A cross-section of the fruit shows the pericarp components, the cuticular layer of
cells, the epidermis of the single layer of regular rectangular cells, the hypodermis
of tangentially oval cells with thick walls in one or more layers, the mesocarp of
parenchyma cells, having oblong, tangentially compressed and greatly varying sizes
and frequently containing droplets of the red oil, occasionally crystals of calcium
oxalate. In the inner regions are the vascular bundles and giant cells causing blisters
on the inner surface of the pericarp, a characteristic of all Capsicum fruits, and the
endocarp of rectangular cells with thick pitted walls.
The diagnostic characteristics of ground Capsicum include all tissue components
described above and fragments of calyx and pedicel, all mixed up.
Jackson and Snowdon (1974) described the following microscopic diagnostic
characteristics of Capsicum annuum L.:
40
1. numerous pale yellow fragments of the epicarp as single layer of polygonal cells
with beaded walls, some showing strongly striated cuticle;
2. the abundant parenchyma of the mesocarp, thin-walled, containing orange to red
oil globules and occasionally microspheroidal crystals of calcium oxalate;
3. the sclerenchymatous endocarp as groups of polygonal cells with thickened sin-
uous walls and having distinct pits;
4. fragments of the epidermis of the testa composed of a layer of very large lignified
and markedly varying cells, unevenly thickened on the inner walls appearing as
balloon-like swellings;
5. endosperm, parenchymatous cells with drops of fixed oil and aleuronic grains;
6. fragments of calyx, the inner epidermis cells with numerous trichomes with stalk
and multicellular head;
7. fragments of pedicel tissues, elongated or small polygonal cells with stomata,
glandular trichomes, cellulosic parenchyma, fibers in isolated groups and paren-
chymatous pith with large central cavity;
8. seed structures, where present, are sinuous with thin outer and thickened, pitted
inner walls.
5.3. General chemical composition
Carbohydrate, protein, fat and fibre are the major components which rapidly increase
from the green stage to the ripe stage.
Fructose, glucose, galactose and sucrose were identified by gas chromatography
and enzymatical methods. Fructose is the major sugar together with glucose amount-
ing to about 70% of total sugars. Free sugar content in seeds was reported to be higher
than in the pericarp or placenta.
The most fat in Capsicums is in the seed. Values for total fat vary from 9 to 16%
depending on the seed content of the variety or processed sample. The high seed fat
content is largely found in cultivated species.
The fibre content varies greatly between the sources and reflects the amount of
pedicel included in the sample.
Three functionally important characteristics in the use of chillies as spice:
- red color and capsaicinoids,
- the pungency stimulants,
- characteristic sweet aroma of paprikas and vegetable Capsicums or the pungent
aroma of chillies.
Other important components of Capsicums are: citric acid is a major acid compo-
nent, the others being succinic, fumaric, malic and quinic acids (Table 6).
Of nutritional importance is the vitamin C content of paprika, the vegetable
Capsicums and chillies. Vitamin C was firstly isolated from paprika by Szent-Gyor-
gyi for which he was awarded the Nobel Prize in 1937. Pound for pound paprika is
higher in vitamin C content than citrus fruit. Contents of vitamin C are up to 340
mg/100 g in some varieties of paprika. The content of vitamin C in dried Capsicums
is only of the order of 30 to 60 mg/100 g, which indicates a large percent of loss of
vitamin C in processing these Capsicums.
T a b l e 6. C h e m i c a l c o m p o s i t i o n of C a p s i c u m s *
Compounds g/100 g fresh f r u i t ( m e a n s S E M )
Water 91 0.6
Glucose 0.85 +0.1
Fructose 0.750.1
Sucrose N.D.
Starch 0.81 0.2
Fibre 2.20.3
Pectin 0.730.1
mg/100 g fresh fruit (means S E M )
Citric acid 28+12
Fumaric acid 1.1 0.4
Malic acid 20818
Oxalic acid 14024
Quininic acid 18312
Vitamin C 2412
Chlorophyll a 7.92
Chrorophyll b 3.40.6
All-trans-lutein 1.40.3
All-trans-ls-carotene 0.920.4
* After Lopez-Hernandez, J . , Oruna-Concha, M. J . , SimaLLozano,)., Vazquez-Bianco, M. E.,

Gonzales-Castro, M. J . (1996b): Chemical composition of Padron peppers (Capsicum annuum L.) grown in
Galicia (N.W. Spain). Food Chemistry 57: 557-559 (with permission)
Abbreviation: N.D. not detected
The vitamins A and B complexes also have been shown in Capsicums. Values
reported of vitamin A are as high as 3315 international units in paprika and 3530 to
6165 IU in chilli varieties. The vitamin E content in fresh and dried paprika depends
on the ripening stage and genetic factors. The presence of 3 to 10 mg/100 g in fresh
ripe Capsicums could be an important source of vitamin E in the human diet. The
loss in drying is about 5%. Oleoresin paprika had earlier been shown to have as much
as 10 mg/g of a-tocopherol.
A total of 14 amino acids in green Capsicum and 16 in paprika, from which
asparagine and proline are dominant, were identified earlier. Recent analysis of
sweet and hot varieties showed no differences in amino acids, but the composition of
the seed was different from that of the pericarp.
Chillies and paprika are evaluated for uniformity of shape, size, color and pun-
gency combination of the trade types. The sweet or mildly pungent paprika is valued
for its bright color and to a minor extent fot its aroma, while the chillies for their
pungency and color.
The paprika group contains less than 0.1% capsaicinoids, the pungency stimu-
lants are 0 to 1.4%.
In addition to water which makes up more than 90% of Capsicum fruit, fibre,
pectin, glucose, starch and fructose represent the main components (Table 6).
6. Chemical taxonomy
of the functional parts
of the Capsicums
6.1 Capsicum: Botanical aspects

Capsicum is a versatile plant used as vegetable, a pungent food additive, a colorant
and a pharmaceutical. The genus Capsicum, which is commonly known as chili,
"red chili", "tabasco", "paprika", "cayenne", etc., is a member of the family So-
lanaceae, and closely related to eggplant, potato, petunia, tomato and tobacco.
Various authors ascribe 25 species to the genus, with new species to be discovered
and named as the exploration of the South American tropics expands. After much
work by taxonomists concerning the classification of the presently domesticated
species, they have been considered to belong to one of five species, namely
Capsicum annuum, Capsicum frutescens, Capsicum baccatum, Capsicum chinense
and Capsicum pubescens (Bosland, 1994). The Capsicum plants grow as perennial
shrubs in suitable climatic conditions. Capsicum fruits are considered to be vegetab-
les, but botanically speaking, they are berries. Capsicum types are usually classified
by fruit characteristics, i.e., pungency, color, fruit shape, as well as by their use.
Capsicum species are commonly divided into two groups, pungent and non-pun-
gent, also called hot and sweet.
The history of chile peppers is one of the enthusiastic acceptances wherever they
were taken. Chile is historically associated with the voyage of Columbus (Heiser,
1976). Columbus is given credit for introducing chile to Europe, and subsequently to
Africa and to Asia. On his first voyage, he encountered a plant whose fruit mimicked
the pungency of the black pepper (Piper nigrum L.). Columbus called it red pepper
because the pods were red. The plant was not the black pepper, but a heretofore
unknown plant that was later classified as Capsicum. Bolivia and Peru are the most
probable ancestral home of the chile pepper, but in the world at large Mexico gets
the credit for this plant, having cultivated the pepper types that entered the world's
cuisines after Columbus's discovery.
Capsicum species are used fresh or dried, whole or ground, and alone or in com-
bination with other flavoring agents. Most of the Capsicum cultivars commercially
cultivated in Europe and Amerika belong to the species of Capsicum annuum.
However, there are a few principal types which belong to other Capsicum species.
The most common types are "Bell", "Cayenne", "Cherry", "Jalapeno", "Mirasol",
"Paprika", "Pasilla", "Pimento", "Serrano", "Squash" and "Wax" (all Capsicum
annuum) as well as "Habanero" (Capsicum chinense), "Long Green" (Capsicum
baccatum), "Manzano" (Capsicum pubescens) and "Tabasco" (Capsicum frutes-
cens).
43
In many European languages, the name of this spice is somehow derived from the
name of pepper, owing to the many confusions of pepper with other spices. A rather
common designation of paprika is "sweet pepper": Spanish "pimiento dulce", French
"piment doux", and Arabic "fulful halu". It should be noted that in most of these lan-
guages, the word for "pepper" may also mean "chile", so it would perhaps be more
accurate to translate these names by "sweet chile" or "sweet chile pepper". Yet other
tongues have names for paprika that mean "red pepper" e.g., Turkish "kirmizi biber",
Bulgarian "cherven piper" and Russian "perets krasni". These may lead to confusion
as in a plethora of other languages, similar names are reserved for chiles.
Other English names are bell pepper and pod pepper because of the shape. Most
confusingly, the English plural peppers always seem to imply some sort of paprika
(also vegetable bell peppers or hot chile peppers), and never true black pepper! For
reasons of clarity, the term "pepper" for Capsicum species will be avoided later in
this chapter. Instead, the word "paprika" throughout for mild or medium-hot varie-
ties and "chile" for hot varieties will be used.
The word "paprika" was borrowed from Hungarian (paprika), it entered a great
number of languages, in many cases probably via German. In the end, also "papri-
ka" is derived from a name of black pepper, in this case Serbian papar. In most lan-
guages, "paprika" denotes the dried spice only, though in some (e.g., German) it is
commonly used for the vegetable bell pepper. The form "paprika" is valid in count-
less European languages, while examples with slightly deviating spelling include
Italian "paprica", Polish "papryka" and Bulgarian "piperka".
6.2. Capsicum: Chemical constituents
Capsicum fruits contain coloring pigments, pungent principles, resin, protein, cellu-
lose, pentosans, mineral elements and a small amount of volatile oil, while seeds
contain fixed (non-volatile) oil. Besides these organic constituents Capsicum fruits
also contain inorganic constituents, mostly potassium and sodium, calcium, phos-
phorus, iron, copper and manganese (Thresh, 1846; Brawer, Schoen, 1962; Brash et
al., 1988; Pruthi, 2003).
The pungent principles capsaicin and its structurally closely related homologes
(so-called capsaicinoids) and analogues, are contained only in small amounts, as
low as 0.001 to 0.005% in "mild" and 0.1% in "hot" cultivars. Apart from capsaicin,
the taste of paprika is mostly due to the fixed oil which is comprised mainly of
triglycerides of which linoleic, linolic, stearinic and other unsaturated fatty acids
predominate. The fixed oil content of the Capsicum seeds also play an important
role in the visual sensing of the paprika powder since it can dissolve and homoge-
neously distribute the colored substances during grinding of the dried fruits. The
characteristic aroma and flavor of the fresh fruit are imparted by the volatile oil con-
taining a range of alkylmethoxypyrazines (e.g., 2-methoxy-3-isobutyl-pyrazine, the
"earthy" flavor) and a structurally diverse group (alcohols, aldehydes, ketones, car-
boxylic acids, and esters of carboxylic acids) of oxygenated hydrocarbons.
Furthermore, the fresh ripe paprika contains sizable amounts (0.1 %) of vitamin C
(ascorbic acid). It was the Hungarian biochemist Albert Szent-Gyorgyi who discov-
44
ered that the Hungarian paprika is a rich source of vitamin C. Later (1937) he won
the Nobel Prize "for his discoveries in connection with the biological combustion
processes, with special reference to vitamin C and the catalysis of fumaric acid"
(Encyclopedia Britannica). During the post-ripening period and processing a sub-
stantial amount of vitamin C undergoes degradation. Paprika powder contains only a
little amount of it.
Paprikas derive their color in the ripe state mainly from carotenoid pigments,
which range from bright red (capsanthine, capsorubine) to yellow (cucurbitene); the
total carotenoid content in dried paprika is 0.1 to 0.5%. A small number of cultivars
do not produce significant amounts of carotenoids; when chlorophyll levels decrease
in the last stages of ripening, these chiles develop a pale hue often referred to as
"white". Due to small amounts of chlorophyll and/or yellow carotenoids, the "white"
is, however, more precisely described as a pale greenish-yellow.
Some varieties of paprika contain pigments of anthocyanin type and develop dark
purple, aubergine-colored or almost black pods; in the last stage of ripening, howev-
er, the anthocyanins get decomposed, and the unusual darkness thus gives way to
normal orange or red colors. The same anthocyanins cause the dark spots which are
sometimes seen on unripe fruits or particularly the stems of paprika plants and which
almost all paprika varieties can develop. In other Capsicum species, anthocyanin
production is a rare phenomenon.
6.2.1. Volatiles
The characteristic flavor and aroma of the fresh fruits is due to their volatile oil con-
tent. The fruits of Capsicum species have a relatively low volatile oil content, ranging
from about 0.1 % to 2.6% in paprika. The total volatiles are generally isolated by
steam distillation. In the case of heat-sensitive compounds present, vacuum distilla-
tion-continuous solvent extraction can be used. The pure volatile oil and concentrated
extracts were analyzed by GC-MS methods. Most compounds of odor significance
have been tentatively identified by their mass spectra, and the identification was con-
firmed by checking the retention time and mass spectra of authentic reference com-
pounds. When Buttery et al. (1969a,b) identified 3-isobutyl-2-methoxypyrazine (1)
(Fig. 3) as a characteristic aroma compound, the alkyl-methoxypyrazines aroused
great interest among flavor chemists. The alkyl-methoxypyrazines have been shown
to be widely distributed in vegetables and with a greenish sweet smell that possibly
plays a significant role in the aroma of salad vegetables (Murray, Whitfield, 1975).
In the volatiles of the green bell fruits (C. annuum var. grossum), different Cap-
sicum fruits with strong odor, the major character-determining component 3-iso-
butyl-2-methoxypyrazine (1) was easily identified by GC-MS technique and sensory
analysis. Other odor impact compounds identified of the bell capsicum volatiles
were hexanal (2), which has a mild nutty odor, cis-3-hexen-l-ol (3) with a green leaf
odor, the C(9) unsaturated ketones (non-l-en-4-one, non-trans-2-en-4-one, non-2-
trans-5-trans-dien-4-one), nona- and decadienals (non-2-trans-6-cis-dienal, non-2-
trans-4-trans-dienal), and a few terpenic compounds (e.g., limonene, trans-beta-
ocimene, linalool (4) (Buttery, et al., (1969a) (Fig. 3).
45
./CH
,o
3
CH,-CH
X
CH, C H , C H O 1 1 CI I C I I -c:
N- ÔCH 3
OH
H,C I
CH C H
3 2 x / C H C H OH
2 2
/
\ / C=CHCH CH CH-CH=CH
2 2 2
H H
CH,
H
3C ,CH
3
,x .CH 3
-CH C H = C H - C - C H
2 3 .C-CH -CH2
CH CH CH CH O S
CH,
3
2\ /<~"2
2 2
(' = ('
/ \
CH,
H 6
7 8 9
Fig. 3. C h e m i c a l structure of t h e m a i n o d o r s i g n i f i c a n t c o m p o u n d s of fresh Capsicum fruits

(see t h e text for d e t a i l e d e x p l a n a t i o n )
The pyrazines were also identified in other chile varieties, but in much smaller
amounts (Murray, Whitfield, 1975; Hoffman et al., 1978). Some common fruit esters,
predominantly C(6)-C(5) and C(6)-C(6) esters and related alcohols were identified
in the volatiles of ripe fruits of the variety of Tabasco (Haymon, Aurand, 1971).
A more elaborate search for aroma-significant compounds in the pungency-stimu-
lating varieties of Capsicum fruits has been reported (Keller et al., 1981). Volatiles
were obtained by steam-distillation from oleoresin of the highly pungent African
chilies (C. frutescens), the partially concentrated hexane extract of Miscella (C.
annuum variety) and the popular fresh mature green Jalapeno - a medium pungent
variety of Capsicum annuum. The 102 compounds identified in the three volatiles
belonged to the structural groups as follows: alcohols (e.g., hexanol, 2-methylbu-
tanol, 4-methylpentan-l-ol, cis-3-hexen-l-ol (3)), carbonyls (e.g., 4-methyl-3-pen-
ten-2-one, 2-hexanone, P-ionone (5), 5-methyl-2-furfural), carboxylic acids (e.g.,
2-methylbutyric acid, 4-methylpentanoic acid, hexanoic acid, octanoic acid), esters
(methyl pentanoate, methyl hexanoate, methyl heptanoate, methyl octanoate, methyl
46
tetradecanoate), pyrazines (e.g., 2,3-dimethylpyrazine, 2,3,5-trimethylpyrazine,
tetramethylpyrazine, 3-isobutyl-2-methoxypyrazine), terpene hydrocarbones (e.g.,
limonene, myrcene, (3-pinene, a-phelladrene, caryophyllene), and a group of miscella-
neous compounds (e.g., p-xylene, 2-pentylfuran, eugenol, pentadecane, hexadecane,
heptadecane).
Based on GC-MS and sensory analysis of volatiles of three cultivars of C. an-
nuum - Jalapeno, Fresno, and Anaheim - nine odor significant compounds were
identified that had been found in one or the other of the earlier studies: cis-3-hexen-
l-ol (3), cis-3-hexenyl-isopentanoate (6) (associated with green and green-fruity
odors); 3-sec-butyl- (7) and 3-isobutyl-2-methoxypyrazine (1) (with green vegetable
and green bell capsicum odors); (3-ionone (5) (only in Jalapeno), linalool (6) (both
with floral character); and the aromatic compounds benzaldehyde (8) and methyl
salicylate (9) (with sweetish, penetrating odors) (Chitwood et al., 1983) (Fig. 3).
6.2.2. Coloring pigments
The coloring pigments of red peppers are comprised of carotenoids. Carotenoids are
widely distributed groups of natural pigments, responsible for the yellow, orange,
and red colors of fruits, roots, flowers, fish, invertebrates, and birds. Only bacteria,
algae, fungi, and green plants can synthesize carotenoids, but humans incorporate
them from the diet. Especially bacterial carotenoids are most diverse. Carotenoid
extracts and fruits rich in carotenoids are now being used in the food industry to
color foods, thus such foods also represent carotenoid sources of the human diet.
The nutritional importance of carotenoids is mostly associated with the provitamin
A activity of (3-carotene and others. Besides its well-established provitamin A activity,
research is under way to study the relationship between [3-carotene intake and occur-
rence of atherosclerosis, cardiovascular diseases, in particular degree of LDL oxi-
dation (van Poppel, Goldbohm 1995; Hinds et al., 1997; Rodriguez-Amaya, 1997;
Woodall et al., 1997; Mailard et al., 1998; Manirakiza et al., 1999, 2003).
The basic carotenoid structure is a symmetrical, linear, 40-carbon tetraterpene
built from eight carbon isoprenoid units joined in such a way that the order is
reversed at the center. Fig. 4 shows the structure of (3-carotene (10), one of the most
typical carotenoid components of capsicum fruits.
4'
Fig. 4. C h e m i c a l structure of 6-carotene (10) (see t h e text for further e x p l a n a t i o n )
47
Carotenoids were for a long time assumed being synthesized by the mevalonate
pathway for isoprenoid biosynthesis. This view was prevalent up until the mid
nineties when it was discovered that the carotenoid precursor isopentenyl-pyrophos-
phate (IPP) was synthesized by two independent metabolic pathways in plants
(Lichtenthaler et al., 1997). The first pathway occurs in the cytoplasmic compart-
ment from mevalonic acid and gives rise to compounds such as sterols and cyto-
kinins. In fungi, carotenoids are derived via the mevalonate biosynthetic pathway.
The second pathway, effective in bacteria and plastids of plants, is responsible for
biosynthesis of gibberellins, carotenoids, abscisic acid, and also contributes to the
biosynthesis of tocopherols as well as chlorophyll A and B. This plastidial pathway
of isoprenoid synthesis is named after its first metabolite 1-deoxyxylulose 5-phos-
phate (DOXP) and has pyruvate and glycerinaldehyde-3-phosphate as precursors
(Fig. 5). The overall pathway of carotenoid biosynthesis can be considered as a series
of stages, as illustrated in Fig. 3. Further details can be obtained from a number of
review articles (Davies, 1980; Spurgeon, Porter, 1983; Britton, 1991).
A proper starting point of the carotenoid biosynthesis is the conversion of ger-
anylgeranyl pyrophosphate (GGPP) (11) to phytoene (12) by the enzyme complex
phytoene synthase (PSY) (Fig. 5). This enzyme takes two units of the C-20 com-
pound GGPP and condensates them in a "head-to-head" manner to yield the C-40
compound phytoene. Phytoene is not a true pigment since it is unable to absorb light
in the visible region. It undergoes four consecutive desaturation steps. In higher
plants these reactions are catalyzed by two related desaturases. The first two steps
are performed by the phytoene desaturase (PDS) and the latter two by zeta-carotene
desaturase (ZDS) (Linden et al., 1991). These reactions lead to formation of the pig-
ment lycopene (13), the main pigment of the red tomato. Lycopene produced by
these two desaturase reactions is in cw-form, also called pro-lycopene (Bartley et al.,
1999). There is one more enzymatic step to produce the all-trans-lycopene, that is
considered to be the main substrate for the subsequent reactions. Lycopene is the
major carotenoid in tomato.
/4/7-frarcs-lycopene can be modified in various manners, such as hydrogenation,
dehydrogenation, cyclization, double bond migration, chain shortening or extension,
rearrangement, isomerization, introduction of oxygen functions or combination of
these processes, resulting in a great diversity of structures. The number of naturally-
occurring carotenoids whose structures have been elucidated now numbers more
than 600. The number of carotenoids so far encountered in foods is much lower,
nevertheless, the carotenoid composition of a given food can be quite complex
(Sandmann, 2001).
Hydrocarbon carotenoids are collectively termed carotens, and those containing
oxygen are called xanthophylls. The most common oxygen functions are hydroxy
(OH) and epoxy groups. Hydroxy groups are common, particularly in position 3 or
4. Many carotenoids possess an epoxy group, usually in positions 5,6 or 5,8 (the 5,8-
epoxides are often referred to as furanoids or furanoid oxides), though some 1,2-
epoxides, 3,6-epoxides and 4,5-epoxides also have been reported. Carotenoids con-
taining aldehyde, keto, carboxy, carbomethoxy, and methoxy groups, acetates and
lactones, are also encountered. Carotenoids, whether carotens or oxocarotenoids
(xanthophylls), may be acyclic, monocyclic, or bicyclic. Cyclization occurs at one
48
Phytoene (12)
PDS
ZDS
OH
HO
OH
HO
HO
Violaxanthin (18)
Fig. 5. S i m p l i f i e d b i o s y n t h e t i c r o u t e of c a r o t e n o i d s (see text, for further d e t a i l e d e x p l a n a t i o n )
or both ends of the molecule, forming one or two six-membered rings (Pfander,
1987).
The next two steps of the simplified carotenoid biosynthetic route (Fig. 5) result
in bifurcation of the pathway, where one path is leading to the synthesis of P-caro-
49
tene (10) by two consecutive cyclization reactions catalysed by the enzyme P-caro-
tene cyclase (LCY-B) (Hugueney et al., 1995), and the other branch leads to a-caro-
tene (14), where two different cyclases are necessary. One of them is LCY-B, and
the other enzyme is e-carotene cyclase (LCY-E) (Cunningham et al., 1996). a- and
p-carotenes only differ in a position of a double bond in one of the end-rings, and
since not all bonds are part of the conjugated system in a-carotene, this leads to a
small but obvious difference in the characteristic carotenoid spectra of the two com-
pounds.
Oxygenated carotenoids are formed by hydroxylation of the precursor hydrocar-
bon carotenoids. For example, two consecutive hydroxylation steps convert P-carotene
first to cryptoxanthin and then to zeaxanthin (15) by the action of P-carotene hydroxy-
lase (BCH). a-Carotene is also twice hydroxylated to yield lutein (16), but by
two different enzymatic reactions. The beta-ring is hydroxylated by BCH, and the
epsilon-ring by -carotene hydroxylase (ECH).
The hydroxylated beta-rings of zeaxanthine are epoxidated in two steps to yield
first antheraxanthin (17) and then violaxanthin (18). In each step one atom of molec-
ular oxygen is incorporated into the substrate molecule while the other is reduced to
water. During light stress violaxanthin can be converted back to antheraxanthin and
zeaxanthin by the enzyme violaxanthin-deepoxidase. This flux between these three
compounds, governed by light intensity, constitutes the xanthophyll cycle. The last
of the ubiquitous carotenoids in higher plants is neoxanthin (19), produced from
violaxanthin by the enzyme neoxanthin synthase (NXS). Neoxanthin with an allenic
bond is widespread and is present in significant amounts in the leaves and green
stages of fruits of higher plants (Fig. 5). Characterization of the key enzymes of the
carotenoid biosynthetic pathways is summarized in a recent review along with their
biotechnological applications which resulted in development of heterologous
carotenoid producing bacterial and fungal systems (Sandmann, 2001).
Carotenoids can be extracted from natural sources by lipid solvents. With fresh
material, ethanol or acetone act both as dehydrating agent and extracting solvents.
When lipids and esterified xantophylls are present (hydroxylated carotenoids gener-
ally occur as esters of fatty acids), the extracts are saponified and the free carotenoids
extracted for analysis. Most carotenoids are unstable in oxygen atmosphere and light
thus careful extractions and separations are generally carried out under inert atmos-
phere, subdued light and low temperature (Rodriguez-Amaya, 1997; Deli, Molnar,
2002). High performance liquid chromatography (HPLC) is the most powerful chro-
matographic technique to separate and - coupled with mass spectrometry (MS) -
identify carotenoids. Recent reviews on the field provide up-to-date summary of
carotenoid analysis (Rodriguez-Amaya, 1997; Wall, Bosland, 1998; Deli, Molnar,
2002; Felt et al., 2005).
The composition of carotenoid pigments produced by paprika have been investi-
gated in detail. Some twenty carotenoids have been isolated so far with capsanthin
and capsorubin representing the most abundant (Deli, Molnar, 2002). The ripening
process is marked by the disappearance of chlorophyll and a rapid rise in the colored
carotenoids (Rahman, Buckle, 1980; Rodriguez-Amaya, 1997; Hornero-Mendez et
al., 2000; Gnayfeed et al., 2001; Deli, Molnar, 2002). The comprehensive analytic
data by Rahman and Buckle (1980) against the background of the biosynthetic rela-
50
tionship among common carotenoids summarized above shows similar formation
and biosynthetic relationship in Capsicum fruits at the green stages and the massive
synthesis at the short ripening stage, except for the large conversion to the new type
of cyclopentanol ketocarotenoids. The data presented in Table 7 are given as mg/100 g
fruit to better follow the synthesis and metabolism of the components and possible
relationships.
The sequence of biosynthesis of formation of P-carotene, its conversion to zeax-
anthin and further formation of its epoxides, the operation of the dynamic xantho-
T a b l e 7. C a r o t e n o i d c o m p o s i t i o n of f i v e v a r i e t i e s of C a p s i c u m a n n u u m at four maturity l e v e l s *
Growth Long red Pacific Ram College Golden

stage cayenne bell horn gold wonder
Total c a r o t e n o i d s M-1 24.0 7.0 5.3 2.9 6.2

M-2 42.4 8.1 8.2 3.1 9.8
M-3 173.0 75.6 65.3 63.1 40.9
M-4 728.0 202.0 196.9 223.4 96.4
P h y t o e n e (12) M - 1 , M-2 A b s e n t f r o m b o t h stages in all v a r i e t i e s
M-3 0.0 0.0 0.0 0.0 0.4

M-4 0.0 0.0 0.1 0.4 1.0
B-Carotene (10) M-1 8.0 2.0 0.8 0.4 2.0

M-2 12.0 2.0 2.0 0.4 3.0
M-3 40.0 3.0 7.0 3.0 9.0
M-4 08.0 16.0 28.0 36.0 16.0
oe-Carotene (14) M-1 1.0 0.0 0.1 0.0 0.0

M-2 3.0 0.0 0.2 0.0 0.0
M-3 5.0 0.4 0.4 0.0 0.4
M-4 9.0 1.0 1.0 0.0 2.0
Z e a x a n t h i n (15) M-1 tr. tr. 0.1 0.1 0.2

M-2 0.4 0.1 0.2 0.2 0.4
M-3 24.0 2.0 4.0 5.0 3.0
M-4 82.0 18.0 16.0 16.0 5.0
Antheraxanthin (17) M - 1 , M-2 A b s e n t f r o m b o t h stages in all v a r i e t i e s
M-3 3.0 0.6 0.4 0.8 0.4

M-4 21.0 7.0 3.0 3.0 5.0
Violaxanthin (18) M-1 5.0 2.0 1.0 0.4 0.6

M-2 11.0 2.0 1.0 0.4 1.0
M-3 16.0 3.0 6.0 6.0 7.0
M-4 41.0 15.0 17.0 19.0 10.0
Lutein (16) M-1 8.0 2.0 2.0 0.5 3.0

M-2 12.0 2.0 2.0 0.6 4.0
M-3 0.6 0.2 0.4 0.1 9.0
M-4 0.0 0.0 0.0 0.0 9.0
Neoxanthin (19) M-1 2.0 1.0 0.2 0.2 0.4

M-2 4.0 2.0 0.4 0.2 0.8
M-3 4.0 1.0 0.8 0.8 0.7
51
T a b l e 7. C o n t ' d
Growth Long red Pacific Ram College Golden

stage cayenne bell horn gold wonder
Capsanthin (20) M - 1 , M-2 A b s e n t f r o m b o t h stages in all varieties
M-3 43.0 29.0 18.0 21.0 0.0

M-4 224.0 65.0 52.0 59.0 0.0
Capsorubin (21) M - 1 , M-2 A b s e n t f r o m b o t h stages in all varieties
M-3 10.0 12.0 7.0 4.0 0.0

M-4 68.0 27.0 15.0 19.0 0.0
* Rahman, F.M.M., Buckle, K.A. (1980): Pigment changes in capsicum cultivars during maturation and ripening,
J . FoodTechnol., 15, 241 (with permission)
Notes: (1) M-1: immaturate green; M-2: mature green; M-3: half ripe; M-4: fully colored ripe stages
(2) All values as mg per 100 g fruit
(3) Numbers in parenthesis after individual carotenoids refer to their numbers in Figs 3-5.
phyll cycle discussed earlier could be visualized to operate in Capsicum fruits. (3-Caro-
tene appears to be continuously formed and metabolized as it forms a substantial
percentage at all stages of maturity and ripening. a-Carotene was found in four of
the five varieties and increasing with ripening, though in very small absolute amounts.
Lutein and neoxanthin, dominant in the green stage, decrease rapidly at the half-
ripened stage and are either absent or dropped to very low levels at the ripening
stage. The yellow fruit variety, Golden wonder, was the only sample in which lutein
increased in actual amounts.
Several specific pathways for carotenoid biosynthesis have evolved in eukaryotes
by novel enzymatic activities. In the Capsicum genus capsanthine-capsorubin syn-
thase (CCS) is able to convert the epoxy-carotenoids antheraxanthin (17) and vio-
laxanthin (18) into the red ketocarotenoids capsanthin (20) and capsorubin (21),
respectively (Fig. 4) (Bouvier et al., 2003). The cyclopentanol structure characteris-
tic of the red ketocarotenoids is conceived to be formed from a 3-hydroxy-5,6-epoxy
end-group [see antheraxanthin (17) and violaxanthin (18) in Fig. 5] by a pinacolic
rearrangement. Since CCS also exhibits lycopene cyclase activity, it is likely to be
related to similarities in the chemical mechanims leading to the formation of beta-
rings in P-carotene (Fig. 3) and kappa-ring in capsanthin (20) and capsorubin (21)
(Fig. 6). In both mechanisms a carbenium ion at C(5) is formed, and both reactions
are probably initiated by proton attack on either a carbon-carbon double bond or the
oxyrane oxygen atom (Deli, Osz, 2004).
The first direct evidence of the conversion of zeaxanthin epoxides into the
cyclopentanyl ketocarotenoids was obtained by Camara et al. A chloroplast-enriched
fraction of the semiripened fruit of Capsicum annuum effected direct conversion of
violaxanthin (18) into capsorubin (21) (Camara, 1980), antheraxanthin (17) essen-
tially into capsanthin (20) and to a small extent to capsorubin (21) (Camara,
Monegar, 1981). In all experiments the control with boiled chromoplast preparation
yielded no transformation of the labeled starting materials to the expected keto-
carotenoids demonstrating the enzymic nature of the conversions.
52
HO Zeaxanthin (15)
Capsanthin-5,6-epoxide (21)
Fig. 6. S i m p l i f i e d b i o s y n t h e t i c route of Capsicum c a r o t e n o i d s (see text for further d e t a i l e d

explanation)
6.2.2.1. Methods for determination of red and total carotenoids
The methods described below (6.3.3) for determining total pigments of Capsicums
are based on measuring the absorbance at the isoabsorption wavelength of the differ-
ent pigments or at the absorbtion maxima of the dominant pigment. The measure-
53
merits give only approximate values sufficient for comparative evaluation, and the
measured absorbencies do not give information on the true pigment concentrations
since the absorption maxima and the molar absorption differ between the red and the
yellow components (Baranyai et al., 1982). The determination of the red and the yel-
low components is desirable for a better understanding of the variations in color that
the eye discerns.
Baranyai and Szabolcs described a simple spectrophotometric method based on
the observation that on reduction of the paprika extracts with sodium borohydride
the red ketocarotenoids yielded an equal amount of yellow components, causing an
increase in the absorption at the maxima of the yellow components (Baranyai, Sza-
bolcs, 1976). This method gave values 20 to 25% higher than the Benedek number
(Benedek, 1958, 1959) usually used in Europe for total pigments since in the new
method the contribution of all components is measured, even that of the cis-com
pounds naturally present.
6.2.3. Capsaicinoids
Seven capsaicinoids and their main chemical structures were identified in Capsicum
species (Table 8).
T a b l e 8. S e v e n c a p s a i c i n o i d s a n d their m a i n c h e m i c a l structures i d e n t i f i e d in Capsicum species*
C h e m i c a l structures N a m e s of capsaicinoids
(CH ) CH-CH=CH(CH ) -CO-R

3 2 2 4 Capsaicin
(CH ) CH(CH ) -CO-R

3 2 2 6 Dihyrocapsaicin
(CH ) CH(CH ) -CO-R

3 2 2 9 Nordihydrocapsaicin
(CH ) CH(CH ) -CO-R

3 2 2 9 Homodihydrocapsaicin
(CH ) CHCH=CH(CH ) -CO-R

3 2 2 5 Homocapsaicin
CH (CH ) -CO-R
3 2 7 Nonanoid acid vanillylamide
CH (CH ) -CO-R
3 2 8 Decanoid acid vanillylamide
* After Anu A., Peter K. V. (2000): The Chemistry of Paprika. Indian Spices 37:15-18 (with permission)
The first five compounds from the seven represent the capsaicin homologues, meanwhile the last two
represent the capsaicin analogues
6.2.3.1. Chemistry of capsaicinoids

Capsicum fruits have been valued for over thousands of years for the piquant taste
they added to the flavorless foods, as well as for the therapeutic effects as a stimu-
lant and counterirritant. These effects have been related to the components stimula-
ting pungency. The chemical study of the active constituents that are responsible for
pungency of the Capsicum fruits have been, however, examined only since the begin-
ning of the 19th century, with the interest in the study of alkaloids in natural products.
Bucholz in 1816 reported to have found that the pungency-stimulating compounds
54
of capsicum are extractable by maceration with organic solvents. Thresh in 1846
reported the crystallization of the active component and named it capsaicin. Micko
improved the isolation method of capsaicin and showed the presence of both hy-
droxyl and methoxy groups and postulated the structural similarity to vanillin
(Micko, 1898). Two decades later, Nelson made the significant contribution to the
structure of capsaicin by identifying the compounds formed by hydrolysis (Nelson,
1919). Nelson and Dawson (1923) further determined the position of the unsatura-
tion of the acid moiety at the C(6)-C(7) position. This established the structure as
8-methyl-6-nonenoyl-vanillylamide ((E)-N-(4-hydroxy-3-methoxybenzyl)-8-me-
thyl-6-nonenamide) (24) (Fig. 7).
Fig. 7. C h e m i c a l structure of c a p s a i c i n ( 2 4 ) (see t h e text, for further d e t a i l e d e x p l a n a t i o n )
The degree of pungency (heat or bite) is determined by the amount of compounds

called capsaicinoids in the fruit. The capsaicinoids are a family of natural products
isolated from the dried fruits of chilli peppers. These substances are the principles
that produce the characteristic sensations associated with ingestion of spicy cuisine
as well as the agents responsible for causing severe irritation, inflammation, erythe-
ma, and transient hypo- and hyperalgesia at sites exposed to capsaicinoids.
Capsaicinoids are particularly irritating to the eyes, skin, nose, tongue and respirato-
ry tract. The nature of the causal components in the spice has been established as a
mixture of acid amides of vanillylamine and C8 to C13 fatty acids, which are known
generally as capsaicinoids. The major capsaicinoids in red peppers are capsaicin,
(E)-N-(4-hydroxy-3-methoxybenzyl)-8-methyl-6-nonenamide (24), and dihydro-
capsaicin (28), the latter being a 6,7-dihydro analogue of capsaicin. Nordihydro-
capsaicin (27) is recognized as the third major capsaicinoid, and the structure is a
mono-nor homologue of the acyl residue of dihydrocapsaicin, that is, N-(4-hydroxy-
3-methoxybenzyl)-7-methyloctanamide (Fig. 8). In commercial capsicums, cap-
saicin generally comprises 33 to 59%, dihydrocapsaicin accounts for 30 to 5 1 % ,
nordihydrocapsaicin for 7 to 15% and the remainder is less than 5% of the capsaici-
noids (Reineccius, 1994). The structures of capsaicin as well as its homologues and
analogues are given in Fig. 6. The seven homologous branched-chain alkyl vanilly-
lamides are capsaicin (24), homocapsaicin I (25), homocapsaicin II (26), nordihy-
drocapsaicin (27), dihydrocapsaicin (28), homodihydrocapsaicin I (29) and homodi-
hydrocapsaicin II (30) (Hoffman et al., 1983; Redly et al., 2001a,b; Karnka et al.,
2002). In addition, three straight-chain analogs, octanoyl vanillylamide (31), nonoyl
vanillylamide (nonivamide) (32) and decyl vanillylamide (33), have also been shown
to occur in Capsicum fruits (Govindarajan, 1986f). Trace amounts of further cap-
saicin homologues and analogues have also been characterized (Suzuki et al., 1957;
55
Suzuki, Iwai, 1984; Govindarajan, 1986a; Collins, Bosland, 1994; Maillard et al.,
1998; Bosland, Votava, 2000; Thompson et al., 2005; Schweiggert et al., 2006).
Capsaicin and its natural homologues are always found in the trans (E) form because
in cis (Z), the -CH(CH ) and the longer chain on the other side of the A6,7 carbon-
3 2
carbon double bond will be close together causing them to repel each other. This
steric hindrance does not exist in the trans isomer. This additional strain imposed
causes the cis isomer to be of a less stable arrangement than the trans isomer.
o
ll
C H , C K ^ r \ ^ C H j - N H - C - R
Name Structure of the R " chain
H CH 3
I I
C a p s a i c i n (24) CH CH CH CH C = C-CH-CH
2 2 2 2 3
I
H
H CH 3
I I
H o m o c a p s a i c i n I (25) CH CH CH CH C = C-CH CHCH
2 2 2 2 2 3
H
D i h y d r o c a p s a i c i n (28) C H 2 _ C H 2 _ C H 2 _ C H 2 _H C H 2 _CH
C H
3
2 _ C H _ C H 3
I I CH 3
H o m o d i h y d r o c a p s a i c i n I (29) C H 2 CH CH CH CHj-CH CH CH-CH

2 2 2 2 2 3
H o m o c a p s a i c i n II (26) C H 2 C H C H C H C = C C H C H C H
2 2 2 2 3
CH 3
H o m o d i h y d r o c a p s a i c i n 11(30)
CH
C H C H C H C H CH C H C H CH CH
2 2 2 2 2
I 3
2 2 3
Ncotradni h
O oyydl rvoacnaipl lsyal iacm (27)
i ni d e (31) C H _ C H _ CHH
C H
2
2 _ C H C H 2 CCH H C H
C H 3
2
C 2
2
C H
2
_qh _ _a-| 2 2 3
N o n o y l v a n i l l y l a m i d e (32) CH CH CH CH CH CH CH CH
2 2 2 2 2 2 2 3
D e c y l v a n i l l y l a m i d e (33) CH CH CH CH CH CH CH CH CH
2 2 2 2 2 2 2 2 3
Fig. 8. C h e m i c a l structures of c a p s a i c i n h o m o l o g s a n d a n a l o g s (see t h e text for further

detailed explanation)
56
The capsaicin biosynthetic pathway has two distinct branches, one of which uti-
lizes L-phenylalanine (34) as the precursor of aromatic residue of capsaicinoids,
presumably via rrafls-cinnamic acid (35) and its hydroxylated derivatives trans-caf-
feic acid (36) and rrans-ferulic acid (37) following the well-established pathways in
other plants (Ishikawa, 2003). Vanillylamine (38) as precursor showed a high level
of incorporation into the capsaicinoids and possibly is the immediate progenitor of
natural capsaicinoids (Fig. 9). The enzymes involved in the formation of the precur-
sors, phenolics, and fatty acids, are similar to those studied for long in other biologi-
cal systems. The second branch forms the branched-chained fatty acids by elonga-
tion of deaminated valine (Fig. 9). The capsaicinoids synthetase, however, has been
found to have narrow specificity in accepting only the /so-C(9:0) to C(11:0) fatty
acids and in the fruit system forming predominantly the vanillylamides of even-
number branched fatty acids, capsaicin (24) and dihydrocapsaicin (28), in all the
cultivated varieties of the Capsicum species (Ravishankar et al., 2003). In the isola-
ted systems, however, the synthetase favors the formation of nordihydrocapsaicin
(27) and capsaicin (24), while at the light-induced activation of the synthetase in
Capsicum annuum cv. grossum, there is higher formation of nordihydrocapsaicin
(27) and dihydrocapsaicin (28) (Govindarajan, 1986 a,b). Synthesis of capsaicinoids
by means of recent development of biotechnological methods has been reviewed in
deatails (Ravishankar et al., 2003).
,4
Kopp and Jurenitsch (1982) have found by administration of C-labeled capsaicin
into the mesocarp of Capsicum fruits, that there was no interconversion of capsaicin
(24) and dihydrocapsaicin (28), confirming earlier observations (Neumann, 1966)
on the stability of formed capsaicinoids. The conversion of the saturated acid into
the unsaturated acid should therefore take place prior to the condensation with vanil-
lylamine to form the different capsaicin homologs. The data confirmed the earlier
suggestion that capsaicinoid composition is regulated besides the substrate specifici-
ty (Fujiwake et al., 1980) more by available precursor amino acids and conditions
controlling the formation of the limited branched acyl acids accepted by the enzyme
capsaicinoids synthetase.
The accumulated data from analysis of fruits from different species varieties and
cultivars (Jurenitsch et al., 1978, 1979a,b; Jurenitsch, Leinmueller, 1980) have
shown considerable variation in the total content and composition of the capsaici-
noids and analogs. Total separation of major and minor capsaicinoids and related com-
pounds have been published by Jurenitsch and Leinmueller (1980) through prelimi-
nary thin-layer chromatography clean-up, separation of capsaicinoids and related
compounds, saponification, methylation and separation of the alkyl acid methyl
esters by gas chromatography (Table 9). Analysis of five cultivated species of
Capsicum showed capsaicin (24) and dihydrocapsaicin (28) to be the dominant con-
stituents together forming about 80% of the total, while nordihydrocapsaicin and
homodihydrocapsaicin, found in all samples, varied widely - from 1 to 12%. Other
higher homologs and straight chain saturated analogs when present are at low levels.
It is also worth mentioning that in spite of the large variations in the total capsaici-
noid content within and between the Capsicum species, the ratio of the principal
components, capsaicin and dihydrocapsaicin, has been found to vary narrowly -
57
<^ \ CH CH COOH
2
L-Phenylalanine (30)
NH,
Phenylalanine-
a m m o n i a lyase
\ C H = C H - C O O H trans-Cynnamic a c i d (31)
frans-Cinnamate-
4-monooxygenase
HOft ^ ^ C H = CH-COOH frans-p-Coumaric acid (32)
frans-p-Coumarate-
3-monooxygenase
HO
K 7 frans-Caffeic acid (33)
Caffeic a c i d O-methyl-
transferase
CH,0
i \CH=CH-COOH frans-Ferulic acid (34)
CH 0.3
HO V a n i l l y l a m i n e (35)
C(10:0)-, C(10:1)-/'soacids <- L-valine

Capsaicinoids
synthetase C(9:0)-, C(11:0)-/'soacids L-Leucine
O
II
hCH NHCR
2 Capsaicinoids
Fig. 9. S i m p l i f i e d p a t h w a y of b i o s y n t h e s i s of c a p s a i c i n o i d s (see t h e text for further d e t a i l e d

explanation)
58
Table 9. C a p s a i c i n o i d c o m p o s i t i o n in fruits of f i v e c u l t i v a t e d s p e c i e s of C a p s i c u m *
P e r c e n t a g e s of c o m p o n e n t c a r o t e n o i d s
Species Sample OV NDC NV C DC DV HC HDC Ratio

No. C:CD
C. annuum 1 0.63 13.96 0.67 49.66 34.52 0.55 _ _ 1:0.7

var.annuum 2 0.09 8.35 1.80 44.63 36.02 4.17 - 4.94 1:0.8
C. annuum 3 _ 3.45 1.29 64.86 29.47 0.93 _ _ 2:2.1

var. p e n d u l u m 4
- 3.75 1.24 37.35 54.64 1.57 - 1.45 1:1.15
C. frutescens 5 - 0.55 0.35 66.97 28.73 0.66 2.32 0.42 2.3:1
C. chinense 6 0.54 6.04 1.06 61.66 24.75 1.44 2.73 1.79 2.5:1
C. pubescens 7 _ 3.47 1.23 36.57 45.97 0.68 _ 12.08 1:1.3

8 0.42 8.42 1.78 29.78 46.62 8.61 - 4.38 1:1.6
* Jurenitsch, J . , Leinmueller, R. (1980): Quantification of nonylic acid vanillylamide and other capsaicinoids in
the pungent principle of Capsicum fruits and preparations by gas4iquid chromatography on glass capillary
columns (in German), J . Chromatogr., 189, 389 (with permission)
Note: The capsaicinoids and analogs are given in the order of increasing gas chromatography retention
of methyl esters of the respective acids obtained by saponification. OV - octanoyl vanillylamide;
NDC - nordihydrocapsaicin; NV - nonoyl vanillylamide; C - capsaicin; DC - dihydrocapsaicin; DV - decyl
vanillylamide; HC - homocapsaicin; DHC - homodihydrocapsaicin
about 1:1 for Capsicum annuum to 2:1 for Capsicum frutescens and Capsicum chi-
nense (Jurenitsch, Kampelmuehler, 1980; Jurenitsch, 1981; Jurenitsch, Leinmueller,
1980).
High performance liquid chromatograpy (HPLC) analysis of capsaicinoids offers
a more simplified but efficient separation of capsaicinoids. The preliminary sample
clean-up is generally not necessary and the separation is done on direct extracts and
mostly at room temperatures, avoiding thermal degradation and loss of materials of
interest. Results of reversed phase HPLC analysis of samples of natural capsaicinoid
mixtures (oleoresins) from different sources and two cayenne extracts are given in
Table 10 (Johnson et al., 1979). As data show, in all samples capsaicin and dihydro-
capsaicin are the main constituents, and the ratio of capsaicin to dihydrocapsaicin
varies from 1:0.5 to 1:1. Kozukue et al. (2005) applied reversed phase HPLC-MS
analysis of eight capsaicinoids in peppers and pepper-containing foods. Quantitation
was based on the UV response at 280 nm. The results also demonstrated that in all
samples capsaicin and dihydrocapsaicin are the main capsaicinoids, the ratio of
them, however, varied in a much wider range.
All capsaicinoid analogs posses a 3-hydroxy-4-methoxy-benzylamide (vanilloid)
pharmacophore, but differ from capsaicin in their hydrophobic alkyl side chain.
Differences in the side chain moiety include saturation of the carbon-carbon double
bond, deletion of a methyl group, and changes in the length of the hydrocarbon chain
59
T a b l e 1 0 . C a p s a i c i n o i d s c o m p o s i t i o n of C a p s i c u m extracts a n d o l e o r e s i n s *
Sample Weight (%)
Total C NDC HC DC HDC C:DC
C a p s a i c i n o i d s (purified mixture) 93.20 40.59 9.39 1.37 39.35 2.50 1:0.52
Oleoresins
Mannheimer 4.00 2.07 0.32 0.05 1.47 0.09 1:0.70
Stange 2.94 1.44 0.39 0.03 0.95 0.13 1:0.66
Kalamazoo 1.52 0.95 0.08 - 0.49 - 1:0.52
N o r d a EP-642 1.85 1.09 0.07 - 0.69 - 1:0.63
Norda 1.40 1.40 - - - - 1:0
Extracts
Cayenne 510 0.24 0.13 0.01 - 0.10 - 1:0.63
Cayenne 970 0.92 0.51 0.07 0.01 0.32 0.01 1:0.63
* Johnson, EX., Majors, R.E., Werum, L., Reiche, P. (1979): The determination of naturally occurring capsaicins
by HPLC, in Liquid Chrom. Anal. Food Beverages, Vol. 1, Charalambous, C, Ed., Academic Press, New York,
pp. 1 7-29 (with permission)
Note: Capsaicinoids in order of their HPLC elution
(Fig. 8). Previous structure-activity studies using models for the study of acute pain
and altered pain sensitivity in mice have demonstrated a strict structural requirement
for both the vanilloid ring pharmacophore and a hydrophobic alkyl chain that may
be saturated or unsaturated, branched or unbranched, and consist of 8 to 12 carbon
atoms for optimal binding and activation of the capsaicin receptor, TRPV1 (Walpole
etal., 1993a,b,c).
With regard to the pungency of capsaicinoids and their related (synthetic) com-
pounds, it can be stated that an aromatic ring with a phenolic hydroxyl group and a
methoxy in ortho position to each other is a basic prerequisite. An aliphatic side
chain is also necessary the length and structure of which are also important. The
pungency is greatly enhanced by an acid amide group, in this instance vanillylamide,
as found in capsaicinoids (Kulka, 1967; Govindarajan, Satyanarayana, 1991). Any
change in one or more of these structural features decreases or even abolishes the
pungency stimulation. Careful sensory testing by threshold tests has shown that the
homologs of capsaicin and dihydrocapsaicin and the straight-chain analogs stimu-
late only 25 to 55% of the pungency stimulated by the two dominant capsaicinoids
(Table 4).
Pungency is an important sensory quality of Capsicum fruits and their processed
forms. As it was shown above, the stimuli in these spices are specific compounds
with certain structural features. Pungency is now being clearly understood as a stim-
ulus response phenomenon. Its evaluation involves both qualitative and quantitative
aspects. In the absence of reliable analytical technique for estimation of capsaici-
noids in the earlier times, spice processing companies and pharmacopeia used sen-
sory testing as a rapid limit value method. The basic principle of pungency evalua-
tion using the organoleptic method was established by Scoville (1912).
60
6.3. Capsicum: Quality control
Capsicum is now one of the two most widely used spices. The wide popularity of the
spice capsicum is attributable to its wide range of shapes and sizes and such sensory
attributes as color, pungency, and distinctive aroma that make generally insipid bulk
nutritive flash and cereal foods more appetizing.
The quality of food should fulfill the consumers' expectations - not necessarily
the maximum of each attribute, but the optimal level and combination appropriate
for each food. Thus a range of quality attributes is required to make different foods
of optimal quality. Quality control which can be exercised through measurement of
the physical and chemical properties of the component stimuli needs to be validated
by a relationship with sensorially perceived responses individually and in combina-
tion. It is obvious that the accuracy and reproducibility of any instrumental method
meaningful for food quality measurement is that which correlates with the sensorial-
ly perceivable differences (Kramer, 1966).
Besides the sensory attributes, capsicum, like other products used as foods and
food additives, should also have certain functional properties for its optimal use in
the industrial sectors, which also have to be considered. The standards of the import-
ing countries are based on the requirements of the food processing industries and
include additional emphasis on cleanliness, which progressively cover, in addition to
insects and rodent parts and extrata, limits of chemical and microbiological contami-
nations, and absence of health-hazard organisms. These specifications assure genu-
ineness, purity, and cleanliness, but they do not give information on the sensory attri-
butes which the consumers require. In the case of some processed products, e.g.,
ground paprika and oleoresin, specifications for total color and capsaicinoids con-
tent are found in standards and manufacturer literature, these being the main selling
factors in this increasing competitive market (Govindarajan, 1986c). Herbs and fruits
that are used as spice, active pharmaceutical ingredients of drugs, or constituents of
food additives should also fulfill even more special requirements described by the
Pharmacopeias and/or other international organizations like EC, FAO, WHO, ISO,
ASTA, etc., and national bodies, which guide the industry concerned in the respec-
tive activities such as manufacturing or trade. The standards are the results of contin-
uous efforts in standardization. For lack of space, it is not possible to cover all such
standards. Only those have been listed that are closely related to the quality of
Capsicum and Ca/xvj'cMm-originated products that can be used for the pharmaceuti-
cal industry.
6.3.1. Capsicum fruits
6.3.1.1. The European Pharmacopeia (Ph. Eur. 5.0)
The 2006 edition of European Pharmacopeia Edition 5.0 lists Capsicum fruit
(Capsici fructus) and describes its Definition, Identification, Nonivamide Test, and
Assay as follows.
61
CAPSICUM
Capsici fructus
(Ph. Eur. 5.0)
DEFINITION
Dried ripe fruits of Capsicum annuum L. var. minimum (Miller) Heise and small-
fruited varieties of Capsicum frutescens L.
Content: minimum 0.4 per cent of total capsaicinoids expressed as capsaicin
( C H N 0 ; Mw.: 305.4) (dried drug).
18 27 3
IDENTIFICATION
A) The fruit is yellowish-orange to reddish-brown, oblong conical with an obtuse
apex, about 1 cm to 3 cm long and up to 1 cm in diameter at the widest part, occa-
sionally attached to a 5-toothed inferior calyx and a straight pedicel. Pericarp
somewhat shrivelled, glabrous, enclosing about 10 to 20 flat, reniform seeds 3
mm to 4 mm long, either loose or attached to a reddish dissepiment.
B) Reduce to a powder. The powder is orange. Examine under a microscope using
chloral hydrate solution R. The powder shows the following diagnostic charac-
ters: fragments of the pericarp having an outer epicarp with cells often arranged
in rows of 5 to 7, cuticle uniformly striated: parenchymatous cells frequently con-
taining droplets of red oil, occasionally containing microsphenoidal crystals of
calcium oxalate; endocarp with characteristic island groups of sclerenchymatous
cells, the groups being separated by thin-walled parenchymatous cells. Fragments
of the seeds having an episperm composed of large, greenish-yellow, sinuous-
walled sclereids with thin outer walls and strongly and unevenly thickened radial
and inner walls which are conspicuously pitted; endosperm parenchymatous cells
with drops of fixed oil and aleurone grains 3 pm to 6 pm in diameter. Occasional
fragments from the calyx having an outer epidermis with anisocytic stomata,
inner epidermis with many trichomes but no stomata; trichomes glandular, with
uniseriate stalks and multicellular heads; mesophyll with many idioblasts con-
taining microsphenoidal crystals of calcium oxalate.
C) Thin-layer chromatography.
Test solution. To 0.50 g of the powdered drug (500) add 5.0 ml of ether R, shake
for 5 min and filter.
Reference solution. Dissolve 2 mg of capsaicin R and 2 mg of dihydrocapsaicin R
in 5.0 ml of ether R.
Plate: TLC octadecylyl I silica gel plate R. Mobile phase: water R, methanol R
(20:80 V/V).
Application: 20 pi, as bands.
Development: over a path of 12 cm.
Drying: in air.
Detection: spray with a 5 g/1 solution of dichloroquinone chlorimide R in
methanol R.
Expose the plate to ammonia vapour until blue zones appear. Examine in day-
light.
62
Results: see the sequence of the zones present in the chromatograms obtained
with the reference solution and the test solution. Furthermore, other zones may be
present in the chromatogram obtained with the test solution (Fig. 10).
Top of the plate
Capsaicin: a blue zone A blue zone (capsaicin)
Dihydrocapsaicin: a blue zone A blue zone (dihydrocapsaicin)
Reference Solution Test s o l u t i o n
Fig. 10. C h a r a c t e r i z a t i o n of t h e t o p of t h e p l a t e
TESTS
Nonivamide. Liquid chromatography.
Test solution. To 2.5 g of the powdered drug add 100 ml of methanol R. Allow to
macerate for 30 min. Place in an ultrasonic bath for 15 min. Filter into a 100 ml vol-
umetric flask, rinse the flask and filter with methanol R. Dilute to 100.0 ml with
methanol R.
Reference solution. Dissolve 20.0 mg of capsaicin R and 4.0 mg of nonivamide R
in 100.0 ml of methanol R.
Column:
- size: 1 = 0.25 m, 0 = 4.6 mm,
- stationary phase phenylsilyl silica gel for chromatography R (5 pm),
- temperature: 30C.
Mobile phase: mixture of 40 volumes of acetonitril R and 60 volumes of a 1 g/1
solution of phosphoric acid R.
Flow rate: 1.0 ml/min.
Detection: spectrophotometer at 225 nm.
Injection: 10 pi.
System suitability: reference solution:
- resolution: minimum 3.0 between the peaks due to capsaicin and nonivamide.
Limit: calculate the percentage content of nonivamide
- nonivamide: maximum 5.0 per cent of the total capsaicinoid content.
ASSAY
Liquid chromatography as described in the test for nonivamide.
Calculate the percentage content of capsaicinoids (Fig. 11).
63
max
1 . nordihydrocapsaicin
70
60 3 2. nonivamide
50 3. capsaicin
40
4. dihydrocapsaicin
30
2 4
20
10 IA
0
25 35
0
10 15 20 mm
Fig. 1 7. C h r o m a t o g r a m of n o n i v a m i d e a n d t h e assay of c a p s i c u m ( P h . Eur. 5.0, p p 1175)
6.3.2. Capsicum extracts - Oleoresin
6.3.2.1. Capsicum extracts

Presently, virtually every commercial spice extraction is carried out by one of two
methods. One method, solvent extraction, involves treating a ground dry spice with
an organic solvent such as hexane, acetone, methanol, ethanol or methylene chlo-
ride. Pursuant to this method, the spice extract is recovered by removal of the sol-
vent, usually by distillation with heat under vacuum. The spice extract recovered in
this way is known as an "Oleoresin" (Eisvale, 1981; Pruthi, 2003). In the case of
oleoresin from Capsicum, the oleoresin is further treated with polar solvent,
methanol, in order to separate the pungent component Oleoresin Capsicum from the
color component Oleoresin paprika (see Table 11). Oleoresins are used almost
exclusively by the food and pharmaceutical industries as a substitute of ground
spices and spice tinctures.
The composition of an oleoresin is affected by the choice of the organic solvent
used in the extraction, but typically will include phospholipids, oils, waxes, sterols,
resins, and a range of non-volatile and volatile compounds which make up much of
the aroma and flavor of the original spice. In its use as food additive, the best oleo-
resin of Capsicum is that which contains the color and flavor components and that
which truly recreates, when appropriately diluted in food formulations, the sensory
qualities of fresh materials (Govindarajan, 1986c).
The other commercial method of spice extraction is the aqueous distillation of the
whole or ground, fresh or dried spice using either boiling water or steam. This
method recovers only the steam volatile components of the spice; i.e. the "essential
oil" which is high in aroma and flavor compounds (Simon, 1990). Many variations
of these two methods are possible. The essential oil may be prepared by distillation
from the original spice, or by distillation from a previously prepared solvent extrac-
ted oleoresin.
These traditional processes have a number of disadvantages. Most organic sol-
vents are toxic, and government food regulations dictate that their residues must be
64
reduced in the oleoresin to very small concentrations, generally in the range of 25 to
30 ppm or less (Pruthi, 2003). The distillation processes used to remove the sol-
vents, or to recover essential oils, lower the content of the very light volatiles which
contribute to aroma and flavor. Of more importance is the growing consumer demand
for food ingredients which are completely natural and free of contact with synthetic
chemicals.
Extraction of spices with supercritical fluid carbon dioxide has been proposed as
a means of eliminating the use of organic solvents and providing the prospect of
simultaneous fractionation of the extract. The use of supercritical fluids (SCF) for
extraction purposes was introduced in the late nineteenth century. A supercritical
fluid is a substance at temperatures and pressures beyond its critical point at which
the liquid phase of the substance will not exist. At these temperatures and pressures,
the supercritical fluid has properties between gas- and liquid-phase characteristics.
These properties make supercritical fluids efficient extraction solvents with high
mass transfer characteristics (McHugh, Krukonis, 1986; Krukonis, 1988). Con-
sequently, supercritical fluids are often used to selectively extract or separate specific
compounds from a mixture by varying fluid density through changes in pressure and
temperature. In food technology, the use of supercritical fluids is essentially limited
to supercritical carbon dioxide (SCF-C0 ) extraction since carbon dioxide has the
2
advantages of being inexpensive and nontoxic and because its critical point is easily
reached.
Oleoresins have several advantages over ground spices, e.g. elimination of micro-
bial contamination, uniformity of color and flavor strength, and optimal utilization.
The Essential Oil Association of America has detailed specifications for three types
of Capsicum oleoresins (Table 11). Oleoresin paprika is mainly used as food color-
ing in meats, dairy products, soups, sauces and snacks. Oleoresin red pepper is used
for both coloring and pungency, mainly in canned meats, sausages, in some snacks
and in a dispersed form in some drinks such as gingerale. Oleoresin Capsicum is the
most pungent and is used for its counter-irritant properties in plasters and some phar-
maceutical preparations.
T a b l e 1 1 . N o m e n c l a t u r e of o l e o r e s i n s of C a p s i c u m s (Essential O i l A s s o c i a t i o n , 1 9 7 5 )
T y p e of o l e o r e s i n Botanical Preparation Color Color Scoville

Capsicums source value description Heat Units
Oleoresin Capsicum C. frutescens Solvent 4,000 max C l e a r r e d , light 480,000 min

( E O A N o . 244) or C. annuum extraction amber,
or dark red
O l e o r e s i n red pepper C. annuum Solvent 20,000 max D e e p red 240,000 min

( E O A N o . 245) var. longum extraction
Oleoresin paprika C. annuum Solvent 40,000- D e e p red N i l or

( E O A N o . 239) extraction 100,000 negligible
65
6.3.2.2. Capsicum Oleoresin
The 2006 edition of the United States Pharmacopeia-National Formulary (USP30
NF25) lists Capsicum Oleoresin and describes it as follows (The USP30-NF25 Page
1611).
Capsicum Oleoresin
(USP30-NF25)
Definition - Capsicum Oleoresin is an alcoholic extract of the dried ripe fruits of
Capsicum annuum var. minimum and small fruited varieties of C. frutescens
(Solanaceae). It contains not less than 8.0 percent of total capsicums [capsaicin
( C H N 0 ) , dihydrocapsaicin (C H 9N0 ), and nordihydrocapsaicin ( C H N 0 ) ] .
18 29 3 18 2 3 ]7 27 3
Caution: Capsicum Oleoresin is a powerful irritant, and even in minute quantities

produces an intense burning sensation when it comes in contact with the eyes and
tender parts of the skin. Care should be taken to protect the eyes and to prevent con-
tact of the skin with Capsicum Oleoresin.
Identification - To about 0.5 g of it in a beaker add 5 ml of water and 10 ml of a
mixture of water, 0.2 M potassium chloride, and 0.2 N hydrochloric acid, and mix.
Add 5.0 ml of 0.5 M sodium nitrite and 5.0 ml of 0.02 M sodium tungstate, and
mix. Heat at 55C to 60C for 15 minutes, allow to cool, and filter. To the fdtrate add
10 ml of 1 N sodium hydroxide: a bright yellow color is produced (presence of cap-
saicin).
Assay
Mobile phase - Prepare a mixture of methanol and 2% acetic acid (56:44), fdter
through a 0.5 pm or finer porosity filter, and degas.
Standard preparation - Prepare a solution of USP Capsaicin RS in methanol having
a known concentration of about 0.5 mg per ml. Filter a portion of this solution
through a 0.2 pm porosity filter, and use the filtrate as the Standard preparation.
Assay preparation - Transfer about 1000 mg of Capsicum Oleoresin, accurately
weighed, to a 100-ml volumetric flask, dissolve in and dilute with methanol to vol-
ume, and mix. Filter a portion of this solution through a 0.2 pm porosity filter, and
use the filtrate as the Assay preparation.
Chromatographic system - The liquid chromatograph is equipped with a 280 nm
detector and a 3.9 mm x 30 cm column that contains packing LI. The flow rate is
about 2 ml per minute. Chromatograph the Standard preparation, and record the
peak responses as directed for Procedure: the tailing factor is not more than 2.0; and
the relative standard deviation for replicate injections is not more than 2.0%.
Chromatograph the Assay preparation, and record the peak responses as directed for
Procedure: the relative retention times are about 0.9 for nordihydrocapsaicin, 1.0 for
capsaicin, and 1.6 for dihydrocapsaicin; and the resolution, R, between the nordihy-
drocapsaicin peak and the capsaicin peak is not less than 1.2.
Procedure: Separetely inject equal volumes (about 10 pi) of the Standard prepa-
ration and the Assay preparation into the chromatograph, record the chromatograms,
66
and measure the responses for the three major peaks. Calculate the percentage of
total capsaicins in the portion of Capsicum Oleoresin taken by the formula:
(CP/WXru/rs)
in which C is the concentration, in mg per ml, of USP Capsaicin RS in the Standard
preparation; P is the designated purity, in percentage, of USP Capsaicin RS, W is
the weight, in mg, of Capsicum Oleoresin taken to prepare the Assay preparation; r v
is the sum of the peak responses for nordihydrocapsaicin, capsaicin, and dihydro-
capsaicin obtained from the Assay preparation; and r is the peak response obtained
s
from the Standard preparation.
6.3.3. Quantitation of capsicum pigments
Appearance and color, the first of the perceived attributes, directly provide a basis
for a decisison of appropriateness. Size, shape, and maximum percentage of defects
are easily measured specifications given in standards. The color of Capsicum fruits
is basically determined by the nature and distribution of the above-described
carotenoids which can be hidden or modified by other pigments such as chlorophylls
and anthocyanides. The major coloring pigments in paprika are capsanthin and cap-
sorubin, comprising the majority of the total carotenoids. Other pigments are p-caro-
tene, zeaxanthin, violaxanthin, neoxanthin and lutein (Anu, Peter, 2000). It is also
worth repeatedly mentioning that the relative amounts of the colored pigments are
changing during the ripening period according to rather well investigated biochemi-
cal pathways (Rahman, Buckle, 1980; Rodriguez-Amaya, 1997; Hornero-Mendez
et al., 2000; Gnayfeed et al., 2001; Deli, Molnar, 2002).
It is also worth mentioning that carotenoid research in the field of plant and food
chemistry is a very extensive area. The interested readers can consult recent reviews
to learn the analytical methods that are currently used to analyse plant and food sam-
ples for their carotenoid contents (Rodriguez-Amaya, 1997; Wall, Bosland, 1998;
Deli, Molnar, 2002; Felt et al., 2005). Some of the methods to measure coloring para-
meters of paprika and oleoresins currently accepted as official are summarized below.
6.3.3.1. The Color Matching Method
This early method for total pigments expressed as Nesslerimeter color value used in
the industry was standardized and adopted by the Essential Oils Association of
America (EOA) for oleoresin capsicum [(Essential Oil Association (1975):
Specification of oleoresin paprika - EOA No. 239, Oleoresin Capsicum - EOA No.
244, Oleoresin red pepper - EOA No. 245, Essential Oil Association, New York)].
The method is based on matching the color of the properly diluted oleoresin acetone
solution with that of potassium dichromate ( K C r 0 ) and cobaltous chloride
2 2 7
(CoC x 6H 0) containing reference solutions.

]2 2
The color values are usually in the range of 40,000 to 100,000 for Oleoresin pap-
rika (see Table 4).
67
6 . 3 . 3 . 2 . Spectrophotometric methods
The alternative method uses the spectrophotometer to measure the total carotenoid
pigments.
6.3.3.2.1. The EOA Method

[Essential Oil Association (1975): Specification of oleoresin paprika - EOA No.
239, Oleoresin Capsicum - EOA No. 244, Oleoresin red pepper - EOA No. 245,
Essential Oil Association, New York],
The absorbance of a 0.01% acetone solution of oleoresin is measured at 458 nm.
The absorbance value is multiplied by 61,000 (an empirical factor worked out to
relate the data from the color matching method) gives the total pigment as the
Nesslerimeter color value.
6.3.3.2.2. The American Spice Trade Association (ASTA) Method

By the above spectrophotometric method, results from different laboratories were
not directly comparable due to differences in the spectrophotometers. In the new
ASTA 20.1 Method (ASTA, 1968) a reference solution of inorganic salts (potassium
dichromate and cobaltous ammonium sulfate in 1.8 M sulfuric acid solution) absorb-
ing in the same region as the carotenoids is used to calculate an instrument factor
which makes interlaboratory comparison possible. In the ASTA Method 20.1 for
extractable color (pigments) in Capsicums absorbance of acetone extract of ground
paprika and other capsicums is measured at 460 nm. The color value (in ASTA) is
calculated using the determined instrument correction factor.
A direct correlation between the earlier ASTA Method 19, measuring absorbance at
450 nm, and the new Method 20.1 cannot be established, an empirical factor (16.4) in
the formula gives values nearly equal to those obtained by the earlier Method No. 19.
6.3.3.2.3. The Hungarian Standard Method

In Hungary, where specified grades of ground paprika are produced (kiilonleges,
csemege, edesnemes, rozsa), the total pigments are determined by similar absorbtion
measurements. Earlier, the total pigment concentration in ground capsicums or in
oleoresins was calculated by using the extinction coefficient in benzene of the major
1%
pigment capsanthin ( 4 7 7 n m = 1826). The results were expressed in grams of
capsanthin per kilogram of dry matter (Hungarian Standard. Examination of Ground
Paprika Spice. Determination of Pigment Content, MSZ 9681/5-76.). At present, the
measurements are performed using acetone extracts similar to the ASTA Method
20.1 (MSZ 9681-5:2002)
68
6.3.4. Quantitation of pungent principles
For the major portion of Capsicum species produced and traded, pungency is the
important quality attribute. The nature of the causal components in the spice has
been established as a mixture of seven homologous branched-chain alkyl vanillyl-
amides, named capsaicinoids. Small amounts of three straight-chain analogues have
also been shown to occur. The chemistry of these compounds has been reviewed
(Suzuki, Iwai, 1984). The structures are given in Fig. 6. The average composition of
these related vanillylamides in the widely traded chillis (Capsicum annuum var.
annuum) varieties is capsaicin 33 to 59%; dihydrocapsaicin, 30 to 51%; nordihydro-
capsaicin, 7 to 15%; and others, in the range of 0 to 5% each. Fruits of the species
Capsicum frutescens, stimulating high pungency and mostly used in the pharmaceu-
tical industry, have higher capsaicin (63 to 77%) and dihydrocapsaicin (20 to 32%)
contents, with other homologues and analogues making up around 10% (Jurenitsch
et al., 1978). The total capsaicinoids varied greatly (0.001 to 0.01% in paprika and
0.1 to ( 1 % in chillis), but the proportion of capsaicin and dihydrocapsain ranged
from 77 to 90% in the fruits of the species C. annuum and from 89 to 98% in those
from species C. frutescens (Govindarajan et al., 1987).
As it was shown above (Table 4), the pungency stimulated by the different alkyl
acyl vanillylamides varied greatly, all much lower compared to capsaicin and dihy-
drocapsaicin, which were equal (Govindarajan et al., 1987). Thus, the estimation of
total capsaicinoids, reproducibly and accurately correlating with the determined
pungency, should be sufficient for quality control. Where the minor capsaicin-relat-
ed vanillylamides make up a larger portion (above 20%), however, their individual
estimation could become necessary because they stimulate much lower pungency. It
has been also known for a long time that synthetic nonoyl vanillylamide (pelargonyl
vanillylamide) has considerable pungency and heat (Kuika, 1967), and has been
found in varying amounts in commercial oleoresins. Therefore, it was necessary to
determine the upper limits of the straight-chain analogs to determine adulteration.
6 . 3 . 4 . 1 . Official methods for organoleptic determination of pungency
6.3.4.1.1. The Scoville Method

A number of methods have been reported from time to time since 1912 for assaying
the pungency or capsaicin content of Capsicum fruits and/or the processed fruits
(Pruthi, 2003). The basic principle of pungency evaluation using an organoleptic
method was established in 1912 by W. L. Scoville (Scoville, 1912). The method is
based on sensory evaluation determining the amount of sugar to neutralize the heat
from the pepper. A solution of the pepper extract variably diluted with sugar solution
is tested in increasing concentration. The highest dilution at which pungency is just
detected is taken as a measure of the heat value. The dilution value, in milliliters per
gram, has since then been called Scoville Heat Units (SHU). The SHU for pure cap-
saicin is reported as 16-17 x 106 (Table 12). The Scoville Heat Units of various
chilli pepper varieties are shown in Table 13. The greatest weakness of the Scoville
organoleptic test is its imprecision, because it relies on human subjectivity.
69
T a b l e 1 2 . P u n g e n c y t h r e s h o l d in S c o v i l l e v a l u e s of c a p s a i c i n h o m o l o g s a n d a n a l o g s *
Capsaicin homolog/analog T o d d et a l . Jurenitsch

(1977) (1981)
Capsaicin 16.1 0.6 17.0
Dihydrocapsaicin 16.1 0.6 10.8
Nordihydrocapsaicin 9.3+0.4 9.6
Homocapsaicin 6.90.5 -
Homodihydrocapsaicin 8.1 0.7 8.3
Vanillyl pelargonamide 9.2 0.4 8.8
Vanillyl capramide 4.5 7.4
Vanyllyl undecanamide 3.5 -
* Govindarajan, VS., Rajalakshmi, D., Chand, N. (1987): Capsicum - Production, technology, chemistry and
quality. Part IV. Evaluation of quality. In: Furia, TE (ed.) CRC Crit. Rev. Food Sci. Nutr., CRC Press, Boca Raton,
25: 266 (with permission)
Pungency is expressed in Scoville Heat Units (millions (106) ml/mg)
T a b l e 1 3 . P u n g e n c y of v a r i o u s C a p s i c u m v a r i e t i e s *
Type H e a t rating (in Scoville H e a t Units)
Habanero 200.000-300.000
Tabasco 30.000-50.000
Cayenne 35.000
Serrano 7.000-25.000
Jalapeno 3.500-4.500
Anaheim 1.000-1.400
Bell & Pimento 0
* Ravishankar, G.A, Suresh, B, Giridhar, P, Ramachandra Rao, S, Sudhakar Johnson, T. (2003): Biotechnological
studies on Capsicum for metabolite production and plant improvement. In: De, A.K. (ed): Capsicum
The genus of Capsicum, Taylor and Francis, London, New York, Chapter 6, pp. 99-128 (with permission)
Note: 0-5.000: mild; 5.000-20.000: medium; 20.000-70.000: hot; 70.000-300.000: extremely hot
6.3.4.1.2. The EOA Method

This method [(Essential Oil Association (1975): Specification of oleoresin paprika
EOA No. 239, Oleoresin Capsicum E A No. 244, Oleoresin red pepper EOA No.
245, Essential Oil Association, New York)] is the codification of the procedure that
was in use by the spice processing industry to check the constancy of pungency of a
usual trade variety and source. The method, based on the approach of the original
Scoville method, specified for oleoresins of capsicum as follows.
A standard solution for testing is made by diluting a stock alcoholic solution of
the oleoresin and it is tested by five trained panelists. If three of the five on a panel
agree on just perceptible pungency at the given dilution (which is equivalent to
70
240,000 ml/g), this value is called the SHU of pungency of the sample. If the pun-
gency response is strong, this first diluted standard is further diluted and the panel
testing is repeated to find the dilution at which three of the five judge the pungency
just perceptible. The Scoville test run shows a rather high correlation to total capsai-
cinoids content (1,500,000 Scoville units = 1% capsaicin).
6.3.4.1.3. The British Standard Method

The British Standard Institution adapted the above industry procedure except that
dilutions were rationalized in more convenient volumes [(British Standards
Institution (1979): Methods for testing spices and condiments: Determination of
Scoville index of chillies, BS 4548 (Part 7), BSI, London)].
6.3.4.1.4. The International Standard Organization (ISO) Method

The British Standard Method adopted and improved by the International Standards
Organization (ISO) requires the testing of a series of dilutions around the anticipated
value by individuals experienced in recognizing pungency [(International Standards
Organization (1981, 1997): Spices and condiments - chillies: Determination of
Scoville index, ISO 3513:1977E, ISO, Geneva)]. Testing of dilutions should be done
from the weakest to the strongest until a level at which three of five panelists agree
on recognition of pungency. There is no published report on the extensive use and
efficiency of these latter two methods, except a few which reported comparison of
experimentally determined SHU values with that of calculated based on capsaici-
noid content of oleoresin capsicum samples (Suzuki et al., 1957).
6.3.4.1.5. The ASTA Method

The ASTA adopted as early as 1968 Method No. 21.0 as an official method for pun-
gency evaluation which took care of many variables that were to be controlled for
good reproducibility [(ASTA (1968): Method 21.0, Pungency of capsicum spices
and oleoresins (Scoville heat test), In: Official Analytical Methods, ASTA,
Englewood Cliffs, N.J.)]. The procedure followed in the industry and EOA was thor-
oughly revised by designing it as a general method applicable to samples of a large
range of capsaicinoid content by careful steps for determining the recognition thresh-
old using an ascending concentration series.
6.3.4.2. Quantitation of capsaicinoids
In addition to pungency, as a bulk characteristic of total capsaicinoids, estimation of

the individual level of each capsaicinoid is also an important quality attribute of
Capsicum fruits. There are over a hundred papers published on the estimation of
total capsaicinoids in Capsicum fruits, the oleoresins and products containing their
extracts. The methods can be grouped into four sets as follows:
71
6.3.4.2.1. Early direct methods
As early as 1931, von Fodor used vanadium oxytrichloride or ammonium vanadate
and hydrochloric acid to react with the phenolic hydroxyl group of capsaicinoids
and measured the blue color formed. The accuracy of determinations based on color
formation reactions of the phenolic moiety could be conveniently used when the
color produced with the chromogenic reagent had absorption maxima far removed
from the absorption range (300 to 550 nm) of the red and yellow carotenoids of
Capsicum fruits. Thus the blue colors formed when the phenolic moiety of the cap-
saicinoids reacted with reagents such as vanadium oxychloride (Palacio, 1977) and
phosphomolybdic or phosphotungstic reagents (Jentzsch et al., 1969) had their
absorption maxima around 725 nm. On the other hand, the chromophore produced
by the more specific 2,6-dichloro-p-benzoquinone-4-chlorimide, (Gibb's phenol
reagent) depending on the reaction conditions, absorbed maximally at 590 or 615
nm (Jentzsch et al., 1969; Rajpoot, Govindarajan, 1981). The extinction coefficients
for the blue colors using different reagents varied greatly and affected sensitivity.
There are also reports using potassium ferricyanide plus ferric chloride (Spanyar,
Blazovich, 1969), sodium nitrite molybdate reagent (Bajaj, 1980) and Folin-
Ciocalteu reagent (Kosuge, Inagaki, 1959) as chromogenic reagents for determina-
tion of total capsaicinoids. It is worth mentioning that the color reactions could also
be applied for visualizaton of thin layer chromatography (TLC) spots of separated
capsaicinoids..
6.3.4.2.2. Methods based on separation of capsaicinoids

The specificity and accuracy of determination of capsaicinoids were improved by a
preliminary separation of interfering pigments and other substances. In the early
methods separation of capsaicinoids from the pigments was accomplished by sol-
vent partition. Several combinations of partition system may be found in the litera-
ture (Spanyar et al., 1957; Benedek, 1959, Chem. Abstr. 1958, 1963a,b; Tirimanna,
1972), none of the methods, however, was validated by pungency tests, without
which the accuracy of the determination cannot be ascertained.
There have been several methods developed for the preliminary separation of cap-
saicinoids from the Capsicum pigments using short column clean-up methods. The
purified capsaicinoids were quantitated by colorimetry after reacting with chro-
mogenic reagents (Hollo et al., 1957, Chem. Abstr. 1958, 1963a,b; Bajaj, 1980) or
directly at the absorption maxima (282 nm) of pure capsaicin (Suzuki et al., 1957;
Brawer, Schoen, 1962; Chem. Abstr., 1963a,b). Suzuki et al. (1957) determined pun-
gency values for a number of chilli and oleoresin samples by threshold testing and
validated the capsaicinoids values determined by the proposed method. After several
reviews, the Joint Committee (Pharmaceutical Society/Society of Analytical
Chemistry) recommended the diethyl ether-alkali partition method for separation
and the spectrophotometric difference method or the method using Gibb's reagent
for measurement [Joint Committee (PS/SAC), 1964]. The method has been adopted
by both the British Standards (BS) and International Standards Institutions (ISO) for
estimation of capsaicinoids (International Standards Organization, 1981).
72
6.3.4.2.3. Newer chromatographic micromethods -
Thin Layer Chromatography
As newer separation methods emerged (e.g. paper chromatography and TLC) which
gave rapid and more efficient separation, they were quickly used in the determina-
tion of total and later individual capsaicinoids. The main body of papers in the 1960s
practically relegated the earlier solvent partition and column methods to the past and
published reliable, rapid micromethods. Most of the early thin layer chromatogra-
phy (TLC) methods used silica gel plates with a wide range of variations in the devel-
oping solvents. The versatility of TLC method for the separation of a complex mix-
ture of compounds could be further improved, however, by using reversed-phase
plates and polar developing solvents containing silver nitrate (Todd et al., 1975,
1977). Methods of visualization applied UV light or chromogenic reagents. The esti-
mation method also varied: visual comparison of size and intensity of spots, direct
densitometry on the plate, collection of the marked spot into a tube, development of
color with a chromogenic reagent and absorption measurements. Quantitation was
by reference to a standard curve using pure capsaicinoids treated under the same
conditions. A comprehensive listing of the methods can be found in some reviews on
capsaicinoids (Suzuki, Iwai, 1984; Govindarajan et al., 1987).
6.3.4.2.4. Newer chromatographic micromethods - Gas Chromatography

Gas chromatography (GC) was early used to detect adulteration of capsaicinoids
with synthetic vanillylamides and individual components in crystalline capsaici-
noids through the analysis of methyl esters of fatty acids derived from them (see
Table 2). As early as 1967, Morrison demonstrated that capsaicinoids can be ana-
lyzed by gas chromatography without derivatization (Morrison, 1967). In order to
improve peak symmetry, prevent degradation of column and improve reproducibili-
ty of measurements, however, most of the GC methods need a derivatization step
to increase volatility of the capsaicinoids, and, furthermore, an efficient clean-up
step is necessary (Todd et al., 1977; Iwai et al., 1979; Krajewska, Powers, 1987;
Manirakiza et al., 1999, 2003) Two types of derivatization procedures have been
reported: trimethylsilylation of capsaicinoids (Lee et al., 1976; Todd et al., 1977;
Iwai et al., 1979; Fung et al., 1982) and hydrolysis of capsaicinoids to yield fatty
acids and subsequent esterification (Jurenitsch et al., 1978; Jurenitsch, Leinmueller,
1980). To overcome the problem of tailing peaks and to avoid the use of derivatization
step, Thomas et al. (1998) and Hawer et al. (1994) have recognized the use of polar
capillary column for interaction with polar functional group of the molecules. In an
earlier work Di Cecco (1976) also used a stable polar analytical column (Carbowax-
Teflon) to analyse column purified capsaicinoids from ground capsicum. Fur-
thermore, the use of a thermoionic selective detector (TSD) instead of flame ionization
detection allowed the elimination of sample clean-up (Thomas et al., 1998).
Although the direct analysis of capsaicinoids could be disandvantageous, direct
GC-MS analysis of commercially available natural capsainoids has been proved to
be a proper method to separate the main capsaicinoids and quantitate capsaicin and
dihydrocapsaicin in Capsicum extracts as well (Kuzma et al., 2006). A typical gas
chromatogram of a commercially available natural capsaicin is shown on Fig. 12.
73
28-Feb-2006 capsaicin 09:34:47
S c a n EI+
v4
17.48 TIC
140 -
1.44e6
-17.68
16.82,
6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00
v4 681 (17.477) scan el+

137 5.35e5
100
138 1 5 2
168 178 195 0 7 0 9 221

2 2 248 262 2 81 3 0 5
i 307 325 341 355 357378 387
100 120 140 160 180 200 220 240 260 280
'r r T"'T""i
300
i :
i f" i i' (
320 340 360 380 400
Fig. 12a: G a s c h r o m a t o g r a p h i c ( G C - M S / T I C ) a n a l y s i s o f a c o m m e r c i a l l y a v a i l a b l e c a p s a i c i n
preparation. Retention times: 16.8 m i n : nordihydrocapsaicin; 17.5 m i n : c a p s a i c i n ;
17.7 m i n : d i h y d r o c a p s a i c i n
12b: G C - M S ( E l , S c a n m o d e ) of c a p s a i c i n ( K u z m a , M o l n a r , Perjesi, 2 0 0 6 : D e v e l o p m e n t
a n d a p p l i c a t i o n of a gas c h r o m a t o g r a p h i c m e t h o d for d e t e r m i n a t i o n of c a p s a i c i n o i d s ,
I n : A b s t r a c t s of Papers, S y m p o s i u m of D r u g R e s e a r c h C o m m i t t e of H u n g a r i a n
P h a r m a c e u t i c a l S o c i e t y , N o v e m b e r 2 4 - 2 5 , D e b r e c e n , H u n g a r y , p. 51)
74
Lee et al. (1976) used selective ion monitoring to identify and quantify individual
capsaicinoids at the nanogram level in partially or fully separated and even mixed
peaks from GC of trimethylsilylated capsaicinoids. Aliquots of fruit extracts were
subjected to TLC or reversed phase (RP) HPLC to separate the capsaicinoids from
other components of the extracts. Iwai et al. (1979) developed a similar method for
determining all homologs in total extracts of capsicum fruits. By this analysis, simi-
lar to other GC-MS methods (Fung et al., 1982; Reilly et al., 2001) the straight-chain
analogs (octanoyl, nonoyl and decyl vanyllilamides) - identified in minor amounts
in other analyzes (Jurenitsch, et al., 1978; Jurenitsch, Leinmueller, 1980) - were not
found in any of the samples. The reason for this was that the analysis was based on
selected m/e values but not on monitoring the mass of octonoyl vanillylamide, and
the method could not differentiate between nonoyl vanillylamide and nordihydro-
capsaicin eluting in the same area.
6.3.4.2.5. Newer chromatographic micromethods - High Performance

Liquid Chromatography
This chromatographic technique has superior and rapid separation capabilities aris-
ing from the use of very fine and highly uniform particles, newer solid phases, and
high pressure to move the eluting solvent and fractions. With all its advantages, high
performance liquid chromatographic analysis is being increasingly used for routine
analyses in both industrial and research laboratories. HPLC has superior separation
capabilities for closely related compounds typically occurring in the case of extract
of natural sources. Combined with additional operational parameters, e.g. reversed-
phase systems, silver-ion complexing of olefinic compounds, optical as well as mass
selective detectors, the separation efficiency, sensitivity, and quantification at submi-
crogram levels of capsaicinoids have been demonstrated in the recent years. HPLC
analysis has made possible the accurate determination of the homologs and analogs
of capsaicin and, combined with mass spectral analysis, has led to identification of
structural isomers of some minor components (Govindarajan, 1986b; Reilly et al.,
2001; Schweiggert et al., 2006) and has made possible the determination of
nanogram levels of the individual capsaicinoids as is required in biosynthetic and
metabolic studies (Kozukue et al., 2005).
Several HPLC methods have been published for the determination of capsaicin
homologs and analogs. Since there is no space to summarize all the methods pub-
lished so far, the attention of interested readers is drawn to recent reviews to get a
comprehensive knowledge on the field (e.g. Govindarajan et al., 1987; Wall,
Bosland, 1998; Manirakiza et al., 2003; Pruthi, 2003). Here only selective papers are
summarized with data obtained.
Lee et al (1976) and Iwai et al. (1979) early used HPLC for the separation of cap-
saicinoids in one or two fractions from total extracts for the subsequent analysis by
mass spectrometry. Sticher et al. (1978) reported separation of four homologs of
capsaicin in purified capsaicinoids using a reversed-phase system. Jurenitsch et al.
(1979) accomplished the separation of the capsaicin homologs and analogs directly
from ground fruit extracts on a reversed-phase system. Detection and quantitation
were done by absorbance at 280 nm. Four samples of Capsicum fruits were analyzed
75
by this HPLC method and also by the TMS-GC method earlier used by the group
(Jurenitsch et al., 1978) for comparison. There was a fairly close agreement between
the methods for total capsaicinoids. Karnka et al. (2002) have reported an optimized
HPLC based method for sample preparation, separation, detection and identification
of the major capsaicinoid compounds in various capsicum samples.
Nonoyl vanyllamide content has assumed importance since more than 3 to 4% of
this analog in a natural sample is considered adulteration unless declared. The
method developed by Jurenitsch et al. (1979) was modified with the inclusion of sil-
ver nitrate in the mobile phase to selectively shorten the retention time of capsaicin,
thus separating it from the coeluting nonoyl vanillylamide (Jurenitsch, Kampel-
muehler, 1980). Constant and coworkers (1995) also used complexation chroma-
tography (AgN0 ) to separate norcapsaicin, zucapsaicin (civamide), capsaicin,
3
nordihydrocapsaicin, nonivamide, homocapsaicin, dihydrocapsaicin and homo-

dihydrocapsaicin-I.
Isocratic reversed phase HPLC analysis performed in the author's laboratory
allowed separation of five main capsaicinoids of a commercially available capsaicin
preparation (see Fig. 13). The validated analytical method has been successfully
applied to quantitate capsaicin and dihydrocapsaicin in commercially available
ground paprikas (Boros et al., 2007, unpublished results).
mAU D A D 1 E, Sig=281,16 Ref= 360,100 (CAPS\CAPS0001.D)
Fig.
0 ~~5 l
~~
A
10 15
1 ' '
13. H P L C a n a l y s i s of a c o m m e r c i a l l y a v a i l a b l e c a p s a i c i n p r e p a r a t i o n .
' 200 min
R e t e n t i o n t i m e s : 9.5 m i n : n o r d i h y d r o c a p s a i c i n ; 10.5 m i n : c a p s a i c i n ;
1 6 . 0 m i n : d i h y d r o c a p s a i c i n ; 17.5 m i n : h o m o c a p s a i c i n ( B o r o s ,
K u z m a , Perjesi, u n p u b l i s h e d results)
The rapid and efficient separation by HPLC directly of extracts of fruits for cap-
saicinoid components combined with the sensitive detection and quantitation by
selective ion mass spectrometry gives an outstanding method for analysis of all the
capsaicin homologs and analogs identified. The use of HPLC-MS (Reilly et al.,
2001a,b) has been reported to differentiate nonivamide and capsaicin by mass-to-
charge (m/e) ratio. The same authors have reported the use of LC-MS-MS with elec-
trospray ionization source operating at selective ion monitoring mode (Reilly et al.,
76
2001a,b). The quantification of capsaicinoids using LC-MS-MS was more sensitive
(in the ng/ml range) and exhibited greater accuracy, even at low analyte concentra-
tions. HPLC coupled with atmospheric pressure chemical ionization mass spectro-
metry has been reported to be a method of choice for separation and identification of
the three groups of capsaicinoids: capsaicins possessing a methyl branched acyl
residue with a carbon-carbon double bond, dihydrocapsaicins analogous to the pre-
vious class, but being saturated compounds, and capsaicin analogues (N-vanillyl-n-
acylamides) composed of saturated, unbranched alkyl chains (Fig. 8) (Schweiggert
et al., 2006).
In summary, HPLC analysis for total capsaicinoids or individual capsaicinoids is
certainly rapid, reproducible, sensitive, and convenient for analysis of capsaicinods
in various capsaicinoid containing matrices.
6.3.4.2.6. The ASTA method for determination of capsaicinoids

In the 1980s it became clear that a more accurate and reproducible method of deter-
mining "heat" in peppers and pepper products was necessary. Under the auspices of
the American Spice Trade Association (ASTA), a new High Pressure Liquid
Chromatography (HPLC) Method was adopted as ASTA Method 21.1. The HPLC
measurement of capsaicin has evolved over the years as new and better instrumenta-
tion has allowed the greater accuracy of analysis. In 1996 AO AC issued Method
995.03: Capsaicinoids in Capsicums and their Extractives. In 1998, in a collabora-
tive effort with AOAC, ASTA issued a revised method of analysis, ASTA Method
21.3 (HPLC Method). Using the revised method, the accepted pungency of pure
capsaicin was re-stated from 15,000,000 to 16,000,000 SHU. AOAC revised its
method to coincide with ASTA Method 21.3, in 1999, and in 2003 AOAC revised
the method once more.
6.3.4.2.7. The United States Pharmacopeia (USP)

The 2006 edition of the USP30-NF25 lists Capsaicin and describes its Definition,
Identification, Melting range, and Content of capsaicin, dihydrocapsaicin, and other
capsaicinoids as follows (The USP30-NF25 Page 1609).
Capsaicin
(USP30-NF25)
Chemical name: 6-Nonenamide, (E)-N-[(4-Hydroxy-3-methoxy-phenyl)methyl]-
8-methyl
Formula: C H 7 N 01 8 2 3
Molecular weight: 305.41

(E)-8-MethyI-N vanillyl-6-nonenamide CAS-number: 404-86-4
Capsaicin contains not less than 90.0 percent and not more than 110.0 percent of
the labeled percentage of total capsaicinoids. The content of capsaicin ( C H N 0 3 )
1 8 2 7
is not less than 55 percent, and the sum of the contents of capsaicin and dihydrocap-
saicin ( C H N 0 ) is not less than 75 percent, and the content of other capsaici-
1 8 2 9 3
noids is not more than 15 percent, all calculated on the dried basis.
77
Packaging and storage - Preserve in tight containers, protected from light, and
store in a cool place.
Caution - Handle Capsaicin with care. Prevent inhalation of particles of it and
prevent its contact with any part of the body.
Solubility - It does not dissolve in water. It well dissolves in alcohols (methanol,
ethanol 96%), ethylacetate and acetonitrile.
Identification - Prepare a test solution of Capsaicin in methanol containing 1 mg

per ml. Prepare a Standard solution of USP Capsaicin RS in methanol containing
1 mg per ml. Separately apply 10-pl portions of the test solution and the Standard
solution to a thin-layer chromatographic plate coated with a 0.25-mm layer of chro-
matographic silica gel mixture. Develop the chromatograms in a solvent system con-
sisting of a mixture of ether and methanol (19:1) until the solvent front has moved
about three-fourths of the length of the plate. Remove the plate from the chamber,
and allow it to air-dry. Spray the plate with a 0.5% solution of 2,6-dibromoquinone-
chlorimide in methanol, allow to stand in a chamber containing ammonia fumes,
and examine the chromatograms: the blue color and the Rvalue of the principal spot
obtained from the test solution correspond to those properties of the principal spot
obtained from the Standard solution.
Melting range: between 57C and 66C, but the range between beginning and
end of melting does not exceed 5C.
Content of capsaicin, dihydrocapsaicin, and other capsaicinoids

Mobile phase - Prepare a mixture of diluted phosphoric acid (1 in 1000) and ace-
tonitrile (600:400). Filter through a fdter having a porosity of 0.5 pm or finer, and
degas. Make adjustments if necessary (see System Suitability under Chromato-
graphy).
Standard capsaicin solution - Dissolve an accurately weighed quantity of USP
Capsaicin RS quantitatively in methanol to obtain a solution having a known con-
centration of about 0.1 mg per ml.
Standard dihydrocapsaicin solution - Dissolve an accurately weighed quantity of
USP Dihydrocapsaicin RS quantitatively in methanol to obtain a solution having a
known concentration of about 0.025 mg per ml.
Test solution - Transfer about 25 mg of Capsaicin, accurately weighed, to a 250
mL volumetric flask, dilute with methanol to volume, and mix.
Chromatographic system - The liquid chromatograph is equipped with a 281 nm
detector and a 4.6-mm x 25-cm column that contains 5 pm packing LI 1 and is main-
tained at a constant temperature of about 30C. Adjust the flow rate to obtain a reten-
tion time of about 20 minutes for the main capsaicin peak. Chromatograph the Stan-
dard capsaicin solution, and record the peak responses as directed for Procedure:
the relative standard deviation for replicate injections is not more than 2%.
78
Procedure - Separately inject equal volumes (about 20 pi) of the Standard cap-
saicin solution, the Standard dihydrocapsaicin solution, and the Test solution into
the chromatograph, record the chromatogram for a period of time that is twice that
of the retention time of capsaicin, and measure the areas of the responses for all of
the peaks. Calculate the percentage of capsaicin ( C H 7 N 0 ) in the portion of
l g 2 3
Capsaicin taken by the formula:
25,^(C/WHry/rs),
in which C is the concentration, in mg per mL, of USP Capsaicin RS in the Stan-

dard capsaicin solution, W is the weight, in mg, of Capsaicin taken to prepare the
Test solution, and r and r are the capsaicin peak responses obtained from the Test
v s
solution and the Standard capsaicin solution, respectively. Not less than 55% is
found. Calculate the percentage of dihydrocapsaicin (CigH^NC^) in the portion of
Capsaicin taken by the formula:
25,000(C/W)( r /r ),
v s
in which C is the concentration, in mg per ml, of USP Dihydrocapsaicin RS in the

Standard capsaicin solution, W is the weight, in mg, of Capsaicin taken to prepare
the Test solution, and r and r are the dihydrocapsaicin peak responses obtained
v s
from the Test solution and the Standard dihydrocapsaicin solution, respectively.
The sum of the percentage of capsaicin found and of the percentage of dihydrocap-
saicin found is not less than 75%. Using the chromatograms obtained from the Stan-
dard capsaicin solution and the Test solution, calculate the percentage of other cap-
saicinoids in the portion of Capsaicin taken by the formula:
25,000(C/W)(rT/r ),
s
in which C is the concentration, in mg per ml, of USP Capsaicin RS in the Standard

capsaicin solution, W is the weight, in mg, of Capsaicin taken to prepare the Test
solution, r is the sum of the peak responses of the capsaicinoids other than cap-
T
saicin and dihydrocapsaicin in the chromatogram obtained from the Test solution,
and r is the capsaicin peak response obtained from the Standard capsaicin solu-
s
tion. Not more than 15% of other capsaicinoids is found.
6.3.4.3. Correlation of pungency and capsaicinoid content
Among the methods published for estimation of total capsaicinoids or individual cap-
saicinoids only a few have been validated by correlating the results to pungency.
Suzuki et al. (1957) compared the pungency of a number of capsicum oleoresin sam-
ples determined by the organoleptic method to that of their total capsaicinoids con-
tent determined by UV absorption. They found a high correlation between the percent
of total capsaicinoids and the estimated Scoville values. Hartman (1970) correlated
SHU to the proportion of capsaicinoid content to GLC. Todd et al. (1977) also com-
pared the pungency in Scoville units calculated from the GC-determined percentages of
79
individual capsaicinoids of the samples and the values determined by the modified
ASTA method and found a high correlation. Similarly, high correlation was obtained
between the Scoville values and the total capsaicinoids estimated by HPLC method
(Woodbury, 1980). Govindarajan et al (1986d) proposed a standardized procedure for
the evaluation of the pungency of SHU by which a linear regression was obtained
between SHU and the capsaicin content of samples. Recently, Korel et al. (2002) used
the electronic nose (EN) technology (Strike et al., 1999) to discriminate ground red
pepper samples by headspace volatiles. The authors report linear correlation between
capsaicin, dihydrocapsaicin, and total capsaicinoids and Scoville scores of the sam-
ples. Based on these literature data it can be concluded that estimation of capsaici-
noids by any reproducible and accurate analytical method truly reflects the pungency
of capsicum fruits or processed products (Govindarajan, 1986e).
6.3.4.4. Stability of capsaicinoids
Information on stability of isolated capsaicin, dihydrocapsaicin as well as of other

capsaicinoids is rather limited. The Material Safety Data Sheet (MSDS) of natural
capsaicin (Formula: C H 7 N 0 , Molecular weight: 305.41, Chemical name: (E)-8-
1 8 2 3
Methyl-N-vanillyl-6-nonenamide, CAS number: 404-86-4) recommends the prepa-

ration between 2 to 8C (Sigma-Aldrich MSDS). The USP30-NF25 prescribes the
capsaicin preparatum to be preserved in tight containers, protected from light, and
stored in a cool place. According to the MSDS of synthetic dihydrocapsaicin (For-
mula: C H 2 9 N 0 , Molecular weight: 307.48, Chemical name: 8-Methyl-N-vanillyl-
1 8 3
nonanamide, CAS number: 19408-84-5), the preparation should be stored at -20C

(Caymanchem MSD). Both preparations are reported to be stable for at least 26 days
at-5C (NMAM, Method 5041, 1996).
Schweiggert et al. (2006) investigated the stability of chili powder. They found
that the capsaicin, dihydrocapsaicin and nordihydrocapsaicin content of the samples
stored at ambient temperature over 6 months dropped by 7 to 12%. Based on their
experience it was recommended that paprika (pepper) samples should be heat-trea-
ted before processing, in order to reduce the number of microorganisms producing
enzymes with peroxidase activity of the fruits.
Kopec et al. (2002) tested the stability of stored ethanol solution of capsaicin rang-
ing in concentration from 0.5 to 128 pM. It was found that the solutions of 4 uM or
higher concentrations are stable for a year if stored at 4C protected from light. The
result has been reinforced by the authors' investigations studying stability of cap-
saicin natural solutions (c = 3.1 mg/ml) stored for a month at 4C protected from
light (Boros et al., unpublished results).
While investigating the stability of a capsaicin containing ointment Jaiarj et al.
(2000) found the preparation to show higher stability stored at 4C other than at
ambient temperature.
In conclusion, the presence of the phenolic hydroxyl group and the carbon-carbon
double bond makes the capsaicinoids sensitive to oxidation. Accordingly, capsaici-
noids should be protected from exposure to light, heat, moisture, and oxidizing
agents, which initiate and/or catalyse the decomposition processes.
80
7. General physiology of retinoids
and carotenoids
All of the main components can be found in the plants in association to the color
materials of the plants and the spices. The chemical structures of these compounds
have been detailed in Chapter 6. The Capsicums as typical characteristics of the spe-
cies were prepared from "chilli" (synonym of chili or paprika) from the whole plants
(e.g. paprika or chilli). These plants contain retinoids and carotenoids beside cap-
sicum. The common biological results of the consumption of these plants appear as
the common results of retinoids, carotenoids and Capsicums.
However, it has to be emphasized that considering their biological effects, the
three components show some similarities and differences. In the forthcoming pages
we will give a short summary of the biological effects of the color chemical com-
pounds (retinoids and carotenoids).
7.1. Biochemistry of retinoids and carotenoids
Retinoids are a class of compounds consisting of four isoprenoid units bound in a

head-to-tail manner. All retinoids may be formally derived from a monocyclic par-
ent compound containing five carbon-carbon double bonds and a functional group at
the terminus of the acyclic portion (IUPAC-IUB, 1982). Vitamin A is a fat-soluble
substance found in animal foods and dairy products. Vitamin A is available as pre-
formed vitamin A, contained in liver, cod liver oil, butter, eggs, or as provitamin A
carotenoids, as found in dark green, red and yellow vegetables (Blaner, Oslon, 1994).
These naturally occurring retinoids exist in the all-trans, 13-cis or 11-cis geometric
configurations, with the great preponderance of the body's retinoids being present in
the all-trans configuration (Blaner, 1993). The alcohol all-trans retinol is the parent
compound for all retinoids. Retinol is precursor for the synthesis of the biologically
active retinaldehyde and retinoic acid forms. Additionally, retinol is the precursor
for the synthesis of retinyl esters (Blaner, 1993). Within target tissues retinol is taken
up by the cells from the circulation and can either be oxidized to retinaldehyde,
retinoic acid or esterified for storage (Goodman, 1984). Retinaldehyde is not known
to play another essential physiological role outside the vision, aside from serving as
an intermediate in the enzymatic oxidation of retinol to retinoic acid (Saari, 1991).
Retinoic acid is the biologically active retinoid form which is needed for mediating
and maintaining cellular differentiation (Ross, Tenuis, 1993).
81
7.1.1. General physiology of retinoids and carotenoids
All retinoids in the body originate in the diet either as provitamin A carotenoids or
as preformed vitamin A (Goodman, 1984). The dietary carotenoids and preformed
retinoids undergo a series of metabolic conversions, extracellularly in the lumen of
the intestine and intracellularly in the intestinal mucosa, which result in the prepon-
derance of the absorbed dietary retinoid being converted to retinol (Semba, 1997).
Provitamin A carotenoids, such as P-carotene, may be converted to retinaldehyde
through the cleavage by carotenoids-15, 15 dioxygenase or by an excentric cleavage
pathway. Approximately 50 of over 600 carotenoids found in nature may be convert-
ed to vitamin A (Semba, 1997). The bioavailability of provitamin A carotenoids is
much less than that of performed vitamin A because of a variety of factors, including
differences in efficacy of absorption and biochemical conversion (Semba, 1997).
Although hundreds of carotenoids have been identified, only few have been found to
exist in appreciable concentrations in the human serum: lutein, zeaxanthin, a- and
P-carotene, a- and P-cryptoxanthin (Bieri et al., 1985).
Retinol is esterified in the intestinal mucosa, packaged as retinyl ester into chy-
lomicra, and carried to the liver via the lymphatic circulation (Blaner, 1993). A very
small portion of the dietary retinoid is converted to retinoic acid (or comes in the
diet as such) and enters the circulation through the portal system bound to serum al-
bumin (Smith et al., 1973).
Approximately 90% of vitamin A in the body are stored in the liver as retinyl
esters (Hendriks et al., 1985; Yamada et al., 1987; Hussian et al., 1989). The liver
has the capacity to store enough vitamin A to last for several months, with a longer
storage capacity among adults than children (West, Sommer, 1984). Retinol is
released from the liver in combination with plasma retinol-binding protein (RBP)
and transthyretin (TTR) (Noy, Blaner, 1991; Soparno, Blaner, 1994). Retinol is poor-
ly soluble in water and is carried in the blood sequestered inside the carrier proteins
(Sziits, Harosi, 1991). Retinol seems to enter cells via specific receptors, although it
is unclear whether all cells contain these receptors (Soparno, Blaner, 1994; Zhang et
al., 1996).
Vitamin A exerts its effects through retinoid receptors, which are found in the
nucleus of cells. These receptors resemble steroid and thyreoid hormone receptors
and support the idea that vitamin A acts much like a hormone (Ross, Ternus, 1993).
Retinol is converted into its active metabolite, all-trans-retinoic acid (ATRA) in the
cells. Retinoic acid can control genes through specific receptors that belong to the
superfamily of thyreoid and steroid receptors (Corpet et al., 1990; Capristo et al.,
1998). Retinoic acid receptors act as transcriptional activators for specific target
genes. The retinoic acid receptor (RAR) is expressed as several isoforms referred to
as RAR a, P and y for which all-trans-retinoic acid acts as a ligand, and retinoid X
receptors (RXR) referred to as RXR a, P and y for which 9-cis retinoic acid acts as a
ligand (Kliewer et al., 1992). 9-Cis retinoic acid seems to be functionally distinct
from ATRA, and interconversion may exist between the two isomers. Each RAR and
RXR has a specific DNA-binding domain, a retinoic acid response element (RARE)
by which these nuclear receptors may affect retinoic acid transcriptional activity.
RAR and RXR receptors form heterodimers which bind to DNA and control gene
82
activity. In addition, RXR receptors also can form heterodimers with vitamin D and
thyreoid hormone receptors (Kliewer et al., 1992). Numerous studies have focused
recently on the role of retinoid receptors in the process of carcinogenesis (Lee, Kumar,
1980; Hunter et al., 1993; Matsushima et al., 1996; Zhang et al., 1996; Benbrook et
al., 1997; Lee et al., 1997; Sherman, Partidge, 1997; Waliszewski et al., 1997).
Retinoids and carotenoids have numerous biological functions such as regulating
the growth, morphogenesis and differentiation of cells, they have a variety of effects
on the cell membrane, they can influence the activity of different enzymes, further-
more, exert also immunomodulatory effects. Carotenoids are non-enzymatic antiox-
idants, therefore they are able to prevent genetic changes by preventing DNA dam-
age caused by free radicals. They modulate membrane functions and stabilize initi-
ated cells in the promotional phase of carcinogenesis (Semba, 1997).
The role of retinoids in GI mucosal prevention has been intensively investigated
in our Department in the last two decades (Rumi et al., 2001). Vitamin A and (3-
carotene were shown to prevent the experimentally induced gastric mucosal lesions
in animals (Javor et al., 1983), and these compounds were found to be effective in
the treatment of patients with gastric peptic ulcer (Patty et al., 1982). Carotenoids
have no inhibitory effects on gastric acid secretion either in animals (Javor et al.,
1983) or in humans (Hunyady et al., 1991), however, P-carotene was able to prevent
the gastric mucosal damage in different experimental models such as ethanol (96%)-,
hydrochloric acid (0.6M)- or indomethacin-induced mucosal damage (Patty et al.,
1982; Javor et al., 1983; Hunyady et al., 1991). Furthermore, the ulcer-healing effect
of vitamin A was proven in randomized, multicenter clinical studies (Patty et al.,
1982). The gastroprotection induced by carotenoids does not depend on the presence
of vitamin A activity, P-ionone ring, number of unsaturated links or chemical struc-
ture of terminal part of molecules (Javor et al., 1983; Mozsik, Javor, 1991).
Moreover, the analysis of gastric mucosal antioxidant mechanisms and free radical
generation during P-carotene-induced gastric mucosal protection suggested that the
scavenger character of P-carotene might partially explain its protective effect (Vincze
et al., 1991). The p-carotene-induced gastric gastroprotection was completely abol-
ished by acute bilateral surgical vagotomy (Vincze et al., 1997). Despite these inves-
tigations, the exact mechanism of action is not fully understood yet.
Although carotenoids, such as p-carotene and vitamin A are popularly often
regarded to be equivalent, there are large differences in the biological functions of
these two nutrients, especially regarding their antioxidant properties, since vitamin
A is a less potent antioxidant than P-carotene (Semba, 1997).
7.1.2. Retinoids and chemical carcinogenesis
A suboptimal diet might be related to approximately 30-60% of all cancer cases

(Peto, Doll, 1981) the major part of which is possibly preventable by adequate
dietary modifications (Doll, 1992). Chemopreventive potentials are conceivable for
antioxidant micronutritients (Block, et al., 1992; Block 1991, 1992, 1996). Particu-
larly intriguing are those with polyene structure, i.e. vitamin A-type retinoids and
carotenoids such as P-carotene (Bertram et al., 1991; Willett, 1991; Ziegler, 1991).
83
Several animal and in vitro studies have now been reported related to the effects
of antioxidants on cell proliferation, including GI epithelial cell proliferation. It was
observed 70 years ago that the deficiency of vitamin A in animals provokes abnor-
mal keratinizations, precancerous squamous metaplasia and cancers of various
epithelia (Wolbach, Howe, 1928). The effectiveness of retinoids as inhibitors of
chemical carcinogenesis of the epithelia of the digestive tract has been somewhat
contradictious, although positive results have been obtained in studies of stomach,
esophageal, liver and pancreatic carcinogenesis. The oral administration of retinyl
ester prevented the occurrence of papillomas and carcinomas in hamsters receiving
polycyclic hydrocarbons (Moon, 1993). Nitrosamine-induced esophageal carcino-
genesis can also markedly be inhibited by administering retinyl ester (Moon,
McCromick 1982). Subsequent studies have shown that synthetic retinoids such as
13-cis-retinoic acid also exert a protective effect against the induction of esophageal
tumors with nitroso compounds, although etretinate was ineffective in inhibiting
esophageal carcinogenesis (Moon, 1993). Only few and contradictious data can be
found concerning the effect of retinoids on chemically induced colon carcinogene-
sis. An antioxidant mixture containing |3-carotene and a-tocopherol reduced cell pro-
liferation in the colon and rectum of mice (Lok et al., 1992). Organic and inorganic
selenium with P-carotene reduced colonic epithelial cell proliferation while con-
comitantly reducing the incidence and multiplicity of colon adenocarcinomas in rats
given chemical carcinogens (Nayini et al., 1991). Colorectal epithelial cell prolifera-
tion was reduced in rats given chemical carcinogens when given a diet low in fat and
protein and high in vitamin E, selenium, vitamin A and fibre (Goettler et al., 1987).
Synthetic retinoids are able to prevent azoxymethane-induced intestinal carcinogen-
esis in rats (Kawaromi et al., 1995), although natural and synthetic retinoids have lit-
tle effect on colon carcinogenesis induced by aflatoxin and dimethylhydrazine
(Moon, 1993). The few studies on liver carcinogenesis indicated that 13-cis-retinoic
acid was highly effective in reducing the incidence of liver tumors induced by 3-
methyl-4-dimethylaminoazobenzene (Daoud, Griffin, 1980; Moon, 1993). Similar
findings have been reported for spontaneous hepatomas in mice given various doses
of retinyl ester (Moon, 1993). Several synthetic retinoids inhibited the development
of azaserine-induced pancreatic tumors in rats when administered during the promo-
tional phase of carcinogenesis (Longnecker et al., 1982). In colorectal carcinoma
cell lines ascorbic acid enhanced the antiproliferative effect of vitamin A (Kan-
daaswani et al., 1983), emphasizing their interdependence.
7.1.3. Effect of antioxidants on colorectal epithelial cell

proliferation, polyp recurrence and carcinogenesis:
Clinical trials in patients
The effect of carotenoids supplementation on precancerous lesions and cancer inci-

dence has been investigated in numerous clinical trials in the last two decades.
Smaller trials in humans suggest that antioxidants can reduce colorectal epithelial
cell proliferation. Vitamin A, combined with ascorbic acid, a-tocopherol, further-
84
more selenium caused a significant reduction in the labeling index (LI) of the upper
colonic crypts and the decrease in polyp recurrence in colorectal adenoma patients
(De Cosse et al., 1975, 1989; Bussey et al., 1982; Mckeown-Eyssen et al., 1988;
Cahill et al., 1991, 1993; Paganelli et al., 1992; Roncucci et al., 1993). Polyp forma-
tion could not be inhibited in larger full-scale trials (De Cosse et al., 1989;
Vogelstein, Knizler, 1993; Greenberg et al., 1994).
Prospective studies, including the particularly conclusive Basle study and the
Finnish cohort study, demonstrated that lower levels of retinol and carotene at entry
were associated with a significantly increased relative risk for bronchial carcinoma
and all other cancers (Salonen et al., 1985; Stahelin et al., 1991).
Few studies indicated a positive correlation between carotene intake and carcino-
genesis. In the P-Carotene and Retinol Efficacy Trial (CARET) (Hennekens et al.,
1994a,b; Omenn et al., 1996) P-carotene (30mg) and vitamin A (25000 IU) were
supplemented for patients with high risk for lung cancer (cigarette smoking history
or asbestos exposure), the trial was finished because of the results of the Alpha-
Tocopherol, Beta-carotene Cancer Prevention Study (ATBC) (Hennekens et al.,
1994a,b).
There was an increase in cardiovascular disease and lung cancer mortality among
those assigned the supplementation combination.
In the Physicians Health Study (PHS) (Steering Committee of the Physician's
Health Study Research Group. Final Report on the Aspirin Component of the ongo-
ing Physician's Health Study, 1989) US male physicians were randomized to alter-
nate daily 50 mg P-carotene and 325 mg aspirin, both active treatment, or both pla-
cebo. There was no significant effect of P-carotene on total cancer incidence.
Numerous problems (Block, 1991, 1992, 1996; Tan, Chu, 1991; Ascherio et al.,
1992; Block et al., 1992; Hunter et al. 1993) have been pointed out with these three
studies.
7.2. Prevalence and importance of the checked human

G I diseases
Upper gastrointestinal (GI) ulceration from drugs and inflammatory bowel diseases
(IBD) as well as GI malignancies are relatively common GI pathologies which have
an appreciable GI mortality and morbidity. Furthermore, the role of environmental,
genetic and nutritional factors in the etiology of various cancers and other GI disor-
ders emphasizes the importance of research in these GI conditions (Rainsford, 1996,
2006).
It has long been recognized that the relatively high incidence of GI ulceration
arises from a variety of causes, among them stressful conditions, ulcerogenic drugs,
infections, smoking and high intake of alcohol (Mozsik et al., 1997b,c). While the
incidence of peptic ulcer disease continues to be high, the international trend is, over-
all, towards a moderate decline (despite some fluctuations in reported statistics in
some countries) except in individuals above 60 years of age in whom the incidence
is increasing. Silent or asymptomatic peptic ulcer disease which is evident in about
85
10% of patients is of particular importance because it often has a fatal outcome
(Rainsford, 1996).
About 10% of the total Hungarian population suffer from peptic ulcer disease dur-
ing their life span. It is true that the peak of disease incidence in patients (including
males and females) appears after 50 years of age, furthermore the costs of therapy
are extremely high, though we still do not know exactly how much (Mozsik et al.,
1997a,b,c; Mozsik, 2006).
The non-steroidal anti-inflammatory drugs (NSAIDs) are widely used in the ther-
apy of inflammatory and pain processes in patients, however, these compounds cause
+
often gastrointestinal mucosal damage (H back diffusion, hemorrhage, bleeding)
and different GI complaints (dullness, epigastric pain, vomiting) in a significant
group of treated patients (Mozsik et al., 2006). The extent of NSAID consumption
has increased significantly in the Hungarian population suffering from different joint
diseases. The number of patients who consumed NSAIDs treating their joint dis-
eases is about 2 to 3 millions (20 to 30% of the nation). It is unambiguous that the
number of patients suffering from NSAIDs-induced gastrointestinal mucosal dam-
age has increased in the last decade (Mozsik, 2006). This fact is enough for doing
research in this field.
GI mucosal lesions induced by NSAIDs are considered to involve multiple patho-
genic elements, such as deficiency of prostaglandins (PGs), gastric hypermotility,
disturbances in microcirculation, oxygen free radicals, neutrophil activation and
direct damaging effect on the gastric mucosa (weak vs. strong acid-induced physico-
chemical properties, like salicylic acid) (Takeuchi et al., 1994, 1996; Mozsik et al.,
2001a,b). However, these components closely interact with each other and it would
be difficult to determine which pathogenic element is of prime importance in the
ulcerogenic response to NSAIDs. Indeed neutrophil activation is caused by alter-
ation of arachidonic acid metabolism, such as PG deficiency; gastric hypermotility
leads to microcirculatory disturbances, resulting the enhancement of neutrophil
adherence to the vascular endothelium; and the production of oxygen radicals is
brought about by neutrophil-endothelium cell interaction as well as by hemodynamic
alterations due to gastric hypermotility (Takeuchi et al., 1996).
Nowadays, the extent and ratio of cyclooxygenase 1 (COX-1) and cyclooxyge-
nase 2 (COX-2) inhibition are dominantly in the international interest (Mozsik et al.,
1997a,b; Karadi, Mozsik, 2000). The extents of actions of NSAIDs on COX-1 and
COX-2 inhibitions can be separated from each other experimentally, but we do not
have enough concrete data on the NSAIDs-induced decrease of gastric mucosal
blood flow (GMBF) in patients, although it is sure that its extent cannot reach such a
level to be able to produce a real tissue hypoxia in the gastric mucosa (Mozsik et al.,
2000; Mozsik, 2006).
Studies performed in animals can help us to understand the mechanisms of GI
mucosal damage and help in prevention. The presence of tissue hypoxia has been
suggested as one of the main etiological factors in the development of gastric muco-
sal injury (in acute ulcer phase and in other injuries in later phase) based on the basic
physiological blood flow observations. (Menguy, Master, 1974a,b,c; Menguy et al.,
1974).
86
A broad biochemical-pharmacological approach of ulcer diseases has been
applied in our Department in the last three decades (Mozsik, Vizi, 1976; Mozsik, Ja-
vor, 1988; Mozsik et al., 1990; 1992; Mozsik, Pfeiffer, 1992) and this shift could
also be observed in international research.
A special experimental approach to this problem has been established by the
simultaneous measurements of the biochemical compounds of membrane-bound
ATP-dependent energy systems such as adenosine triphosphate (ATP), adenosine
diphosphate (ADP), adenosine monophosphate (AMP) and cyclic adenosine
monophosphate (cAMP) which, together with the lactate level, give an excellent
biochemical "cross-section" of the target (stomach) organ (Mozsik, Vizi, 1976;
Mozsik, Javor, 1988; Mozsik et al., 1990, 1992; Mozsik, Pfeiffer, 1992).
The energy is stored in form of ATP in the gastric mucosa and the measurement of
its actual tissue level informs us about the dynamic equilibrium between the ATP
resynthesis and breakdown. The gastric mucosal ATP can be splitted in direction of
ADP (by membrane ATPase) or cyclic AMP (by adenylate cyclase) liberating a free
energy source for the tissue functions, while ADP and cAMP transform to AMP sup-
plying further energy liberation. AMP is a common energy-storing adenosine com-
pound originating from both pathways of ATP-ADP and ATP-cAMP breakdowns
(transformations). The liberated energy is used for the regulation of the different
functions of the cells (Mozsik, Vizi, 1976; Mozsik, Javor, 1988; Mozsik et al., 1990,
1992; Mozsik, Pfeiffer, 1992).
If the energy of different cellular functions originates from the gastric mucosal
ATP breakdown, the results of activity of plasma membrane enzymes (membrane
ATPase, adenylate cyclase) can be observed in the changes of mucosal ADP, cAMP
and AMP. Furthermore, different biochemical parameters can be calculated from the
values of ATP, ADP and AMP:
a) adenylate pool (ATP+ADP+AMP),
b) ratio of ATP/ADP and
c) "energy charge" [(ATP+0.5 ADP) / (ATP+ADP+AMP)] (Atkinson, 1968).
The value of energy charge is theoretically 1, when all of adenosine compounds
are in phosphorylated form, and 0, when these compounds are in dephosphorylated
form (Atkinson, 1968).
After the basic observations of Menguy et al. (1974) and Menguy and Master
(1974a,b,c), experimental and clinical researchers accepted these results as a strong
correlation existing between the decrease of gastric mucosal blood flow and the
decrease of gastric mucosal ATP level was considered to be a consequence of tissue
hypoxia in the gastric mucosa (without the presence of any scientifically proved
arguments) (Mozsik et al., 2000). The biochemical measurement of the decrease of
gastric mucosal ATP is not enough proof for the existence of tissue hypoxia alone,
because the simultaneous elevation of tissue lactate is also basically necessary
(Mozsik et al., 2000). The biochemical measurements of adenosine compounds offer
an excellent biochemical research information on the extent of tissue oxygenation,
when the observations are simultaneous from the same tissue sample. Furthermore,
the existence of tissue hypoxia can be characterized well by the extremely low va-
lues of tissue ATP, "energy charge" and increased levels of tissue lactate (Mozsik et
al.,2000, 2001a,b).
87
It is very important to emphasize that the observations of Menguy et al. were car-
ried out in rats with hemorrhagic shock (Menguy, Master, 1974a,b,c; Menguy et al.,
1974).
+
Earlier Davenport proved that weak acids induce gastric H back diffusion in pres-
ence of a strong acid like HC1 (Davenport, 1970, 1973). Later Mozsik et al. (1981)
demonstrated the decreased breakdown of adenosine triphosphate (ATP) into adeno-
sine diphosphate (ADP) in rats treated with sodium salicylate plus pylorus-ligation,
while its value increased only in pylorus-ligated rats (Mozsik, Vizi, 1976). These
and other observations proved the role of ATP-dependent energy systems in the
development of gastric hyperacid secretion in this experimental model.
Na-salicylate and indomethacin are both NSAIDs; however, their actions are sig-
nificantly different, i.e. salicylate acts as weak acid, while indomethacin inhibits
prostaglandin synthesis (Mozsik et al., 1996a,b). These previous data suggest a sig-
nificant difference in the metabolism of the stomach, in time of development of
the gastric mucosal damage produced by sodium salicylate and indomethacin
(Davenport, 1970, 1973). However, we have only few data on drugs-induced intra-
cellular metabolic changes in the GI mucosa, therefore our aim was to investigate
this matter on the membrane-bound ATP dependent energy systems (ATP-ADP,
ATP-cAMP) and to study the changes in the feedback mechanism between them
(Mozsik, 1969; 2006; Mozsik etal., 1981, 1990).
The incidence of IBDs is definitely on the rise in Europe and USA though few
studies consider this fact partly to be the consequence of improved efficiency in their
diagnosis (Monterio, Tavarela, 1995). With a total number of approximately 80 ill-
nesses per 100,000 inhabitants they are comparatively rare. These illnesses are not
only a burden for the individual but also put them under considerable psychological
strain. The etiology of IBDs still remains unclear. Although hereditary factors,
microorganisms harmful to the mucosa and certain nutritional (eating) habits are
assumed to be causal factors, there is little hard evidence to substantiate these
assumptions (Jewell, 1995; Travis 1995). The familiar association of IBDs, rein-
forced by the demonstration of increased incidence of both Crohn's disease and
ulcerative colitis in monozygotic twins, is a quite compelling evidence for an inher-
ited component in the etiology of both diseases (Farmer, Michener, 1986; Jewell,
1995). Genetic markers, such as HLA class II antigens and cytokine polymorphisms,
have provided interesting data and may well prove additional factors in the predis-
position to IBD. Of the HLA associations observed, that for the DR2 system is rela-
tively strong. Within this system, however, there appear to be variations that may be
due to ethnic origins (e.g. high frequency of DRB 1.502 alleles in Japanese and
Jewish patients with ulcerative colitis but not in white non-Jewish subjects) (Jewell,
1995; Satsangi, Jewell, 1996).
Crohn's disease (CD) is frequently complicated by various nutritional distur-
bances (Ladefoged et al., 1996; Capristo et al., 1998). Although it is important to
correct these disturbances in treating CD patients, the nutritional status of CD
patients has been poorly documented, especially concerning their vitamin status.
Among various reports of vitamin A deficiency in different gastrointestinal and liver
diseases (Jensen, Gluud, 1994; Whitington et al., 1994), relatively few studies
have investigated CD (Fernandez-Banares et al., 1989; Zurakowski et al., 1997).
88
There has been inadequate documentation of the prevalence of vitamin A deficiency
in patients with CD who are a population at a risk of protein malnutrition and fat
malabsorption as a result of diseased or resected small bowel. No observations can
be found in the literature describing the changes of all serum carotenoids in patients
with IBD or GI cancer.
Colorectal adenocarcinoma is the second most common malignancy in Europe
and the USA and leads to considerable mortality (Lakatos, 1994; Bousvaros,
Zurakowski, 1998). The disease is rare in Asia and Africa, this difference is thought
to be largely environmental rather than racial. 45% of all GI malignancies are co-
lorectal compared with 25% stomach cancer, 15% pancreatic cancer, 11% esophageal
cancer and 4% liver and bile duct cancer in West-European Countries (Jankowsky,
1994). The prevalence of GI cancers is increasing in Hungary with the exception of
gastric cancer. The incidence of colorectal cancer increases with age, the average
age at diagnosis is between 50 and 60 years (Lakatos, 1994).
Colorectal cancer has a multifactiorial etiology; numerous environmental and
genetic aspects have been investigated. It is estimated that perhaps 35% (range 10 to
70%) of all cancer mortality in the United States could be attributable to dietary fac-
tors (Willett, 1991; Potter et al., 1993). Ecologic and migration studies indicate the
importance of environmental factors in colon cancer (Potter et al., 1992). Diet
appears to have a particularly strong association with occurrence of this cancer, and
thus it offers promise for intervention (Willett, 1991; Potter et al., 1993).
The observation that higher colon cancer mortality rates occur in the countries
where meat and fat consumption was higher led to the hypothesis that these food
items contributed to an individual risk of developing colon cancer and was a stimu-
lus for the current interest in dietary intake in most analytic studies of the etiology of
colon cancer (Potter, 1992):
1. The oldest hypothesis asserts that fat intake increases bile acid production, ulti-
mately increasing the exposure of the bowel mucosa to the toxic, trophic and can-
cer-promoting effects of bile acids. The capacity of colonic flora to transform bile
acids into potential carcinogens has been found to be greater in populations with
high rates of colon cancer and among meat-eating populations than in vegetarian
populations (Potter, 1992);
2. A more recent hypothesis is the cooked food hypothesis. High-fat diets contain
greater amounts of carcinogenic heterocyclic amines (from meat proteins) and
promoters as a consequence of cooking at high temperature (cooking in fat pro-
duces higher temperature than cooking in water) (Corpet et al., 1990);
3. A third and recent hypothesis is that high consumption of meat, particularly red
meat, may increase fecal concentrations of iron, which catalyzes oxidative reac-
tions, leading to increased lipid peroxidation and oxidative DNA damage and to
an increased risk of colorectal cancer (Babbs, 1990). Furthermore, the protective
role of vegetable and fruit consumption, dietary fibres, calcium, vitamin D and
the potential carcinogenic effect of sucrose have been proved in the last decades
(Potter etal., 1993).
The role of dietary components is well known in other types of GI malignancies,
too. Consumption of salted, pickled and smoked foods has been associated with
increased risk of gastric cancer in case-control studies (Fontham, 1997). The strong,
89
consistent inverse association between consumption of fruits and vegetables and
colorectal carcinogenesis is clear. Relatively high intake of P-carotene and vitamin C
is consistently associated with reduced risk of gastric cancer, the Basle study found
a negative correlation between serum carotene levels and risk of gastric cancer.
Similar results were found in case of esophageal cancer (Fontham, 1997).
7.3. Gastric cytoprotection, as a special form of defensive

mechanisms to gastrointestinal (GI) mucosal injury
produced by retinoids
A special mechanism of actions of prostaglandins was described by Robert et al.
(1979) on gastric mucosa in rats, namely the prostaglandins were able to prevent the
gastric mucosal damage without presence of any decrease of gastric acid secretion.
This phenomenon was named "cytoprotective action". In the early 1980s many
researchers believed that this phenomenon is only the specific action of PGs, mean-
while later the existence of gastric cytoprotection has been proven for different
agents (such as atropine, cimetidine, growth factors - for reference see Moron et al.,
1983). We proved the cytoprotective effects of retinoids in 1983 (Javor et al., 1983).
This phenomenon was special and surprising because the existence of gastric cyto-
protection was produced by different nutritional components. That is why this phe-
nomenon was termed as "nutritional cytoprotection". Below we give a short critical
summary of our observations with retinoids obtained in different studies (animal
experiments, malignant cell lines, human observations).
The carotenoids are chemical components responsible for the color of plants, the
number of naturally occurring carotenoids is about 600 (Mozsik et al., 1986). To study
their mechanisms of actions different compounds (vitamin A, P-carotene, zeaxanthin,
lutein, cryptoxanthin, capsanthol, and capsorubin) were tested. The selection of these
retinoids was done on their significance of chemical properties, namely vitamin A
activity, number of unsaturated double bonds and terminal (P) chemical structure:
1. Gastric cytoprotection does not depend on vitamin A activity. For example, the vi-
tamin A activity of P-carotene (P,P-carotene) is completely eliminated by modifi-
cations such as introduction of a hydroxyl group at C-3(3'), substituting the E-end
group for the a-end group. Although zeaxanthin (3,3'dihydroxy-P,P-carotene) and
lutein (3,3'dihydroxy-P-carotene) are not precursors of vitamin A, they do exert a
cytoprotective effect similar to that of vitamin A precursors P-carotene and P-cryp-
toxanthin (3-hydroxy-P,P-carotene);
2. Gastric cytoprotection cannot be related exclusively to a conjugated polyene chain
present in carotenoids, because, for example, acyclic lycopene (P,P-carotene) with
an undecene chromophore, and cyclic capsorubin (3,3'-dihydroxy-K,K-carotene-
6,6-dione) with a nonene-dione chromophore, are inactive. Furthermore, cytopro-
tective zeaxanthin and lutein have different chromophore systems;
3. Gastric cytoprotection by asymmetrical C40 carotenoids cannot be explained by a
splitting of the molecules at the central double bond, which would result two C 2u
units with or without cytoprotective properties. According to such conversion, one
90
might expect that capsanthin (3,3'-dihydroxy-(3, K-carotene-6'-one) would be
cytoprotective, as its molecule can be built up from an active (1/2 zeaxanthin) and
an inactive (1/2 capsorubin) C o unit. The lack of gastric cytoprotection by cap-
2
santhin argues against such a conversion. It should be noted that capsanthol, the
reduction product of capsanthin, is also inactive (Javor et al., 1983) (Table 14).
T a b l e 1 4 . C o r r e l a t i o n s b e t w e e n t h e gastric c y t o p r o t e c t i v e efects of r e t i n o i d s , their c h e m i c a l

structure a n d v i t a m i n A a c t i v i t y in rats*
Retinoids Terminal chemical Vitamin A Gastric mucosal

structure activity prevention
Vitamin A R=a yes yes
P-carotene X=Y=a yes yes
fj-cryptoxanthin X=a, Y=b yes yes
Zeaxanthin X=Y=b none yes
Lutein X=b, Y=c none yes
Capsorubin X=Y=d none none
Capsanthin X=b, Y=d none none
Capsanthol X=b, Y=e none none
Lycopene X=Y=f none none
* from Javor, T., Bata, M., Lovasz, L., Moron, F., Nagy, L., Patty, I., Szabolcs, J . , Tarnok, F., Toth, Cy., Mozsik,
Cy. (1983): Gastric cytoprotective effects of vitamin A and other carotenoids. Int. J . Tiss. React. 5: 289-296
(with permission)
7.4. N e w results in the biological actions of retinoids

and carotenoids in animals, healthy human subjects
and patients with different gastrointestinal disorders
The number of patients with gastrointestinal mucosal damage (inflammatory dis-
eases, chemicals-, drugs-, bacterial- and virus-induced infections, precancerous
states and cancers) is extremely high over the world. The role(s) of some genetic and
environmental factors in the clinical manifestation of these disorders has (have) been
emphasized. The results of different randomized studies are very controversial con-
cerning these disorders.
Many observations have indicated the essential role(s) of plants (as nutritional
factors) in the protection (prevention) of GI mucosal damage in animal experiments
and in human observations. The retinoids (as color materials of plants) were thor-
oughly studied in these disorders, however, their mechanisms of action remained to
be unclear up to now.
The different suggested mechanisms of retinoids were analyzed and summarized
in animals under different experimental circumstances, in human healthy subjects
and in patients with different GI diseases (inflammatory, precancerous states and
cancers) (Mozsik, 2004) The potential roles of gastrointestinal mucosal protective
effects of retinoids were studied in healthy human subjects, in patients with gastric
91
(GU) and duodenal (DU) ulcer (n=131), Crohn's disease (n=40), ulcerative colitis
(n=30), hepatitis C (n=90), colorectal polyps (n=59), stomach- (n=21), pancreas-
(n=10), hepatocellular- (n=14) and colon- (n=44) cancer.
The effect of vitamin A was measured on the gastric basal acid output (BAO) and
pentagastrin stimulated maximal output (MAO) and indomethacin (3 x 25mg)-
induced gastric mucosal microbleeding in healthy human subjects. The serum levels
of vitamin A, a- and P-carotene, a- and P-cryptoxanthin and lutein were measured
by HPLC method in patients with different gastrointestinal chronic inflammatory
precancerous states and cancer diseases.
7.4.1. Gastrointestinal mucosal protective effects produced

by retinoids in animal experiments
Many experiments have been carried out with retinoids in different animal models
(for details see Ref. of Mozsik, 2006).
It has been found in animal experiments that: 1) The retinoids prevent the drug
(EtOH, HC1, sodium salicylate-, indomethacin)-induced gastric mucosal damage
without any presence of inhibition of gastric acid secretion ("nutritional gastric cyto-
protection"). 2) The gastric cytoprotective effects of retinoids do not depend on the
a) vitamin A activity; b) number of unsaturated double bonds; c) the presence of a
characteristic chemical structure of their terminal components; d) the presence of vi-
tamin A activity; e) modification of vascular permeability. Their gastrointestinal
mucosal prevention depends on 1) intact vagal nerve and 2) intact adrenals in the
experimental animals; 3) gastric biochemical changes (the retinoids produce a dose-
dependent inhibition on the extent of ATP-transformation into ADP, meanwhile they
produce an increase in the transformation of ATP into cAMP; 4) intact function of
sulfhydryl groups.
These gastric cytoprotective effects of retinoids differ: 1) the retinoid-induced
gastric mucosal preventive effects differ from those of PGs; 2) the cAMP is an intra-
cellular signal for the development of gastric mucosal damage produced by chemi-
cals (EtOH, HC1, NaOH, concentrated NaCl, sodium salicylate, indomethacin) and
for the prevention in gastric mucosa induced by retinoids (but not by PGs); 3) the
gastric mucosal protection induced by retinoids and gastric mucosal permeability
can be separated in time (Mozsik et al., 1991, 1995, 1996a,b, 1997a,b, 2001a;
Mozsik, 2004, 2006).
7.4.2. Effects of vitamin A on the gastric secretory responses

and indomethacin-induced gastric microbleedings
in healthy human subjects
The existence of gastric mucosal prevention can be proved in healthy persons (against
IND treatment), in patients with gastric ulcer (GU) and duodenal ulcer (DU) without
presence of any inhibitory effect on gastric acid secretion (Mozsik et al., 2007b).
Orally administered indomethacin (IND) (a nonspecific cyclooxygenase 1 and 2
enzyme inhibitor) produces gastric mucosal damage in human healthy subjects. This
injury can be detected by the measurement of gastric microbleedings (Moron et al.,
1984; Nagy et al., 1984). Recently, the capsaicin-induced gastric mucosal protection
was observed by the direct measurement of the changes in the gastric transmucosal
potential difference (GTPD) produced by intragastric administration of ethanol in
human healthy subjects (Mozsik et al., 2004, 2005a).
7.4.3. Ulcer healing effect of vitamin A in patients with chronic

gastric ulcer (multiclinical randomized, prospective study)
A randomized, prospective study was carried out in healthy human subjects (n = 12

in each group) to test the cytoprotective effects of atropine (0.125 mg/kg i.m.), cime-
tidine (12.5 mg/kg i.m.) and vitamin A (100.000 IU i.m.) on the gastric basal (BAO)
and maximal (MAO) acid output, and on gastric microbleeding from indomethacin
(4 x 25 mg per os)-induced gastric microbleedings (Moron et al., 1984; Mozsik et
al., 1986).
7.4.4. Changes in serum levels of retinoids in patients

with different gastrointestinal inflammatory diseases
The serum levels of vitamin A and zeaxanthin were decreased significantly in
patients with chronic gastrointestinal inflammatory diseases (gastritis, hepatitis,
ileitis terminalis, ulcerative colitis), colorectal polyposis, and different (esophageal,
gastric, pancreas, hepatocellular and colorectal) malignant diseases, meanwhile the
serum levels of vitamin A provitamins were unchanged and their GI mucosal pre-
ventive effects did not depend on the presence of vitamin A activity.
It has been concluded that: 1) many experimental and human observations proved
clearly the presence and nature of the defensive role of retinoids in the GI tract; 2)
there is a correlation between the: a) scavenger properties of retinoids vs. intact vagal
nerve; b) scavenging properties vs. intact adrenals; and 3) GI mucosal preventive
effects of retinoids and their biochemical changes in GI mucosa; 4) the decrease of
extent of ATP-ADP transformation in association with increased extent of ATP-
cAMP transformation vs. retinoids produced gastrointestinal mucosal prevention
only partly as scavengers in chemicals-induced release of oxygen free radicals; 5)
sulfhydryl groups of GI tissues take place in the development of GI mucosa (without
and with application of retinoids). A lot of clinical observations were carried out in
human healthy subjects and patients with different gastrointestinal disorders in con-
nection to retionoids and carotenoids (Rumi et al., 1999, 2000, 2001; Mozsik et al.,
2001a, 2005b; Mozsik, 2004, 2005, 2006).
93
According to these results, the serum level of the retinoids decreased in patients
with Crohn's diesase, ulcerative colitis, hepatitis, colorectal polyps and different
gastrointestinal tumors (oesophageal, stomach, pancreas, hepatocellular, and colon)
(Table 15).
T a b l e 1 5 . C h a n g e s o f s e r u m c a r o t e n o i d s in patients w i t h different C I d i s e a s e ( c o m p a r e d t o t h e
results of h e a l t h y s u b j e c t s ) *
Hepatocellular cc.
Ulcerative colitis
Disease
Colorectal polyp
Oesophagus cc.
Crohn's disease
Pancreas cc.
Stomach cc.
Hepatitis C
Colon cc.
Carotenoid
Vitamin A u i u 11 11 1 NS 11 Ill
a-Carotene 1 NS NS NS NS NS NS NS NS
P-Carotene 1 1 NS NS NS NS NS NS NS
Lutein 1 NS NS NS NS NS NS NS NS
Zeaxanthin 11 NS NS 1 11 1 111 11 -1LL"-

a-Cryptoxanthin NS NS NS NS NS NS NS NS NS
P-Cryptoxanthin NS NS NS NS NS NS NS NS NS
111= p<0.001; ll=p<0.01; l=p<0.05; NS= not significant

* Rumi, Gy. jr., Par, A., Matus, Z., Rumi, Gy., Mozsik, Gy. (2001): The Defensive Effects of Retinoids in the
Gastrointestinal Tract (Animal Experiments and Human Observations). Akademiai Kiado, Budapest, Hungary
(with permission)
7.4.5. Changes in serum levels of retinoids in patients

with different gastrointestinal cancers
The retinoids are chemical compounds of color materials of plants. The literature
uniformly agrees that the increased intake of plants as foods prevents the different
types of GI cancers. We studied the possible role of different retinoids (vitamin A, (3-
and p-carotene, a- and p-cryptoxanthin, zeaxanthin, lutein) in patients with different
GI cancers, and these present observations were based on our studies published ear-
lier (Mozsik et al., 1994, 1995, 1998, 2001b; Rumi et al., 1999; 2000, 2001; Mozsik
2004). GI tumors had different locations (oesophagus, stomach, pancreas, liver, and
colon). We suggest that different aetiological factors are involved in the develop-
ment of these types of different GI cancers (Barrett's oesophageal metaplasia, chron-
ic atrophic gastritis, viral infection in liver, chronic inflammatory bowel diseases).
The serum levels of vitamin A and zeaxanthin were decreased significantly in all
of the groups of GI cancer patients (vitamin A was not decreased in patients with
pancreatic cancer). Surprisingly, the serum levels of provitamins of vitamin A were
found to be normal in patients with different GI tumors. These results together indi-
cate that probably the transformation of provitamins into vitamin A is impaired by
94
some reason(s) at the hepatic level, which suggests a key role of the liver in the tu-
mor development of patients with different GI cancers. Similar correlations were
obtained in the serum levels of retinoids in patients with hepatocellular cancers,
which offers a further proof for the existence of this hypothesis.
It was also interesting to evaluate the possible correlation between the terminal
chemical structure, vitamin A activity and GI mucosal prevention. Our results clear-
ly proved that there is no close correlation between the terminal chemical structure,
vitamin A activity and GI mucosal protection (Javor et al., 1983; Mozsik, 1994,
1996; Mozsik et al., 2005b, 2007a) (Table 16).
T a b l e 1 6 . S u m m a r y of t h e c h a n g e s of s e r u m l e v e l of retinoids in patients w i t h different

gastrointestinal cancers*
Oesophageal Gastric Hepatocellular Pancreatic Colorectal I n situ

cancer cancer cancer cancer cancer colon
cancer
~~-____Patients
Retinoids ~~~~~ 8 21 15 10 44 9
Vitamin A u u in NS III
ce-Carotene NS NS NS NS NS NS
fl-Carotene NS NS NS NS NS NS
oc-Cryptoxantin NS NS NS NS NS NS
fi-Cryptoxantin NS NS NS NS NS NS
Zeaxanthin 11 I III ill
Lutein NS NS NS 11 NS NS
P values: between healthy controls vs. each group; -l=P<0.05 J4=P<0.01 111=P<0.01
* Mozsik, Cy, Rumi, Cy., Domotor, A., Figler, M., Casztonyi, B., Rapp, E., Belagyi, J . , Matus, Z., Melegh, B.
(2005b): Involvement of serum retinoids and Leiden mutation in patients with esophageal, gastric, liver,
pancretic, and colorectal cancers in Hungary. World J . Gastroenterol. 11: 7646-7650 (with permission)
Similar results were obtained in the animal experiments published earlier (Javor
et al., 1983; Mozsik, 1994, 1996; Mozsik et al., 2001b, 2005b) (Table 17, Fig. 14).
At present we have no correct information on the correlation between the serum
vs. tissue levels of retinoids in patients with different GI tumors.
In animal observations we proved the presence of P-carotene in the gastric mucosa
in indomethacin-treated rats after acute surgical vagotomy (Mozsik et al., 1991,
2001a, 2005b, 2007a), however, no gastric mucosal protection was found, indicating
the necessity of intact vagal nerve for the development of P-carotene-induced gastric
cytoprotection (Mozsik et al., 2001). The mechanisms of action of GI mucosal defen-
sive effects of retinoids are very complex. Our earlier observations indicated that the
GI mucosal protection of retinoids depends on 1) intact vagal nerve, 2) intact adren-
als in the experimental animals, 3) gastric mucosal biochemical changes (the
retinoids produce a dose-dependent inhibition on the extent of ATP-transformation
into ADP in association with a simultaneous increase in the transformation of ATP
into cAMP), 4) intact function of sulfhydryl groups, and 5) scavenger properties
(Mozsik et al., 1991, 1994, 1995, 1998, 2001a, 2005b, 2007a; Rumi et al., 2001).
T a b l e 1 7 . C o r r e l a t i o n s b e t w e e n t h e gastric c y t o p r o t e c t i v e effects of retinoids, their c h e m i c a l
structure a n d v i t a m i n A a c t i v i t y in patients w i t h t h e different gastrointestinal disorders
Retinoids Terminal chemical Vitamin A Gastric mucosal

structure activity prevention
Vitamin A R=a Yes Yes
a-Carotene X=Y=a Yes Yes
(3-Cryptoxanthin X=a, Y=b Yes Yes
Zeaxanthin X=Y=b None Yes
Lutein X=b, Y=c None Yes
Capsorubin X=Y=d None None
Capsanthin X=b, Y=d None None
Capsanthol X=b, Y=e None None
Lycopene X=Y=f None None
The results were based on the results obtained in animals Uavor, X, Bata, M , Lovasz, L., Moron, R, Nagy, L.,
Patty, I., Szabolcs,)., Tarnok, R, Toth, Gy., Mozsik, Gy. (1983): Gastric cytoprotective effects of vitamin A and
other carotenoids. Int. J . Tiss. React. 5: 289-296] and patients with different gastrointestinal disorders [Mozsik,
Gy., Rumi, Gy., Domotor, A., Figler, M., Gasztonyi, B., Papp, E., Belagyi, J . , Matus, Z., Melegh, B. (2005b):
Involvement of serum retinoids and Leiden mutation in patients with esophageal, gastric, liver, pancretic,
and colorectal cancers in Hungary. World J . Gastroenterol. 11: 7646-76501
Zeaxanthin, |imol/l
Healthy (57)
0.14
0.12 -|
0.10
Y = A + B*X
S t o m a c h (21)
A = 0.0107 0.0037
0.08
B = 0.063 0.005
C o l o n (53) R = 0.88 0.014
0.06 P = 0.006
Liver (15)
0.04
In situ c o l o n (9)
0.02
0.0 0.5 1.5 2.0
Vitamin A, |imol/l
Fig. 14. C o r r e l a t i o n b e t w e e n t h e s e r u m l e v e l s of z e a x a n t h i n a n d v i t a m i n A a n d patients w i t h

different gastrointestinal t u m o u r s ( t h e n u m b e r of e x a m i n e d patients in parenthesis).
( M o z s i k , G y . , R u m i , G y . , D o m o t o r , A . , Figler, M . , G a s z t o n y i , B., P a p p , E., B e l a g y i , J . ,
M a t u s , Z . , M e l e g h , B., 2 0 0 5 b : I n v o l v e m e n t o f s e r u m retinoids a n d L e i d e n m u t a t i o n
in patients w i t h e s o p h a g e a l , gastric, liver, p a n c r e t i c , a n d c o l o r e c t a l c a n c e r s in H u n g a r y .
W o r l d J . Gastroenterol. 1 1 : 7646-7650) (with permission)
96
The retinoid-induced GI mucosal protections do not depend on the 1) inhibition
of gastric acid secretory responses 2) vitamin A activity, 3) number of unsaturated
double bonds, 4) the presence of a characteristic chemical structure of their terminal
components, and 5) modification of vascular permeability (see updated review paper
of Mozsik 2004, 2006; Mozsik et al., 2005b, 2007a). These results clearly proved
that the beneficial effect of retinoids is much more complex than only those depend-
ing on their scavenger properties. The results of biochemical observations suggested
the existence of different cAMP-dependent cellular regulatory mechanisms (inclu-
ding the functions of retinoid receptors, gene expressions) (Mozsik et al., 2005b,
2007a).
7.4.5.1. Leiden mutation in patients

with different gastrointestinal tumors
The presence of Leiden mutation (replacement of Arg by Glu of residue 506 in the
factor V molecule, FVR, 506 Q) has been proven in thrombophilia (Bargen, Parker
1936; Dahlback et al., 1993; Bertina et al., 1994; Dahlback 1995) as well as in
Crohn's disease and ulcerative colitis (Best et al., 1976; Talbot et al., 1986; Nagy et
al., 2000; 2001; Papa et al., 2003), meanwhile no higher prevalence of Leiden muta-
tion has been observed in patients with acute gastritis and hepatitis (Nagy et al.,
2001). The presence of Leiden mutation (APC) is responsible for blood coagulation
in thrombophilia.
The involvement of vascular events was suggested in the development of different
acute inflammatory processes in the GI tract (Mozsik, 2004). That was the reason
why we studied the potential role of Leiden mutation in the acute and chronic gas-
trointestinal inflammatory processes {Helicobacter /ry/on'-induced gastritis, viral hep-
atitis, Crohn's disease, ulcerative colitis). The prevalence of Leiden mutation was
increased in chronic inflammatory bowel diseases, but no changes were observed in
gastritis and hepatitis. It was also significantly higher in the patients with oeso-
phageal, gastric, hepatocellular, pancreatic and colorectal cancers. These results may
indicate that the increased prevalence of Leiden mutation alone resistance does not
take place directly in the tumor genesis of the human GI cancers. We compared the
different results in the examined parameters in patients with acute and chronic gas-
trointestinal inflammatory diseases, and we suppose a time-sequence process
between the inflammatory diseases and GI cancers in patients, suggesting a key role
of retinoids in the progress of precancerous states to cancerous states (Rumi et al.,
1999, 2001; Mozsik et al., 2001b, 2005b; Mozsik, 2004).
7.4.5.2. Correlation between the prevalence of Leiden mutation

and decrease of serum levels of vitamin A and zeaxanthin
in patients with different gastrointestinal tumors
Different correlations were found between the genetic (Leiden mutation) and envi-
romental factors (vitamin A, zeaxanthin) in patients with different gastrointestinal
tumors (Figs 15-17).
97
We found a positive and significant correlation between the following parameters:
1) prevalence of Leiden mutation vs decrease in serum vitamin A level, 2) preva-
lence of Leiden mutation vs. decrease of serum level of zeaxanthin, 3) decrease in
serum level of zeaxanthin vs. the decrease in serum level of vitamin A (Fig. 14), 4)
the increased prevalence of Leiden mutation represents a genetic factor in the differ-
ent chronic precancerous states 5) interestingly the positive correlations were found
between the prevalence of Leiden mutation vs. decrease in serum levels of vitamin A
and zeaxanthin, which suggest the promoter roles of retinoids in the human GI tu-
mor genesis, 6) these tumor promotor properties of retinoids do not depend on the
nature of tumor histology (planocellular, adenocarcinoma and hepatocellular can-
cers) (Mozsik et al., 2005b) (Figs 14-17).
p<0.001
Healthy Oesophag. Gastric Liver Pancreas Colorectal In situ c c .
600 8 21 15 10 44 9
Fig. 75. P r e v a l e n c e of L e i d e n m u t a t i o n in patients w i t h different gastrointestinal t u m o r s .

T h e n u m b e r o n t h e abscissa i n d i c a t e s t h e n u m b e r of patients ( 6 0 0 b l o o d d o n o r s u s e d as
c o n t r o l ) ( M o z s i k , G y . , R u m i , G y . , D o m o t o r , A . , Figler, M . , G a s z t o n y i , B., P a p p , E.,
B e l a g y i , J . , M a t u s , Z . , M e l e g h , B., 2 0 0 5 b : I n v o l v e m e n t of s e r u m retinoids a n d L e i d e n
m u t a t i o n in patients w i t h e s o p h a g e a l , gastric, liver, p a n c r e t i c , a n d c o l o r e c t a l c a n c e r s
in H u n g a r y . W o r l d J . G a s t r o e n t e r o l . 1 1 : 7 6 4 6 - 7 6 5 0 ) ( w i t h p e r m i s s i o n )
98
Vitamin A, n.mol/1
2.0
J E
Y = A + B*X
A = 3.166 0 . 5 3 6
Pancreatic (10) B = - 0 . 1 3 0 0.025
1.6 R = - 0.94 0 . 3 7
P = 0.015
1.2
Stomach (21)
0.8
f Liver (15)
0.4
C o l o n (53)
"~i
25 30
Prevalence of Leiden, %
Fig. 76. C o r r e l a t i o n b e t w e e n t h e p r e v a l e n c e of L e i d e n m u t a t i o n a n d s e r u m level of v i t a m i n A w i t h

different gastrointestinal t u m o r s (the n u m b e r of e x a m i n e d patients in parenthesis)
( M o z s i k , G y . , R u m i , G y . , D o m o t o r , A . , Figler, M . , G a s z t o n y i , B., P a p p , E., B e l a g y i , J.,
M a t u s , Z . , M e l e g h , B., 2 0 0 5 b : I n v o l v e m e n t of s e r u m retinoids a n d L e i d e n m u t a t i o n
in patients w i t h o e s o p h a g e a l , gastric, liver, p a n c r e t i c , a n d c o l o r e c t a l c a n c e r s in H u n g a r y .
Zeaxanthin, (j,mol/l
0,16 Y =A+ B*X
A = 0.151 0.034
B = -0.0043 0.0018
0,14 Healthy (57)
R = -0.94 0.28
P = 0.0101
0,12
0,10H
Stomach (21)
j
0,08
O e s o p h a g e a l (8) j C o | o n
0,06
LiverÔsT
0,04
0,02
10 12 14 16 18 20 22
Fig. 17. C o r r e l a t i o n b e t w e e n t h e p r e v a l e n c e of L e i d e n m u t a t i o n a n d t h e s e r u m level of z e a x a n t h i n

w i t h different gastrointestinal t u m o r s (the n u m b e r of e x a m i n e d patients in parenthesis)
( M o z s i k , G y . , R u m i , G y . , D o m o t o r , A . , Figler, M . , G a s z t o n y i , B., P a p p , E., B e l a g y i , J . ,
M a t u s , Z . , M e l e g h , B., 2 0 0 5 b : I n v o l v e m e n t of s e r u m retinoids a n d L e i d e n m u t a t i o n
in patients w i t h e s o p h a g e a l , gastric, liver, p a n c r e t i c , a n d c o l o r e c t a l c a n c e r s in H u n g a r y .
99
8. Animal and human observations
with capsaicinoids
8 . 1 . Physiological and pharmacological research tool

by capsaicin (Short overview)
8.1.1. The chemistry of capsaicinoids
According to the USP30-NF25 (see Section 6.3.4.2.7) Capsaicin contains not less
than 90.0% and not more than 110.0% of the labeled percentage of total capsaici-
noids. The content of capsaicin (C H 7N0 ) is not less than 55%, and the sum of
18 2 3
the contents of capsaicin and dihydrocapsaicin ( C H N 0 ) is not less than 75%,

18 29 3
and the content of other capsaicinoids is not more than 15%, all calculated on the
dried basis.
Chemical composition of Natural Capsaicin

Natural Capsaicin contains a mixture of a series of structurally related compounds
called capsaicinoids (see Section 6.2.3.1). They all share the common vanillin struc-
tural moiety, which is considered to be associated with their effect on the TRPV]
receptors (Bevan, Szolcsanyi, 1990). The two main components of Natural Capsaicin
are capsaicin and dihydrocapsaicin. There are, however, some other minor ones as
well. The structures of the main components of Natural Capsaicin are shown in Fig. 7.
Stability of capsaicinoids
Information on the stability of isolated capsaicin, dihydrocapsaicin as well as on
other capsaicinoids is rather limited (see Section 6.3.4.4).
The recommended storage temperature of capsaicin and dihydrocapsaicin is
2-8C and -20C, respectively. According to the MDSD sheet, the Sigma-Aldrich
Capsaicin natural (product number 21750, CAS number: 404-86-4) (-65% cap-
saicin) should be stored at 2-8C.
Literature data
Kopec et al. (2002) tested the stability of (100%) ethanol solution of capsaicin of
different concentrations. The 4 mM or more concentrated solutions protected from
light and stored at 4C have been proved to be stable for a period of 12 months.
While investigating the stability of a capsaicin containing ointment Jaiarj et al.
(2000) found the preparation to show higher stability stored at 4C than at ambient
temperature.
Schweiggert et al. (2006) investigated the stability of chili powder. They found
that the capsaicin, dihydrocapsaicin and nordihydrocapsaicin content of the sam-
ples decreased by 7-12%. Based on their experience it was recommended that pap-
rika (pepper) samples should be heat-treated before processing, in order to reduce
100
the number of microorganisms producing enzymes with peroxidase activity of the
fruits.
The presence of phenolic hydroxyl group and that of the carbon-carbon double
bond make the capsaicinoids sensitive to oxidation. Accordingly, natural capsaicin
should be protected from exposure to light, heat, moisture, and oxidizing agents,
which initiate and/or catalyse the decomposition processes.
8.1.2. Source of capsaicin
The term Capsicum refers to the fruit of numerous species of the solanaceous genus
Capsicum. The genus name Capsicum is either derived from Greek "Kapso" mean-
ing to bite, referring to its pungency or from the Latin "Capso" or box referring to
the fruit pod. Members of the genus vary widely in size, shape, flavor, and much
more importantly in pungency. Red hot peppers, also called chilies, paprika, and
sweet non-pungent peppers are widely consumed by humans (Maga, 1975; Rozin,
1990). Capsaicin (8-methyl-N-vanillyl-6-nonenamide) is the major pungent ingredi-
ent of hot peppers.
8.1.3. Selective sensory effects of capsaicin
Apart from being used as a food additive, the compound has wide important phar-
macological actions. Capsaicin is uniquely selective for mammalian small afferent
neurons of dorsal root ganglia C and A8 fibers, a property which led to its use to
investigate the role of these afferent fibres in a number of physiological processes
(Jancso, Jancso-Gabor, 1959; Szolcsanyi, 1982; 1984a,b). In the skin the polymodal
sensory receptors, chemoreceptors and warmth receptors are affected by capsaicin
(Szolcsanyi, 1977, 1982, 1983a,b, 1984a,b, 1985, 1990a,b, 1993, 1996, 2004; Be-
van, Szolcsanyi, 1990; Szolcsanyi et al., 1994; Holzer, 1988, 1990, 1991a,b, 1992 a,b,
1998; Holzer, Sametz, 1986; Holzer, Lippe, 1988; Holzer et al., 1989). Most of these
afferents contain substance P (SP) and/or calcitonin gene-related peptide (CGRP)
(Holzer, 1991a,b; Maggi, 1995).
The action of capsaicin expresses itself as an initial short-lasting stimulation that
can be followed by desensibilization to capsaicin itself and to other stimuli of af-
ferent sensory neurons. Capsaicin in ng-pg/kg doses applied to the peripheral or
central endings or cell bodies of sensory neurons induces transient excitation of
these sensory neurons. In response to stimulation peptide mediators are released
from the central and peripheral nerve endings (Szolcsanyi, 1984a,b, 1996; Maggi, 1995;
Holzer, 1998, 1991a).
In the periphery neuropeptide release exerts local neuroregulatory tissue respons-
es (Szolcsanyi, 1984a,b, 1991, 1996). Neuropeptides are stored in sensory vehicles
(Gulbekian et al., 1986; Merighi et al., 1988) and are released on stimulation with
2+
capsaicin in a Ca -dependent manner (Maggi et al., 1989). In this way the peripher-
al terminals of capsaicin sensitive nerves are not only sensory receptors for convey-
ing impulses in the afferent direction but also effector sites from where mediators
101
are released for neurotransmission (Szolcsanyi, 1984, 1996). With large doses
(mg/kg) there is an initial stimulation, the duration of which is not yet defined, fol-
lowed by sensory desensitization which is restricted to those types of neurons but
are also activated by the drugs (Szolcsanyi, 1984a,b, 1993, 2004).
Four response stages of the capsaicin-sensitive primary afferents to capsaicin have
been introduced by Szolcsanyi (1984a,b, 1985) depending on the dose and duration
of exposure to the drug. These are excitation, sensory blocking effect, long-term
selective neurotoxic impairment and irreversible cell destruction.
8.1.4. Mechanism of action of capsaicin on sensory receptors
In explaining the mechanism of these sensory effects of capsaicin it has been hypoth-
esized that in the polymodal nociceptive primary afferent neurons there exists a cap-
saicin-gated cation channel which operates at the peripheral receptive terminals at
the level of the cell body. Capsaicin exerts its excitatory effect by activation of this
+ + 2+
cation channel which is permeable to a wide range of cations as Na , K and Ca ,
+
but not to anions as CI" (Winter, 1987; Wood et al., 1988). The influx of Na and
2+
C a induces a membrane depolarization which, at a certain level, opens
tetrotodotoxin-sensitive fast sodium channels at the regenerative region of the senso-
ry receptor and in this way triggers nerve terminal spike and direct stimulation of
2+
transmitter release by Ca .
Opening of the capsaicin-gated cation channel for shorter or longer periods of
time triggers a chain of intracellular events (Bevan, Szolcsanyi, 1990). Functional
blockage with reversible intracellular molecular changes (stage 2) or neurotoxic
degeneration (stages 3 and 4) develops depending on the concentration and the con-
tact time at the site of action on different parts (receptor, axon, cell body, central ter-
minal) of the capsaicin-sensitive primary afferent neurons. Prolonged stimulation
and consequently prolonged opening of this cation channel result in osmotic swelling
due to intracellular NaCl accumulation together with intracellular accumulation of
2+ 2+
C a at the exposed sites of the nerve terminal which activate Ca -dependent
enzymes and impair mitochondrial functions (Bevan, Szolcsanyi, 1990; Chard et al.,
2+
1995). Prolonged activation of C a is therefore the first step in sequence of events
ultimately leading to cell death.
In mammals, the long-lasting sensory blocking or neurotoxic effects of capsaicin
on primary afferent neurons have been described following topical, periaxonal, or
systemic pretreatment in adults or neonates. Autonomic efferent neural mechanisms
are not affected by drug (Szolcsanyi, 1982; Maggi, Meli, 1988; Holzer, 1991a). This
selective sensory blocking effect of capsaicin has been used as an experimental tool
for the elimination of the capsaicin-sensitive subset of primary afferent neurons and
consequently for identifying those tissue responses that are mediated by capsaicin-
sensitive afferent neurons (Szolcsanyi, 1996; Mozsik et al., 1997a). The later tissue
responses are absent in sensory desensitized animals.
102
8.1.5. Capsaicin actions in the gastrointestinal tract of animals
In their experiments, Makara et al. (1965) found that intragastric capsaicin (1 mg/0.5 ml
volume/rat) enhanced the development of Shay ulcer 12 hours later. After four con-
secutive daily doses of reserpine (1.0 mg/kg, sc.), the above dose of capsaicin given
simultaneously with reserpine enhanced gastric ulceration by the latter. On the other
hand, daily administration of paprika oil containing 1.0 mg/kg of capsaicin tended to
accelerate rather than retard the healing process of reserpine-induced gastric ulcer
in rats.
This was attributed by the authors to the capsaicin-induced local hyperemia or the
carotenoids and other pigments present in paprika oil. When Lee (1963) fed rabbits
for 12 months on various diets (high fat, high carbohydrate or high protein) supple-
mented with large doses of ground hot pepper, ulcers developed in the stomach of all
animals, and cirrhosis of the liver occurred in animals fed either high fat or high car-
bohydrate diets supplemented with capsaicin.
In the study of Nopanitaya (1974) the effect of capsaicin (1.0 mg/kg) and its com-
bination with various diets on the morphology of the duodenal mucosa of young rats
was investigated for periods of 28 and 56 days. The author reported ultrastructural
alterations in the mitochondria of the absorptive cells of rats fed a low protein diet
and also those supplemented with capsaicin. The changes, however, were less pro-
th th
nounced at the 56 day than at the 2 8 day, indicating some sort of adaptation to
capsaicin.
In 1981, Szolcsanyi and Bartho proved that capsaicin protects against experimen-
tal gastric ulcer. Introduction of capsaicin into the stomach of pylorus-ligated (Shay)-
rats in small doses (5 to 50 pg) and low concentration (10 pg/ml) markedly reduced
the ulcer formation in these 18 hours later. On the other hand, acute gastric ulcera-
tion induced by pylorus-ligation (Shay ulcer) or acid distension was aggravated in
rats desensitized 2 weeks earlier with systematic capsaicin in high doses which selec-
tively impairs capsaicin sensitive sensory nerves.
In capsaicin desensitized rats the aggressive side of the balance remained appar-
ently unchanged since hypersecretion of the pylorus-ligated rats did not differ from
+
that of the controls with respect to volume, H and pepsin concentration. This sug-
gested that it is the gastric defense mechanism which was impaired in capsaicin
desensitized rats. As a result of these data a role of capsaicin-sensitive afferents in
modulating gastric mucosal defenses was forwarded by the authors. In explaining
the nature of this novel gastroprotective action of low-dose capsaicin, it was postu-
lated that intragastric capsaicin exerts opposite effects on gastric ulcer formation
depending on the concentration in which it is introduced into the gastric lumen. Low
concentrations tend to inhibit the development of ulceration and high concentrations
promote ulcer formation. Release of vasodilatation mediators from capsaicin-sensi-
tive sensory nerve endings with the consequent enhancement of the microcirculation
was proposed as the mechanism responsible for the mucosal protective effects of
intragastrically administered capsaicin in low concentrations.
This mechanism seems to operate under physiological conditions and provides a
type of resistance or defense against ulcer formation. In capsaicin-desensitized rats,
mucosal sensory receptors will be unresponsive to stimuli and consequently no
103
release of vascular dilatator mediators will take place upon challenge with noxious
agents. The result is that the gastric ulcer will be aggravated (Szolcsanyi, Bartho,
1981).
These observations and the drawn conclusions were confirmed after a few years
by a number of investigators. Intragastric application of capsaicin in small doses has
been shown to protect the rat gastric mucosa against experimental gastric ulcerations
induced by ethanol (Holzer, Lippe, 1988; Esplugues, Whittle, 1989; Esplugues et
al., 1990, 1992), acidified aspirin (Holzer et al., 1989) and indomethacin (Gray et
al., 1994). Furthermore, after desensitization rats exhibited more severe gastric
mucosal damage than their sensory intact controls in response to chemical challenge
with ethanol (Holzer, Lippe, 1988; Lippe et al., 1989; Esplugues, Whittle, 1990;
Esplugues et al., 1992; Pabst et al., 1993), cysteamine (Holzer, Sametz, 1986; Gray
et al., 1994), platelet activating factor (Esplugues et al., 1989; Pique et al., 1990),
endothelin-1 (Whittle, Lopez-Belmonte, 1991).
Capsaicin desensitization 6 days prior to cold restraint stress, however, was report-
ed to have had a little effect on gastric ulcer. Only the number of lesions was higher
in capsaicin-treated rats restrained for 3 hours (Dugani, Glavin, 1986). Acute intra-
gastric capsaicin (40 mg/kg) followed immediately by stress resulted in significantly
more frequent and more severe ulceration relative to controls or rats given a 1, 2 and
3 hours delay between capsaicin administration and application of restraint stress
(Dugani, Glavin, 1985). Cysteamine-induced duodenal ulcers (Holzer, Sametz,
1986), and gastric mucosal damage evoked by 0.6 M HC1 (Takeuchi et al., 1994),
were not changed after capsaicin desensitization.
Studies have shown that gastric mucosal barrier disruption is accompanied by an
increase of gastric mucosal blood flow (GMBF), which appears to be triggered by
+
H rediffused through the breached mucosal defenses (Bruggeman et al., 1979;
Starlinger et al., 1981). Such an increase in GMBF is thought to be a defense mech-
anism, whereby the increased blood flow prevents the accumulation of injurious
+
concentration of H in the submucosa. Capsaicin-sensitive nerves which are unduly
+ +
sensitive to H (Bevan, Yeats, 1990) have been shown to respond to H back-dif-
fused through breached mucosal defenses and signal for an increase in gastric
mucosal blood flow (Holzer, 1991a,b). This further strengthened the role of these
nerves in maintaining mucosal integrity. Holzer (1991a,b) postulated that the acid-
induced mucosal hyperemia results from local axon reflex between collaterals of
afferent nerve fibres within the gastric wall. Li et al. (1992) found that gastric mucos-
al hyperemia in response to perfusion of the rat stomach with 0.15 M HC1 in 15 v/v
ethanol could to be completely blocked by close arterial infusion of a hCGRP recep-
tor antagonist.
Lippe and Holzer (1992) reported that in rats NG-nitro-L-arginine methyl ester
(L-NAME) (an inhibitor of endothelium derived nitric oxide formation) depressed
the increase in GMBF produced by gastric perfusion with dilute ethanol in 0.15 M
+
HC1. The loss of H from the lumen under these circumstances was also markedly
enhanced by L-NAME. Considerable controversy still exists in the literature, however,
as regards the mediators of the hyperemic response to back-diffusion of acid
(Whittle, 1977; Ritchie, 1991; Gislason etal., 1995).
104
Whether capsaicin-sensitive sensory nerves are involved in repair mechanisms of
the injured mucosa has been also investigated. In anaesthetized rats, 180 min after
exposure to 50 v/v ethanol, rapid repair of the injured mucosa (assessed by reduction
of deep mucosal damage and partial re-epithelialization of the denuded surface) was
reported to be similar in sensory denervated and sensory intact groups. This suggest-
ed that nociceptive neurons control mechanisms of defense against acute gastric
mucosal injury, but they are not required for the rapid repair of the injured mucosa
(Pabst et al., 1993). On the contrary, the different status of sensory neurons delayed
healing of gastric ulcers provoked in rats by 0.6 M HC1.
In addition, in stomachs damaged by 0.6 M HC1, capsaicin-desensitized not sen-
sory intact animals showed no hyperemic responses to intragastric instillation of 50
mM HC1. The conclusion was that capsaicin-sensitive sensory nerves contribute to
healing of gastric ulcer (lesions) by mediating the hyperemic responses associated
with acid back-diffusion following injury (Takeuchi et al., 1994). Similarly, sensory
denervated rats showed markedly increased area of acetic acid-induced ulceration at
1 and 2 weeks following the acetic acid injection indicating that the injury of senso-
ry function adversely affected the healing of gastric ulcer (Tramontana et al., 1994).
Capsaicin exerts protective effects on the chemical-induced mucosal injury not
only in the stomach, but also in the colon. Evangelista and Meli (1989) found that
systemic (one week) capsaicin neonatal pretreatment enhanced trinitrobenzene sul-
fonic acid-induced colic colitis in the rat. This pretreatment, however, did not affect
acute colitis (24 hours) caused by trinitrobenzene sulfonic acid, ethanol or acetic acid.
Reinshagen et al. (1994) employed systemic capsaicin pretreatment as a tool to
investigate the role of sensory nerves in an immune-complex model of colitis in rab-
bits. They found that capsaicin pretreatment per se caused no histological evidence
of inflammation. Meanwhile, colitis was more severe in sensory denervated than in
sensory intact rabbits. The increase in ulcer index and neutrophil infiltration was
more marked in the capsaicin pretreated control group at both 48 and 96 hours,
respectively. The difference in neutrophil infiltration between the two groups was,
however, more marked at 48 than 96 hours (Reinshagen et al., 1994).
Endoh and Leung (1990) reported that topical capsaicin application protected
against acetic acid-induced colitis. In the trinitrobenzene sulfonic acid-induced coli-
tis rat model, however, partial and transient protective effect was seen by Goso et al.
(1993) after topical capsaicin administration. Co-administration of 640 pM capsaicin
reduced the ulcerative area from 9 1 % to 64% only when the colon was examined
1 hour later. An approximately 8-fold higher dose of capsaicin (5000 pM) yielded
similar protection, while 100 pM had no protective effect. No protection by capsaicin
was seen, however, when the colon was examined 24 hours after noxious challenge.
8.1.6. Capsaicin-sensitive sensory nerves

and gastric acid secretion
Several studies in rats have indicated the involvement of capsaicin-sensitive sensory

nerves in the regulation of gastric acid secretion, however, contradictory data were
reported. In most studies, the indirect approach through functional ablation of cap-
105
saicin-sensitive afferent nerves with systemic neonatal (Evangelista et al., 1989;
Esplugues et al., 1990), adult (Alfoldi et al., 1986; 1987; Dugani, Glavin, 1986;
Robert et al., 1991) or peripheral capsaicin (Raybould, Tache, 1989) treatment was
used as a tool to investigate the role of capsaicin-sensitive afferent nerves in the re-
gulation of gastric acid secretion.
Adult rats treated with systemic capsaicin (60 mg/kg, sc.) showed depressed pen-
tagastrin-stimulated gastric acid secretion (Dugani, Glavin, 1986). On the contrary,
adult systemic capsaicin pretreatment with 300 mg/kg sc. did not modify gastric
acid secretion elicited by pentagastrin, carbachol or by a small dose of histamine
(0.1 mg/kg). However, the gastric acid secretory response to 0.5 and 5.0 mg/kg his-
tamine was greatly reduced in capsaicin-desensitized rats. It was suggested that the
histamine-induced increase in gastric acid secretion involves a capsaicin-sensitive
mechanism, while these mechanisms are not required for the effects of pentagastrin
and cholinergic stimulation of gastric acid secretion (Alfoldi et al., 1986, 1987).
Similar data were reported by Raybould and Tache (1989) using topical capsaicin
application into the cervical vagus. The gastric acid secretory response to distension
(5 ml for 6 min) was reduced in capsaicin-treated rats. This mechanism by which
capsaicin-sensitive vagal afferent fibres play a role in the secretory response to his-
tamine was explained by histamine acting in part by increasing vagal C-fibres dis-
charge resulting in a vago-vagal reflex increase in gastric acid secretion or by that
histamine stimulates vagal afferent C-fibres which results in the release of peptides
from sensory nerves terminals.
However, the authors admit that up to date a peptide that increases gastric acid
secretion and that is localized in vagal afferent nerves has not been identified. Adult
rats treated neonatally with systemic capsaicin (300 mg/kg, sc.) did not show any
reduction in their gastric acid secretory to histamine, pentagastrin or carbachol, while
acid secretion in response to distension was abolished. On the other hand, capsaicin
desensitization (neonatal treatment) substantially reduced the gastric acid secretion
to 2-deoxy-D-glucose (Evangelista et al., 1989), while it did not modify that stimu-
lated by insulin (Esplugues et al., 1990).
The conflicting observations regarding the effect of capsaicin continued to be seen
when it was given into the stomach. It was reported that intraduodenal (but not intra-
gastric) instillation of capsaicin (1.0 mg in 2 ml saline solution) in pylorus-ligated
rats induced a significant rise in total acidity 12 hours later (Makara et al., 1965).
The results with capsaicin in the gastrointestinal tract were contradictory, as men-
tioned above. Small attention was paid to the applied doses of capsaicin. Szolcsanyi
and Bartho (1981), however, emphasized that capsaicin protects against the chemi-
cal-induced gastric ulcer formation, when the capsaicin was given in 5 and 50 pg
doses (10 pg/ml concentration) intragastrically, meanwhile capsaicin in high dose
aggravated the ulcer formation by the production of desensitization.
We carried out systematic observations with capsaicin concerning its small and
high concentrations (or pretreatment produced desensitization of capsaicin sensitive
afferent nerves) on different experimental models (aspirin, HC1, indomethacin,
ethanol, cysteamine). The changes of gastric acid secretion, gastric mucosal dam-
+
age, gastric H back-diffusion, gastric mucosal blood flow were measured and cal-
culated, when the capsaicin was applied in small doses and in high doses producing
106
capsaicin desensitization (Mozsik, et al., 1997a,b). The results of these observations
clearly demonstrated that: 1. capsaicin, given in small doses, dose-dependently
inhibited all of the parameters in all experimental models; 2. the gastric mucosal
protective effects of capsaicin remained besides those of the other drugs (acting at
the level of efferent nerves, e.g. atropine, cimetidine or topically, such as sucralfate,
retinoids). Consequently, capsaicin enhanced the other drugs-induced gastric mucos-
al protective effects (Mozsik et al., 1997a,b); 3. after denervation of capsaicin-sensi-
tive afferent nerves (by pretreatment with high dose of capsaicin) the gastric muco-
sal ulcer formation was enhanced. These results offered to conclude that the gastric
mucosal protective effects can be obtained only with capsaicin, when it is given in
small doses, but not by its application in higher doses (Abdel-Salam et al., 1994,
1995a,b,c,d,e,f,g, 1996a,b, 1997a,b,c,d; Mozsik etal., 1993, 1996a,b, 1997a,b,c).
8.1.7. Molecular-pharmacological studies
Recently, the molecular-pharmacological observations were carried out (and calcu-

lated, based on the dose-response curves of drugs) with capsaicin, atropine, cimeti-
dine, omeprazole, PGI , vitamin A, P-carotene, studying their effects on the gastric
2
acid secretion in 2 and 4 hours pylorus-ligated rats alone, or in combination of

betanechol (7.6 and 15.4 nmol/kg), histamine (2.7 and 13.6 pmol/kg) and pentagas-
trin (65.1 and 325.6 nmol/kg) and on the gastric mucosal damage produced by intra-
gastrically applied ethanol, HC1, acidified aspirin and subcutaneously applied
indomethacin (alone and in combination with application of 7.6 and 15.2 pmol/kg
betanechol, 13.6 and 54.3 pmol/kg histamine and 6.51 and 325.6 nmol/kg pentagas-
trin) (calculating the number and severity of gastric mucosal damage) in rats (Figs
18-21, Tables 18-25).
The necessary doses for producing 50% inhibition on the gastric acid secretion
and gastric mucosal damage were calculated in molar values/kg body weight (ED ). 50
The values for affinity (pD) and intrinsic (a-values) were calculated according to
standard procedures employed in molecular pharmacology (Csaky, 1969). The val-
ues of the pD (necessary dose to inhibit the gastric acid secretion and gastric muco-
2
sal damage in 50%) and pA (necessary dose to produce 50% decrease in gastric
2
acid secretion and on gastric mucosal damage) were calculated from the affinity and
intrinsic activity curves.
The doses of drugs (compounds) were calculated as molar values for the determi-
nation of their biological effects. The affinity (pD values) and intrinsic activity (re-
2
values) are shown as molar values. The intrinsic activity of atropine (a) was taken as
1.00 for comparing the effects of agents on gastric acid secretion and gastric muco-
sal damage (Figs 18-21, Tables 18-25) (Mozsik et al., 2006).
The results obtained allowed the following conclusions:
1. Only the values of pD and pA (expressed in [-] molar doses) can be used for the
2 2
evaluation of physiological and pharmacological regulations of the target organ(s)

in animal experiment(s) (Mozsik et al., 2006);
2. The following pD (ED %) values were obtained for the different drugs (com-
2 50
pounds) for actions in inhibiting gastric acid secretion: atropine 5.75; omeprazole
107
meansSEM
"i 1 1 1 1 1
9 8 7 6 5 4 3 - g l l
|o M
Fig. 18. Affinity c u r v e s for different drugs a n d c o m p o u n d s i n h i b i t i n g t h e gastric a c i d s e c r e t i o n of

4 h pylorus-ligated rats ( M o z s i k , G y . , D o m o t o r , A . , A b d e l - S a l a m , O . M . E . , 2 0 0 6 : M o l e c u l a r
p h a r m a c o l o g i c a l a p p r o a c h to d r u g a c t i o n s o n t h e afferent a n d efferent fibres of t h e v a g a l
n e r v e in t h e gastric m u c o s a l p r o t e c t i o n in rats. I n f l a m m o p h a r m a c o l o g y 1 4 : 2 4 3 - 2 4 9 )
(with permission)
i.ooH
9 8 7 6 5 4 3
-loglM]
Fig. 19. Intrinsic affinity c u r v e s for different drugs a n d c o m p o u n d s i n h i b i t i n g t h e gastric a c i d

s e c r e t i o n of 4 h pylorus-ligated rats e x p r e s s e d in relation to that of a t r o p i n e ( 1 . 0 0 ) ( a a t r o p i n e )
(Mozsik, Gy., Domotor, A., Abdel-Salam, O . M . E . , 2 0 0 6 : M o l e c u l a r pharmacological
a p p r o a c h to d r u g a c t i o n s o n t h e afferent a n d efferent fibres of t h e v a g a l n e r v e in t h e
gastric m u c o s a l p r o t e c t i o n in rats. I n f l a m m o p h a r m a c o l o g y 1 4 : 2 4 3 - 2 4 9 ) ( w i t h permission)
108
Fig. 20. Affinity c u r v e s for different drugs a n d c o m p o u n d s i n h i b i t i n g t h e gastric m u c o s a l d a m a g e
p r o d u c e d b y v a r i o u s c h e m i c a l agents in rats ( M o z s i k , C y . , D o m o t o r , A . ,
A b d e l - S a l a m , O . M . E . , 2 0 0 6 : M o l e c u l a r p h a r m a c o l o g i c a l a p p r o a c h to d r u g a c t i o n s o n t h e
afferent a n d efferent fibres of t h e v a g a l n e r v e in t h e gastric m u c o s a l p r o t e c t i o n in rats.
I n f l a m m o p h a r m a c o l o g y 14: 2 4 3 - 2 4 9 ) (with permission)
Fig. 21. Intrinsic a c t i v i t y c u r v e s for different drugs a n d c o m p o u n d s i n h i b i t i n g t h e gastric m u c o s a l

d a m a g e p r o d u c e d by c h e m i c a l agents e x p r e s s e d relative to that of a t r o p i n e (oc tropine 1 -00)
a
=
in rats ( M o z s i k , G y . , D o m o t o r , A . , A b d e l - S a l a m , O . M . E . , 2 0 0 6 : M o l e c u l a r
p h a r m a c o l o g i c a l a p p r o a c h to d r u g a c t i o n s o n t h e afferent a n d efferent fibres of t h e v a g a l
n e r v e in t h e gastric m u c o s a l p r o t e c t i o n in rats. I n f l a m m o p h a r m a c o l o g y 1 4 : 2 4 3 - 2 4 9 )
(with permission)
109
T a b l e 1 8 . I n h i b i t o r y effects of c a p s a i c i n a n d resiniferatoxin ( R T X ) o n basal gastric
a c i d s e c r e t i o n in rats.*
Compounds Models ED 5 0 values
Capsaicin 2 h p y l o r u s ligated rats 3.27 nmol/kg
Capsaicin 4 h p y l o r u s ligated rats 3.27 nmol/kg
RTX 4 h p y l o r u s ligated rats 0 . 9 5 4 nmol/kg
* Mozsik, Cy., Domotor, A., Abdel-Salam, O.M.E. (2006): Molecular pharmacological approach to drug actions
on the afferent and efferent fibres of the vagal nerve in the gastric mucosal protection in rats.
Inflammopharmacology 14: 243-249 (with permission)
T a b l e 1 9 . I n h i b i t o r y effects of resiniferatoxin o n t h e s t i m u l a t e d gastric a c i d s e c r e t i o n in rats*
Capsaicin I h pylorus-l igated rats + b e t a n e c h o l ( 7 . 6 u.mol/kg) 0 9 5 4 nmol/kg
Capsaicin 1 h pylorus- i gated rats + b e t a n e c h o l (15.2 umol/kg) 0 9 5 4 nmol/kg
Capsaicin 1 h pylorus-l igated rats + h i s t a m i n e ( 1 3 . 6 nmol/kg) 0 9 5 4 nmol/kg
Capsaicin 1 h pylorus-l igated rats + h i s t a m i n e (54.3 pmol/kg) 0 9 5 4 nmol/kg
Capsaicin 1 h pylorus- igated rats + pentagastrin (65.1 nmol/kg) 0 9 5 4 nmol/kg

Capsaicin 1 h pylorus- igated rats pentagastrin ( 3 2 5 . 6 nmol/kg) 0 9 5 4 nmol/kg
RTX 4 h pylorus-l igated rats + b e t a n e c h o l ( 7 . 6 nmol/kg) 0 9 5 4 nmol/kg
RTX 4 h pylorus-l igated rats + b e t a n e c h o l (15.2 nmol/kg) 0 9 5 4 nmol/kg
RTX 4 h pylorus-l igated rats + h i s t a m i n e (2.7 pmol/kg) 0 9 5 4 nmol/kg
RTX 4 h pylorus-l igated rats + h i s t a m i n e ( 1 3 . 6 pmol/kg) 0 9 5 4 nmol/kg
RTX 4 h pylorus-l igated rats + pentagastrin (65.1 nmol/kg) 0 9 5 4 nmol/kg
RTX 4 h pylorus-l igated rats pentagastrin ( 3 2 5 . 6 nmol/kg) 0 9 5 4 nmol/kg
* Mozsik, Gy., Domotor, A., Abdel-Salam, O.M.E. (2006): Molecular pharmacological approach to drug actions
4.88; cimetidine 3.00; capsaicin 8.50, whereas no effects were observed with
PGI , vitamin A and P-carotene;
2
3. The intrinsic activity values obtained in relation to atropine 1.00 were cimetidine
0.64, capsaicin 0.75, omeprazole 1.00; no effects were observed for PGI , vitamin 2
A and P-carotene;
4. The following p D (ED ) values were obtained for the different drugs or com-
2 50
pounds inhibiting the gastric mucosal damage produced by chemicals: capsaicin

8.48, PGI 7.45, atropine 5.75, cimetidine 3.00, omeprazole 4.88, vitamin A 5.45
2
and P-carotene 5.73;

w e r e
5. The intrinsic activity values ( c % r o p i n e 1-00) obtained: capsaicin 0.76, cime-
at
=
tidine 0.64, those of other components were 1.00 on the gastric mucosal damage;
6. The values of pA were obtained as follows: capsaicin 8.50, PGI 7.44, atropine
2 2
5.80, cimetidine 3.00, omeprazole 4.90, vitamin A 5.44 and p-carotene 5.70.
110
T a b l e 2 0 . P r o t e c t i v e effects of c a p s a i c i n a n d resiniferatoxin o n t h e gastric m u c o s a l d a m a g e
c a u s e d b y e x o g e n o u s agents i n rats*
Capsaicin 0.6 M H C I (2 ml) (4 h) 0.135 pmol/kg

Capsaicin a s p i r i n ( 2 0 0 mg/kg) + 0 . 1 5 M H C I (4 h) 0.13 p m o l / k g
Capsaicin e t h a n o l ( 9 6 % in 1 m l ) (1 h) 0.13 p m o l / k g
Capsaicin 4 h pylorus-ligated rats + I N D ( 2 0 mg/kg) 1.98 n m o l / k g
RTX 4 h pylorus-ligated rats + I N D ( 2 0 mg/kg) 0.95 nmol/kg
Capsaicin 4 h pylorus-ligated rats + I N D ( 2 0 mg/kg) + 0 . 1 5 N H C I (2 m l ) 0.25 pmol/kg
RTX 4 h pylorus-ligated rats + I N D ( 2 0 mg/kg)+ 0 . 1 5 N H C I (2 m l ) 0.95 n m o l / k g
Capsaicin 4 h pylorus-ligated rats + I N D ( 2 0 mg/kg) 0.3 N H C I (2 m l ) 2.78 p m o l / k g
RTX 4 h pylorus-ligated rats + I N D ( 2 0 mg/kg) 0.3 N H C I (2 m l ) 0.15 p m o l / k g
T a b l e 2 1 . P r o t e c t i v e effects of resiniferatoxin o n gastric m u c o s a l d a m a g e c a u s e d b y e x o g e n a m

a n d e n d o g e n o u s agents in rats*
RTX 4 h pylorus-ligated rats +

b e t a n e c h o l (7.6 p m o l / k g ) + I N D ( 2 0 mg/kg) 0.20 p m o l / k g
b e t a n e c h o l (15.2 p m o l / k g ) + I N D ( 2 0 mg/kg) 0.20 p m o l / k g
h i s t a m i n e ( 1 3 . 6 p m o l / k g ) + I N D ( 2 0 mg/kg) 0.13 p m o l / k g
RTX 4 h pylorus-ligatedrats +
h i s t a m i n e (54.3 pmol/kg) + I N D ( 2 0 mg/kg) 0.13 p m o l / k g
pentagastrin (6.51 nmol/kg) + I N D ( 2 0 mg/kg) 0.13 p m o l / k g
RTX 4 h pylorus-ligated rats+
pentagastrin ( 3 2 5 . 6 nmol/kg) + I N D ( 2 0 mg/kg) 0.13 p m o l / k g
The results of these molecular pharmacological observations clearly proved that

the capsaicin-sensitive afferent nerves have an essential role both in the regulation of
gastric acid secretion and in the defense of the gastric mucosal damage produced by
the different chemical agents. It has been emphasized that capsaicin exerts its gastric
acid inhibitory and gastric mucosal protective effect in smaller molar concentrations
than other compounds (atropine, cimetidine, omeprazole and other compounds with-
111
T a b l e 2 2 . T h e p D , intrinsic a c t i v i t y ( a ,
2 a r o p i n e=1.00) and p A 2 v a l u e s for different drugs
( c o m p o u n d s ) i n h i b i t i n g t h e gastric a c i d o u t p u t s in 4 h pylorus-ligated rats*
Compounds pD 2 Intrinsic activity pA 2
Capsaicin 8.48 0.76 8.50

Atropine 5.75 1.00 5.80
Cimetidine 3.00 0.64 3.20
Omeprazole 4.88 1.00 4.90
* For further information, see Figs 18 and 19.

* Mozsik, Cy., Domotor, A., Abdel-Salam, O.M.E. (2006): Molecular pharmacological approach to drug actions
T a b l e 2 3 . D o s e r a n g e s of t h e tested n u t r i t i o n a l c o m p o u n d s ( v i t a m i n A a n d (3-Carotene), P G I 2 and

PGE 2 o n t h e gastric a c i d s e c r e t i o n of 4 h pylorus-ligated rats, w i t h o u t p r e s e n c e of a n y
gastric i n h i b i t o r y a c t i o n s *
V i t a m i n A : 3.49 x 10 8
to 3.49 x 10~ M ( 0 . 0 1 - 1 0 . 0 mg/kg): n o i n h i b i t o r y a c t i o n
5
o n t h e gastric a c i d s e c r e t i o n
P-Carotene: 1.86 x 1 0 8
to 1.86 x 1 0 " M ( 0 . 0 1 - 1 0 . 0 mg/kg): n o i n h i b i t o r y a c t i o n
5
o n t h e gastric a c i d s e c r e t i o n
P G I : 2.8 x 10-'' to 1.42 x 1 0
2
8
M ( 1 . 0 - 5 . 0 pg/kg): n o i n h i b i t o r y a c t i o n o n t h e gastric
a c i d secretion
P G E : 1.33 to 1.99 x 1 0 " M ( 5 0 . 0 - 1 5 0 . 0 pg/kg): n o i n h i b i t o r y a c t i o n o n t h e gastric
2
7
a c i d secretion
** Mozsik, Gy., Domotor, A., Abdel-Salam, O.M.E. (2006): Molecular pharmacological approach to drug
actions on the afferent and efferent fibres of the vagal nerve in the gastric mucosal protection in rats.
T a b l e 2 4 . T h e p D , intrinsic a c t i v i t y ( a
2 a t r o p i n e =1.00) and p A 2 v a l u e s for different drugs,
c o m p o u n d s i n h i b i t i n g t h e gastric m u c o s a l d a m a g e p r o d u c e d by c h e m i c a l agents in rats*
Compounds pD 2 Intrinsic activity pA 2
Capsaicin 8.48 0.76 8.50

PGI2 7.45 1.00 7.44
Atropine 5.75 1.00 5.80
Cimetidine 3.00 0.64 3.20
Omeprazole 4.88 1.00 4.90
Vitamin A 5.45 1.00 5.44
P-Carotene 5.73 1.00 5.73
* For further information, see Figs 18-21. (*M6zsik, Gy, Domotor, A., Abdel-Salam, O.M.E. (2006): Molecular
pharmacological approach to drug actions on the afferent and efferent fibres of the vagal nerve in the gastric
mucosal protection in rats. Inflammopharmacology 14: 243-249 (with permission)
112
T a b l e 2 5 . S u m m a r y of t h e affinity ( p D ) a n d intrinsic a c t i v i t y ( e x p r e s s e d in v a l u e of
2
âtropine
1 . 0 0 ) ( p A ) v a l u e s of c a p s a i c i n , a t r o p i n e , p i r e n z e p i n e , c i m e t i d i n e , r a n i t i d i n e ,
2
f a m o t i d i n e , o m e p r a z o l e a n d e s o m e p r a z o l e o n t h e gastric basal a c i d o u t p u t ( B A O ) in
healthy h u m a n subjects*
Compounds M.W. pD 2 Intrinsic activity pA 2
Capsaicin 305.4 5.88 0.76 5.87
Atropine 289.38 5.40 1.00 5.40
Pirenzepine 424.34 3.93 0.89 3.93
Cimetidine 252.34 2.23 1.00 2.23
Ranitidine 314.41 3.33 1.00 3.33
Famotidine 337.43 3.77 1.00 3.77
Nizatidine 331.47 3.34 1.00 3.34
Omeprazole 345.42 3.97 1.00 3.97
Esomeprazole 345.42 3.97 1.00 3.97
* Mozsik Cy., Szolcsanyi, J . , Domotor, A. (2007b): Capsaicin research as a new tool to approach of the human
gastrointestinal physiology, pathology and pharmacology (review). Inflammopharmacology 15: 232-245 (with
permission) (For further information, see Figs 26-27)
out any gastric acid inhibitory effects). That is a clear explanation for why the essen-
tial role of capsaicin-sensitive afferent nerves in the physiological and pharmacolo-
gical regulation of gastrointestinal tract is emphasized.
8.1.8. Capsaicin actions in healthy human subjects

and in patients with different gastrointestinal disorders
Early works regarding the effect of peppers on the human stomach have yielded con-
flicting results. In peptic ulcer patients, Schneider et al. (1956) studied the influence
of a variety of spices on sensation of pain and healing peptic ulcer. Ulcer patients
placed on established treatment with anticholinergics and diet were given different
pungent species in capsules that contained the average quantity habitually consumed
by Americans with three of their daily meals for six weeks. With the exception of
black pepper, which resulted in distressing pain after one or two days, none of the
tested spices have modified pain sensation or delayed healing of ulcers.
In another study, instillation of red chilli powder was reported to be associated
with a significant increase of DNA from gastric aspirates (Desai et al., 1973).
Viranuvatti et al. (1972) studied the local effect of capsicum in twenty human sub-
jects by instillation of a 3% capsicum solution through an intragastric tube or via the
lumen of gastrofiberscope. There was no change in 13 cases, edema and/or hyper-
emia developed in three cases, hemorrhagic spots occurred in another three cases,
and bleeding occurred in one case.
Capsaicin was reported to increase the gastric acidity in human subjects receiving
intragastrically aqueous suspensions of hot peppers (Berkesy, 1934; Varga, 1936;
113
Ketusinh et al., 1966; Solanke, 1973). Solanke (1973) studied the effect of red pep-
per suspension (200 ml of 4% solution) instilled through a nasogastric tube on gas-
tric acid secretion in patients with proven duodenal ulcer and non-duodenal ulcer
subjects. Patients were allocated into two treatments: fresh red pepper suspension
and a red pepper suspension with pH adjusted to 7.4 with 0.01 N sodium hydroxide.
They found a significant increase in gastric acid secretion after treatment with either
form of the red pepper suspension. Many other observations were carried out in
human beings with different extractions of chilli, paprika.
The Good Clinical Practice (GCP) was introduced in the clinical research of
human beings and patients.
From 1997, our clinical studies with capsaicin were carried out in prospective and
randomized, multiclinical conditions as officially accepted in the multiclinical phar-
macological studies over the World.
The following principles were accepted in these human observations:
1. Pure capsaicin (Sigma, U.S.A., later on Sigma-Aldrich, U.S.A.) was used in the
studies with healthy human subjects and in patients with different diseases
(instead of different extractions of different capsaicin containing plants);
2. The clinical observations were carried out according to the medical rules of clin-
ical pharmacology (in random allocation and in prospective and randomized
studies);
3. The clinical observations were carried out according to the criteria of GCP;
4. All of the persons participating received all the different doses of capsaicin, how-
ever, in a random allocation.
We have to emphasize that the classical pharmacological studies were carried out
with capsaicin obtained from Sigma (U.S.A.), or later on from Sigma-Aldrich
(U.S.A.).
The main results of the human multiclinical pharmacological studies with cap-
saicin:
1. Capsaicin (in the dose range of 100 to 800 pg given orally to one person) dose-
dependently inhibited the gastric basal acid output (BAO) in healthy human sub-
jects (Fig. 22) (Mozsik et a l , 1999, 2005a);
2. Capsaicin dose-dependently enhanced the gastric transmucosal potential differ-
ence (GTPD) in healthy human subjects (Fig. 23) (Mozsik et al., 2005a);
3. The ethanol-induced decrease of GTPD can be dose-dependently reversed by to-
pical application of capsaicin (given in doses of 100, 200, 400 and 800 pg orally)
(Fig. 24) (Mozsik et al., 2005a);
4. Indomethacin (3x25 mg/day given orally, plus 25 mg given immediately before
measuring of gastric blood loss) produced a significant increase of gastric micro-
bleeding in comparison to control (untreated) conditions (Fig. 25) (Mozsik et al.,
2005a, 2007b);
5. The extent of basal and indomethacin-induced gastric microbleeding remained
unchanged before and after 2-weeks treatment with capsaicin (3 x 400 pg/ per-
son/day) (Mozsik, et al., 2005a, 2007b);
6. The dose-dependent gastroprotective effect of capsaicin on the indomethacin-
induced gastric microbleeding remained the same after two-weeks capsaicin treat-
ment (3 x 400 pg given orally /day/person) (Mozsik et al., 2005a, 2007b).
114
These observations proved that capsaicin (dose-dependently) prevents the ethanol-
and indomethacin-induced gastric mucosal damage in healthy human subjects
(Mozsik et al., 2005a, 2007b) before and after two-weeks treatment with capsaicin
(3x400 pg orally /day/ person) (Mozsik et al., 2005a, 2007b), however, we have to
emphasize that indomethacin (3x25 mg given orally, plus 25 mg given immediately
before the measuring of gastric microbleeding) produced the same extent of gastric
microbleeding. We have also to emphasize that the extent of baseline of gastric
microbleeding remained the same before and after two-weeks treatment with cap-
saicin (Mozsik et al., 2004, 2005a, 2007b).
Effect of capsaicin on gastric basal acid output ( B A O ) in healthy h u m a n subjects
meanSEM
7 6 5
Doses of capsaicin (-iog[M])
Fig. 22. D o s e - r e s p o n s e c u r v e of t h e effect of c a p s a i c i n o n t h e gastric basal a c i d o u t p u t ( B A O ) in

healthy h u m a n subjects ( M o z s i k , C y . , R a c z , I., S z o l c s a n y i , J . , 2 0 0 5 a : G a s t r o p r o t e c t i o n
i n d u c e d by c a p s a i c i n in h e a l t h y h u m a n s u b j e c t s . W o r l d J . G a s t r o e n t e r o l . 1 1 : 5 1 8 0 - 5 1 8 4 )
(with permission)
8.1.8.1. Results of the comparative molecular-pharmacological studies

of capsaicin, atropine, omeprazole, famotidine, ranitidine
and cimetidine on the gastric basal acid output (BAO) in
human subjects
The affinity (pD values) and intrinsic activity (pA values) curves of capsaicin,
atropine, omeprazole, famitidine, ranitidine and cimetidine (applied in their physio-
logical and human therapeutic doses) were determined in patients with gastrointesti-
nal disorders, according to the method of Csaky (1969) (Figs 26-27, Table 25).
115
Effect of capsaicin on G T P D (-AmV) in healthy human subjects
Doses of capsaicin (-log[M|)
Fig. 23. D o s e - r e s p o n s e c u r v e of t h e effect of c a p s a i c i n o n t h e gastric t r a n s m u c o s a l potential

d i f f e r e n c e ( G T P D ) in h e a l t h y h u m a n s u b j e c t s . M o z s i k , G y . , R a c z , I., S z o l c s a n y i , J . ,
2 0 0 5 a : G a s t r o p r o t e c t i o n i n d u c e d by c a p s a i c i n in h e a l t h y h u m a n s u b j e c t s . W o r l d J .
Gastroenterol. 1 1 : 5180-5184) (with permission)
meanSEM
6 5
Doses of capsaicin (-log[M])
Fig. 24. D o s e - r e s p o n s e c u r v e of t h e effect of c a p s a i c i n o n t h e gastric t r a n s m u c o s a l potential

d i f f e r e n c e after intragastric a d m i n i s t r a t i o n of e t h a n o l in h e a l t h y h u m a n subjects.
M o z s i k , G y . , R a c z , I., S z o l c s a n y i , J . , 2 0 0 5 a : G a s t r o p r o t e c t i o n i n d u c e d by c a p s a i c i n in
healthy h u m a n subjects. W o r l d J . Gastroenterol. 1 1 : 5180-5184) (with permission)
116
meanSEM
= P<0.01
= P<0.001
6 5
Doses of capsaicin (-Iog[M])
Fig. 25. D o s e - r e s p o n s e c u r v e of t h e effect of c a p s a i c i n o n t h e i n d o m e t h a c i n ( 3 x 2 5 + 2 5 m g g i v e n

orally) i n d u c e d gastric m i c r o b l e e d i n g in h e a l t h y h u m a n s u b j e c t s . M o z s i k , G y . , R a c z , I.,
S z o l c s a n y i , J . , 2 0 0 5 a : G a s t r o p r o t e c t i o n i n d u c e d by c a p s a i c i n in h e a l t h y h u m a n subjects
Fig. 26. Affinity c u r v e s for t h e i n h i b i t o r y a c t i o n s of different drugs o n t h e gastric basal a c i d o u t p u t

( H + output/h) in h e a l t h y h u m a n subjects [ M o z s i k G y . , S z o l c s a n y i , J . , D o m o t o r , A . ,
2 0 0 7 b : C a p s a i c i n research as a n e w t o o l to a p p r o a c h of t h e h u m a n gastrointestinal
physiology, p a t h o l o g y a n d p h a r m a c o l o g y ( r e v i e w ) . I n f l a m m o p h a r m a c o l o g y 1 5 : 2 3 2 - 2 4 5 ]
(with permission)
117
Fig. 27. Intrinsic a c t i v i t y c u r v e s for t h e i n h i b i t o r y effects of different drugs o n t h e gastric basal
a c i d o u t p u t ( H + output/h) in h e a l t h y h u m a n subjects, c o m p a r e d to t h e a c t i o n of a t r o p i n e
(1.00)(a a t r o p i n e ) [ M o z s i k G y . , S z o l c s a n y i , J . , D o m o t o r , A . , 2 0 0 7 b : C a p s a i c i n research
as a n e w t o o l to a p p r o a c h of t h e h u m a n gastrointestinal p h y s i o l o g y , p a t h o l o g y
a n d p h a r m a c o l o g y (review). I n f l a m m o p h a r m a c o l o g y 1 5 : 2 3 2 - 2 4 5 ] (with permission)
8.1.9. Side effects of aspirin and other nonsteroidal

anti-inflammatory drugs (NSAIDs) in the gastrointestinal
tract of patients
NSAIDs treatment is necessary for the patients with myocardial infarction, throm-
boembolic episodes, stroke, cancers, for persons in whom the development of dif-
ferent diseases should be prevented (reinfarction after myocardial infarction, throm-
boembolic episodes produced by atrial fibrillation, cancers, after different surgical
interventions and immobilization), and for healthy subjects in order to prevent the
development of colorectal cancers.
The number of these groups of patients reaches up to 50-60% of the total popula-
tion in Hungary, an extremely large number of patients with these diseases is
involved in the different countries of the World.
The patients who underwent cardiac surgeries have to treated permanently with
aspirin (in doses of 100 mg/day/person). This is the basic stand-point of the different
consensus meetings of Europe and the World (Todd, Clissold, 1990; Expert
Consensus Document on the Use of Antiplatelet Agents 2004; Mcgettigan, Henry
2006).
The administration of aspirin is absolutely indicated from medical points of view
in patients mentioned above, accepting the opinion of cardiologists; however, we
have to emphasize that aspirin very frequently produces gastrointestinal bleeding.
Consequently there is a contradictory medical (and evidence-based proved) stand-
point between the cardiologists and gastroenterologists (during the treatment of only
one patient, as well as in the treatment of populations of patients mentioned above).
Another large population of patients suffers from different degenerative joint dis-
eases, trauma, acute and chronic pain producing states. These patients have to receive
118
permanently treatment with NSAIDs. The NSAIDs are not gastrointestinal protec-
tive agents either in healthy person or in patients with these diseases. The patients
appearing at the ambulance of Gastrointestinal Units are suffering from the drug-
induced side effects in the gastrointestinal tract.
The number of patients with NSAIDs-induced gastrointestinal disorders (blood
loss, bleeding, peptic ulcer) also represents a significant fraction of the population.
Furthermore, these patients have to be treated permanently by different NSAIDs.
The actions of NSAIDs are associated with the selective and non-selective inhi-
bitory properties of the cyclooxygenase system (emphasizing the key role of COX-1
and COX-2).
Aspirin is a specific COX-1 inhibitor, meanwhile the other NSAIDs applied in the
clinical practice represent the compounds acting as non-selective COX-1 and COX-
2 inhibitors. Recently the specifically acting compounds, inhibiting the COX-2
enzyme, have been produced, however, it was also observed that during the treat-
ment an extremely large number of patients suffered myocardial infarction (Couzin,
2004a,b; Lenzer, 2004; McGettigan, Henry, 2006).
We have to emphasize that the small doses of capsaicin are able to prevent gastric
mucosal bleeding in humans, produced by the inhibition the COX-1 and COX-2
enzymes. This discovery opened a new pathway in the physiological and pharmaco-
logical regulation of different tissues (see later).
119
9. Toxicological studies
with capsaicin
9 . 1 . Animal observations
9.1.1. Acute toxicology studies of capsaicin

in animal experiments
The only reported acute toxicity study with capsaicin was in ears which were admi-
nistered four increasing s.c. doses of capsaicin to a cumulative amount of 21.0-66.0
mg/rat, eight of 17 rats died (Cabanac et al., 1976). Additionally, Molnar (1965) and
Molnar and Gyorgy (1967) reported that capsaicin aministered i.v. at a dose higher
than 10 pg/kg to cats caused a rapid fall in the mean arterial pressure which was fol-
lowed either by a pressor phase or by death. These results called our attention to
methods of administration of capsaicin (intravenously or orally in animal experi-
ments9.
Capsaicin had an i.p. L D (7.65 mg/kg) in adult female and male mice (please
50
note that capsaicin was not given as pure agent, but as an extract) (Glinsukon et al.,
1980; Ato, Yamamoto, 1996). The toxicity of capsaicin present in the capsicum
extract was approximately fourfold higher than that of pure capsaicin given intraperi-
toneally to mice. Capsaicin had a slightly elevated L D in pregnant female rats
50
when administered in propylene glycol as compared to that administered intraperi-

toneally in dimethylsulfoxyde (DMSO) (P< 0.05) (Glinsukon et al., 1980). Guinea
pigs are the most susceptible species to capsaicin toxicity with L D of 1.10 mg/kg,
50
whereas hamsters and rabbits are less susceptible (Glinsukon et al., 1980).
The relative lethality caused by capsaicin administered by various routes in the
mouse (Glinsukon et al., 1980):
0.56 (0.36-0.87) mg/kg intravenously,
1.60 (1.03-2.48) mg/kg intratracheally,
7.65 (5.28-11.09) mg/ kg intraperitoneally,
7.80 (5.53-10.99) mg/kg subcutaneously,
60 to 190 mg/kg intragastrically,
> 218 mg/kg intrarectally and
> 512 mg/kg dermally.
At the autopsy, only hyperemia without hemorrhage was observed in the visceral
organs and the muscular wall of the peritoneal cavity with a slight increase in the
amount of peritoneal fluid in the rats treated intraperitonally with capsaicin. A similar
observation was also found in mice treated intraperitoneally with capsaicin. His-
topathological changes seen in the gastric mucosa of mice treated intragastrically with
capsaicin were desquamatic necrosis with increased mucous material (PAS stain).
Some of the chief and parietal cells showed pale basophilic cytoplasm and vacuoliza-
tion. No significant histopathological changes were observed in the other organs.
120
The pattern of the electrocardiogram and heart rate did not change for 5 min after
capsaicin administration. Respiratory rates were slightly increased during the first
min, whereas a small increase of the tidal volume was also observed. The tidal vol-
ume then decreased to 10 to 20% of the control within 3-4 min, and the respiration
stopped. During this time, the heart rate gradually decreased, the electrocardiograph-
ic signals disappeared much later (in about 6-14 min). Mean arterial pressure was
somewhat variable in the rats treated with capsaicin. At the beginning, capsaicin
caused a transient hypotension and then hypertension. Mean arterial pressure gradu-
ally decreased along with the decrease in the tidal volume. Convulsions were not
observed in these rats anesthetized with a lethal dose of capsaicin. This finding was
confirmed in mice anesthetized with sodium barbital and subsequently given a sin-
gle lethal dose of capsaicin.
The L D values indicate a high susceptibility in guinea pigs, rats and mice,
50
whereas hamsters and rabbits are less susceptible to capsaicin. Capsaicin is a highly
toxic compound when administered by all routes except gastric, rectal and dermal.
Cabanac et al. (1976) published a report on the acute toxicity of capsaicin in which
adult male rats were given four increasing subcutaneuos doses of capsaicin (cumula-
tive amount of 21.0 to 66.0 mg/kg).
The lethality caused by capsaicin administered gastrically to the mouse is much
less than that caused by the intraperitoneal administration route. The minimum lethal
dose of capsaicin per kg was 100 mg, which would be contained in 32.4 g dry weight
of fruits. For a 60 kg person, this toxic level would be comparable to the consump-
tion of about 1.94 kg of dry weight of capsicum fruits (Molnar, 1965).
9.1.2. Acute toxicity studies with pure trans-capsaicin derivates

in dogs after intravenous administration
The trans-geometric isomer of capsaicin, or trans-8-methyl-N-vanillyl-6-nonen-

amide, is the most abundant pungent molecule in chilli peppers and thus represents
the most important ingredient in spicy foods. Although there are two geometric iso-
mers of capsaicin, only trans-capsaicin occurs naturally (Cordell, Araujo, 1993).
The capsaicin content of chilli peppers ranges from 0.1 to 1.0% w/w (Govin-
dajarajan, Sathyanarayna, 1991). Furthermore, this food additive has been widely
used to evaluate the different physiological or pathological regulatory mechanisms
in the human observation in the form of non-prescription (in U.S.A.) or prescription
(in Europe) topical analgetics, and self-defense products (e.g. pepper spray).
The trans-geometric isomer of capsaicin is a highly selective agonist for the tran-
sient receptor potential vanilloid receptor 1 (TRVP1 or VR1, according to older
nomenclature) (Caterina et al., 1997).
TRVP1 is a ligand-gated, non-selective, cation channel preferentially expressed in
small-diameter, primary afferent neurons (C-fibres and A8-fibres), especially noci-
ceptive sensory nerves. TRPV1 responds to noxious stimuli including capsaicin,
heat and extracellular acidification, and integrates simultaneous exposures to these
stimuli (Tominata et al., 1998). Based on the highly selective agonistic property of
121
capsaicin toward TRVP1 receptors, drug products containing pure synthetic trans-
capsaicin are under evaluation as topical and injectable therapies (Bley, 2004).
Former studies of the toxicological potential of capsaicin in vivo began in 1935,
when De Lille and Ramirez (1935) reported that administration of a capsaicin extract
into dogs produced a fall in blood pressure accompanied by variable effects on the
respiration, an increase in salivary secretion, and a relatively small increase in gas-
tric secretion. Capsaicin really can increase the buffering ("non-parietal compo-
nent") of the gastric secretion, in association with the decrease of "parietal compo-
nent" of gastric secretion by the application of pure capsaicin (Sigma, U.S.A.) in
healthy human subjects (Mozsik et al., 2004, 2007b).
The capsaicin materials tested in the studies cited above were either natural
extracts or racemic mixtures, and may not exhibit the same toxicological profile as
pure trans-capsaicin. Although the exact content and nature of impurities in the test
articles used in these studies are often not explicitly stated, a typical capsaicin extract
is a mixture of trans-capsaicin (cis-capsaicin does not occur naturally) and other
capsaicinoids (including capsaicin, nordihydrocapsaicin, dihydrocapsaicin, homo-
capsaicin and homodihydrocapsaicin). Earlier Buck and Burks (1986) clearly proved
that there is no physiological difference between the application of capsaicin or dihy-
drocapsaicin in animal experiments.
The actual percentage of capsaicin and other capsaicinoids will vary depending
on the peppers used and method of extraction: In fact, the United States Pharma-
copeia defines capsaicin as a product which contains > 55% capsaicin and in
combination of capsaicin and dihydrocapsaicin > 75%; total capsaicinoid con-
tent may be as little as 90% (United States Pharmacopoeia, 2005). Additionally,
extracts are expected to contain chemical entities other than vanilloid compounds.
Chanda et al. (2004, 2005) made observations with pure trans-capsaicin in dogs.
The objectives of this study were to evaluate the possible cardiovascular and respira-
tory effects of pure capsaicin, and to evaluate the potential of any target organ toxic-
ity that might occur as a result of introduction of pure trans-capsaicin given into the
systemic circulation in dogs.
These studies were carried out in approximatively 10-17 months old dogs weigh-
ing between 19-21.8 kg at the time of the observations. Capsaicin for this study was
dissolved in 10% v/v hydroxypropyl B-cyclodextrin (Aldrich Chemical Gillingham,
U.K.). The studies were carried out in acute observations, and after two weeks of
capsaicin treatment (in doses of 0.03, 0.1 and 0.3 mg/ kg given intravenously).
Different biochemical (glucose, urea nitrogen, creatinine, total protein, albumin, al-
bumin/globulin ratio, cholesterol, alanine aminotransferase, alkaline phosphatase,
calcium, gamma glutamyltransferase, inorganic phosphorus, sodium, potassium,
chloride, total bilirubin) and hematological parameters (red blood count, hemoglo-
bin, hematrocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean
corpuscular hemoglobin concentration, platelet count, leukocyte count, differential
blood cell count, blood smear, prothombin time, activated partial thromboplastin
time) were determined. Urine samples were tested for appearance/color parameters,
volume, specific gravity, pH, protein, glucose, ketones, bilirubin, blood, microscop-
ic examination of urine sediments, and urobilinogen. At the necropsy, the macro-
scopic observations were recorded, the organs were weighed, and selected tissues
122
were collected and preserved. Microscopic examinations were carried out from all
the tissue samples.
The capsaicin concentration of the plasma samples was determined by high per-
formance liquid chromatography (HPLC).
All of these observations were carried out in acute experiments and after two
weeks of capsaicin treatment.
The studies reported were conducted according to the principles of Good
Laboratory Practice (GLP). The trans-capsaicin (CAS 404-86-4) used in both stud-
ies decribed was manufactured under the Current Good Manufacturing Practice
(cGMP) conditions. The two batches of trans-capsaicin used for the studies had >
99% agreement.
The main results of these observations with trans-capsaicin in acute administra-
tion (before a chronic capsaicin treatment) are as follows.
9.1.2.1. Acute effects on cardiovascular and respiratory parameters
Administration of capsaicin (given in vehicle, 0.03 and 0.1 mg/kg) had no detectable
effect on the cardiac and respiratory systems. No effect was observed on arterial
blood pressure, rate change of the force of ventricular contraction (dP/dt ), heart
max
rate ECG waveform, and femoral blood flow. However, administration of 0.3 mg/kg
capsaicin elicited a rise in mean arterial blood pressure from a base line of 967 to
13821 mmHg within 2 min of starting the infusion. This effect peaked at 14617
mmHg at the end of infusion (15 min). The hypertensive effect was accompanied by
increases in heart rate (from 71 3 at baseline to 11925 bpm), d P / d t max (from
405091 at baseline to 66791027 mmHg/s) and femoral blood flow (from 117+27
at baseline to 17434 ml/min). These changes were statistically significant {P<0.05)
in comparison with the results obtained after giving vehicle, 0.03 or 0.1 mg/kg cap-
saicin injection (Chanda et al., 2005).
The change in heart rate was also associated with decrases in the RR and QT inter-
vals of the ECG. However, the corrected QT intervals (both Q T and Q T ) were
C F CB
unchanged. After administration the vehicle elicited a decrease in the depth of respi-
ration. This was reflected as decreases at 30 min post-infusion in tidal volume (TV)
(from 14319 to 118+20 ml), peak inspiratory flow (PIF) (from 253 12 to 198+10
ml/s) and peak expiratory flow (PEF) (from 29747 to 24624 ml/s). The rate of
respiration was unaffected. The administration of 0.03 and 0.1 mg/kg trans-capsaicin
did not elicit any further changes in respiration. The administration of high (0.3
mg/kg) dose of trans-capsaicin (given intravenously) elicited increases in PIF, PEF
and TV. The increase of in PIF and PEF following 0.3 mg/kg trans-capsaicin is sig-
nificantly different from the decrease in these parameters following the vehicle treat-
ment (P<0.05). However, these increases were transient, lasting only 5 to 10 min
after the end of infusion.
123
9.1.2.2. Plasma levels of capsaicin
No detectable levels of capsaicin were found 5 min after administration of 0.05
mg/kg capsaicin. Following the administration of the intermediate dose (0.1 mg/kg
intravenously), 2-A dogs showed detectable levels of capsaicin (in ranges of approxi-
matively 17 and 11 ng/ml). The high dose (0.3 mg/kg) produced an increase in the
plasma levels in all dogs (ranging from 32.2 to 65.6 ng/ml, mean 47.96.4 ng /ml).
9.1.3. Results of subacute toxicology of capsaicin in dogs
9.1.3.1. Two weeks treatment with trans-capsaicin
All dogs survived to the scheduled termination on Day 15. The only capsaicin solu-
tion-related sign observed during the study was vacuolization during dosing, which
was noted in all dogs.
In general, the observation was made more frequently in male than in female dogs.
Clear nasal discharge was seen in all groups, and the daily incidence was slightly
higher in males than in females given 0.3 mg/kg trans-capsaicin (intravenously),
although the daily incidence in males given 0.1 or 0.3 mg/kg/day of trans-capsaicin
in females 0.3 mg/kg/day was slightly higher than in controls. Slight tremors (head,
limbs, and/or body) were seen in all groups during this study. The majority of these
observations were noted during the study.
As the study progressed, dogs given 0.3 mg/kg trans-capsaicin demonstrated an
apparent tolerance to the general anesthetic and analgesic, as indicated by a general
vacuolization during the dosing period.
There were no statistically significant differences in the body weights and food
consumption values between the groups of dogs treated with capsaicin chronically.
The body weights, however, were slightly lower (in about 7%) in males treated with
0.3 mg/kg trans-capsaicin for 14 days. Over the duration of the study, males in this
group lost approximately 0.4 kg, whereas the control gained 0.1 kg. Though this
value was statistically not significant, the food consumption of males and females
given 0.3 mg/kg/ day was slightly lower than that of controls (approximately 11 and
12%, respectively).
9.1.3.2. Clinical chemistry and hematology
The only difference considered related to test article was the minimally higher ALT
for males and females given 0.3 mg/kg/day trans-capsaicin intravenously after 14
days' treatment. Other statistically significant differences for clinical chemistry test
results were considered incidental because they exhibited no dose relationship or
were present before initiation of the treatment. In hematology, in female dogs in the
0.3 mg/kg/day treated group WBC was statistically significantly (P<0.05) lower. A
few animals, including controls, had notably high neutrophil counts, which were
likely secondary to inflammated lesions at injection sites.
124
9.1.3.3. Organ weights, macroscopic Snd microscopic observations
There were no capsaicin solution-related organ weight changes or macroscopic or
microscopic observations.
The statistically significant differences of prostate, brain and adrenal weight val-
ues with respect to controls were considered incidental because there were no corre-
lating macroscopic and microscopic findings. Thrombosis, due to administration of
vehicle, was noted at the intravenous injection sites in all groups. Other lesions
observed were thrombosis-induced inflammation, fibrosis, edema and hemorrhage.
9.1.3.4. Pharmacokinetic data after 14-days treatment

with trans-capsaicin in dogs
After intravenous administration, the peak of the plasma concentration ( C ) was

max
reached in all cases immediately at the end of infusion. Capsaicin was rapidly elimi-
nated and measurable values were only obtained immediately after the end of infu-
sion (0.25 h) in 0.03, 0.1 and 0.3 mg/kg/day. In the 0.3 mg/kg/day group, measur-
able values were obtained up to 0.5 h in all dogs on Day 1, but they were very close
to the limit of quantitation (10 ng/ml). On Day 15, only one dog had still a measur-
able value.
Females generally had higher than, or similar, mean C values compared to
m a x
males, but the largest difference did not exceed 44%. The increases in mean C for
m a x
males and females were proportional to the increase of dose levels from 0.03 to 0.3
mg/kg/day.
9.1.3.5. Absorption and metabolism of oral application

of capsaicinoids in animal experiments
Because of their increasing experimental use and planned drug production in a very
wide field of medical research and medical treatment, we have to gain correct knowl-
edge on the absorption, metabolism and excretion of capsaicinoids.
It is known that capsaicin given directly into the stomach of rats has only minimal
excitatory effects visible on immediate blood pressure responses (Lippe et al., 1989)
in contrast to intravascular or subcutaneous administration (Donnerer, Lembeck,
1983). On the other hand, it has been shown that capsaicin disappears within a rather
short time from the intestinal lumen (Kawada et al., 1984; Kawada, Iwai, 1985) and
should therefore reach the circulatory system.
+
Since biotransformed products of capsaicin are difficult to detect, the use of [ H]-
+
labelled dihydrocapsaicin ([ H]-DHC) allows to determine the percentage of
unchanged compound in the total extracted radioactivity. Dihydrocapsaicin (DHC)
has been shown to display pharmacodynamic and pharmacokinetic properties com-
parable with those of capsaicin (Burk, Bucks, 1982; Kawada et al., 1984).
3 3
[ H]-Dihydrocapsacin ([ H]-DHC and unlabelled capsaicin were readily absorbed
125
from the gastrointestinal tract but were almost completely metabolized before reach-
ing the general circulation. A certain degree of biotransformation already took place
in the intestinal lumen. Unchanged compounds (identified by chromatography) were
present in the portal vein blood. There seems to be a saturable absorption and degra-
dation process in the gastrointestinal tract and a very effective metabolism limited to
the liver (Donnerer et al., 1990).
Less than 5% of the total amount of extracted radioactivity consisted of unchanged
3
[ H]-DHC in blood and brain 15 min after gastrointestinal application. On the other
3
hand, approximately 50% unchanged [ H]-DHC was detected 3 min after intra-
venous application or 90 min after subcutaneous application of capsaicinoids
3
(Donnerer et al., 1990). Dihydrocapsaicin (DHC) or [ H]-DHC were metabolized
when incubated in vitro with liver tissue but not with brain tissue (Donnerer et al.,
1990). The metabolic product(s) did not show capsaicin-like biological activity
(Donnerer et al., 1990). These results clearly indicate that the rapid hepatic metabo-
lization limits the systemic pharmacological effects of enterally absorbed capsaicin
in rats (Donnerer et al., 1990).
Mean C m a xvalues for males increased 1: 3.3: 10- fold on Day 1 and 1: 2.9: 9.2
fold on Day 14-fold increase in administered dose. These results clearly indicate that
no accumulation exists for capsaicin after multiple dosing by this route of its admi-
nistration (Chanda et al., 2005).
9.1.3.6. Summary and conclusions of the administration of different

doses of trans-capsaicin in acute and subacute experiments
in dogs
In the acute study, surface Lead II ECG was monitored to determine the QT inter-
C
vals and the duration of cardiac repolarization. However, there were no observable
changes in the QT F (Fridericia's correlation, Q T F = Q T / A T (RR interval). Such a
C C
3
change would have been theoretically possible, as capsaicin has been reported to
block voltage-activated potassium channels in rat ventricular myocytes (Castle,
1992). Because many drugs able to block voltage-activated channels actually short-
en the duration of cardiac action potentials, there are limited correlations between
the potassium channel blocking activity and QT interval prolongation (Martin et al.,
2004). The lack of measurable effects on the cardiac action potential makes it likely
that the hemodynamic effects of capsaicin measured during the acute study with 0.3
mg/kg/day capsaicin are due to agonist activity at TRVP1 receptors; this is because
the reported potency of capsaicin appears to be several fold higher than that of either
calcium and potassium ion channels (Castle, 1992; Cheng et al., 2003). Thus, it is
likely that capsaicin receptors expressed on pericardiac sensory nerves induced the
transient increases in the heart rate observed during the acute dosing study, in the
course of performing their role to sense cardiac ischemia (Pan, Chen, 2004). For
longer administration of capsaicin, it is possible that the putative antihypertensive
actions of capsaicin result from either prolonged desensitization of pericardiac sen-
sory fibres, including the release of vasoactive peptides (CGRP and SP) from the
126
perivascular C-fibres, or activating an endogenous system which counterbalances
hypertension caused by sodium salt loading (Vaishnava, Wang, 2003).
Aside from the cardiac effects, capsaicin has also been studied in animals for other
possible target organ toxicity. Almost all of these studies used pepper plant extracts,
which are likely to display varying degrees of capsaicinoids content and possible
diverse impurity profiles. These impurities may be the contributing factor in some of
toxicities observed. Chanda et al. (2004) observed that the pure capsaicin displays a
different genotoxicity profil than that described in some previous literature.
Additionally, in contrast to the high dose levels used in toxicological studies, human
exposure to dietary capsaicinoids (a mixture of capsaicin, dihydrocapsaicin, nordi-
hydrocapsaicin, homocapsaicin and homodihydrocapsaicin) in U.S.A. and in
European countries is about 1.5 mg/day, which translates into, at most, 0.025
mg/day/ day (Chanda et al., 2005).
When capsaicin was dissolved in diethylene glycol monoethyl ether and
Dulbecco's phosphate-buffered saline and administered in intravenous infusion for
14 days (in a 15 min time period to anesthetized dogs), the vehicle itself caused
marked vascular irritation at the administration sites. There were no deaths, no test
article-related organ weight changes, and no macroscopic and microscopic observa-
tions. The only test article-related clinical sign observed in the study was vacuoliza-
tion in the dogs treated with 0.3 mg/kg /day. The only test article-related clinical
pathology finding was higher ALT in male and female dogs receiving 0.3 mg/kg/day
capsaicin. This may indicate the liver as a possible target organ when capsaicin is
delivered at high doses directly into the systemic circulation. When capsaicin was
given in a dose of 0.1 mg/kg/day for 14 days, it was rapidly eliminated in dogs.
Capsaicin dissolved in dimethyl sulfoxide (DMSO) has been studied by Glinsukon
et al. (1980) for determination of L D values by several administration routes in
50
mice. The authors also determined the L D values of capsaicin for one administra-
50
tion route (intraperitoneal) in different species. The order of sensitivity ( L D va-

50
lues) for species, from the least to most, by the intraperitoneal route using DMSO as
the delivery vehicle was reported to be: hamsters (> 120 mg/kg), rabbit (> 50 mg/kg),
rat (9.5 mg/kg), mouse (6.5 to 7.65 mg/kg) and guinea pig (1.1 mg/kg). This study
and those described above were mixed with respect to gender. Electrocardiograms
(ECGs), mean arterial pressure and resporiratory rates were also measured by
Glinsukon et al. (1980) in anesthetized rats after treatment with a lethal intraperi-
toneal dose of capsaicin. Saito and Yamamoto (1996) reported the oral L D values
50
for capsaicin extract for both genders in mice and rats, when propylene glycol was
used as vehicle. They did not find any significant difference in the L D values of
50
the two genders. The major toxic signs in mice and rats included salivation, stagger-
ing gait, bradypnoe and cyanosis. Tremors, clonic convulsions, dyspnoe, lateral or
prone position was observed, then the animals died 4 to 26 min after oral dosing by
gavage.
The cause of death was proposed to be due to hypotension and respiratory paraly-
sis in rats and mice although the authors noted that the pathophysiology of these
deaths was not clearly understood.
In a study with Monsereenusorn (1983), 50 mg /kg capsaicin was given orally by
gavage to rats for up to 60 days. The effects of capsaicin on body weight, rectal
127
temperature, food and water consumption, hematological parameters, plasma bio-
chemistry, urine concentration and dilution tests were evaluated at 10, 20, 30, 40, 50
and 60 days. The major finding was decreased body weight gain starting at 40 days,
despite an increase in food consumption. Minor changes in the clinical chemistry
(reduced plasma urea nitrogen, glucose, phospholipids, triglycerides, total choles-
terol, free fatty acids, glutaminic pyruvic transaminase, and alkaline phosphatase)
were noted after one-month treatment; however, these differences were not biologi-
cally significant,
The results of observations done by Chanda et al. (2005) indicate that trans-cap-
saicin, given into the sytemic circulation, induces transient increases in heart rate
and blood pressure which are rapidly eliminated, and do not cause alterations in cardiac
repolarization.
9.1.3.7. Chronic toxicity studies in animals
Chronic toxicological studies are absolutely required to test drug candidates in spe-
cies (one from the rodents and dog) for a period of 6 months. No observations of
these types could be found in the literature, consequently these studies should be
done in the forthcoming time with our preparation.
9.1.4. Metabolism of capsaicin
Early studies by Lee and Kumar (1980) showed that phenobarbital-induced rat liver
microsomes converted capsaicin and dihydrocapsaicin to corresponding catechol
metabolites, N-(4,5-dihydroxyl-3- methoxybenzyl)-acidamides via hydroxylation
on the vanillyl moiety. This finding was further confirmed by Miller et al. (1983)
3
who demonstrated the covalent binding of [ H]-dihydrocapsaicin to hepatic micro-
somal proteins following in vitro incubation or administration to rats. Based on these
results, it has been postulated that capsaicin is activated by the liver mixed-function
oxidase system to an electrophilic intermediate, most likely a ring epoxide, capable
of covalently interacting with nucleophilic sites of hepatic protein (Fig. 28).
This irreversible interaction of capsaicin with liver microsomal protein may
account for the binding of capsaicin which was observed in the spinal cord or brain,
and it was concluded that capsaicin-induced neuropathy in rodents might be mediat-
ed by mechanisms other than covalent interaction (Miller et al., 1983). The alkyl
side chain of capsaicin is also considered to be susceptible to enzymatic oxidation.
Thus, when capsaicin was incubated with NADPH and the liver S9 fraction from
phenobarbital pretreated rats, it was hydroxylated at the terminal carbon of the side
chain (Surh, Lee, 1995, 1996; Surh et al., 1995).
128
CH NHCO-R-OH
2 CH NHCO-R
2 CH NH 2 2
COVALENT INTERACTION W I T H CRITICAL CELLULAR NUCLEOPHILES

(e.g., PROTEINS, DNA, RNA...)
TOXICITY
(NECROSIS, MUTAGENESIS, CARCINOGENESIS, ETC.)
R = (CH ) CH = CHCH
2 2
^ C H 3
Fig. 28. M e t a b o l i s m of c a p s a i c i n ( S u r h , Y.J., L e e , S.S., 1 9 9 5 : C a p s a i c i n a d o u b l e - e d g e d s w o r d :

toxicity, m e t a b o l i s m , a n d c h e m o p r e v e n t i v e p o t e n t i a l . Life S c i . 5 6 : 1 8 4 5 - 1 8 5 5 )
(with permission)
9.1.4.1. The potential routes of metabolism of capsaicin
9.1.4.1.1. Enzymatic oxidative metabolism of capsaicin
One-electron oxidation of capsaicin has been investigated by means of electrochem-

ical, enzymatic and chemical procedures (Lawson, Gannett, 1989; Boersch et al.,
1991). Lawson and Gannett (1989) reported that incubation of capsaicin with micro-
129
somes or its non-enzymatic reaction with potassium ferricyanide resulted in the for-
mation of a dimer, 5,5'-bis-capsaicin. A phenoxy radical was proposed to be involved
in the mutagenesis by capsaicin. Formation of phenoxy radical intermediates has
been often observed with certain plant phenolics (Newmark, 1984, 1987), which
plays a critical role in lignin biosynthesis in the process of wood formation
(Freudenberg, 1962). Formation of dimeric tyrosine by oxidation of tyrosine with
horseradish peroxidase-catalyzed coupling reaction has been proposed as a mecha-
nism for the dimerization of two diiodotyrosyl residues in thyroglobin to form the
thyroid hormone, thyroxine (Taurog et al., 1994). Boersch et al. (1991) have demon-
strated that incubation of capsaicin with peroxidase and hydrogen peroxide pro-
duced a fluorescent dimer analogue to that previously reported by Lawson and
Gannett (1989). The formation of this fluorescent oxidation product was also
observed by chemical or electrochemical oxidation of capsaicin (Lawson, Gannett,
1989). Gannett et al. (1997) have shown that the liver cytochrome P450 2E1
(CYP2E1) activity is responsible for conversion to the reactive phenoxy radical
which, in turn, dimerizes or covalently binds to CYP2E1, thereby interacting with
the enzyme (Fig. 28).
9.1.4.1.2. Non-oxidative metabolism of capsaicin

Cell-free extracts of various tissues of rats contained enzyme activity for hydrolyz-
ing capsaicin or its dihydro analogue at the acid-amide bond to produce vanillyl-
amine and the corresponding fatty acyl moieties (Kawada et al., 1984; Kawada, Iwai,
1985; Oi et al., 1992). The highest enzyme activity was found in the liver followed
by such extrahepatic tissues as kidney, lung and small intestine (Kawada, Iwai,
1985), after oral administration of capsaicin (Oi et al., 1992). The splitting of the
side chain of dihydrocapsaicin also occurred in vivo (Kawada, Iwai, 1985), which is
considered to be the rate-limiting step in the overall metabolism of this compound.
Hydrolysis of the amide linkage of capsaicinoids will thus lead to the formation of
vanillylamine as a common metabolite regardless of the type of fatty acid functional
in their side chain. Indeed, the systemic vanilloid, olvanil [N-(3-methoxy-4-hydroxy-
benzyl)oleamide] which has a longer side chain than capsaicin, has been found to be
susceptible to hydrolysis of the amide bond as determined in various metabolic
model systems including cell-free extracts of liver and intestine, isolated hepato-
cytes and enterocytes, and isolated perfused intestine, and also in whole animal stu-
dies (Wehmeyer et al., 1990). Oxidative deamination of the resulting vanillylamine
produces the aromatic alcohol for excretion as a free form or a glucoronic conjugate
(Kawada, Iwai, 1985; Wehmeyer et al., 1990). Capsaicin hydrolyzing enzymes have
been purified from the rat hepatic microsomes (Park, Lee, 1994), and identified as
previously known isoenzymes of carbylesterase based on such biochemical and bio-
physical parameters as Mr (relative mass), pi (inhibitory concentration in logarith-
mic scale), pH-dependency, mode of inhibition and subcellular toxicity. The
enzymes are likely to be present either free in the lumen of endoplasmatic reticulum
or loosely bound to the terminal surface of the membrane (Park, Lee, 1994).
Capsaicinoids, when administered to rats intragastrically, were readily absorbed
from the gastrointestinal tract but were further metabolized to a great extent in the
130
liver before reaching the general circulation (Park, Lee, 1994). As a result, gastroin-
testinally absorbed capsaicinoids are expected to reach the central nervous system or
other extrahepatic organs almost exclusively as degradation products (Donnerer et
al., 1990).
9.1.4.2. Role of metabolic activation in capsaicin-induced toxicity
There is no clear-cut mechanism which can solely explain the toxicity exerted by
capsaicin. Bioactivation to an electrophilic intermediate with subsequent covalent
modification of critical cellular macromolecules such as DNA, RNA and proteins
has been thought to play a role in cell death (Miller et al., 1983; Anonymous, 1986),
fueling interest in the role of metabolism in these observed processes. Based on the
results of previous metabolism studies, the following activation pathways can be
postulated which may account for the capsaicin-induced cellular damages (Fig. 29):
a) cytochrome P450-catalyzed epoxidation of the vanillyl ring to produce an arene
oxide;
b) one-electron oxidation of the hydroxy 1 group to form a phenoxy radical;
c) O-demethylation at the aromatic ring and subsequent oxidation of the resulting
catechol to the semiquinone and quinone derivates.
The possible involvement of an electrophilic epoxide is not proven (Miller et al.,
1983), but the presumed oxirane epoxidation of capsaicin is expected to occur
because of the formation of arene oxide. Nonetheless, ring epoxidation of capsaicin
is expected to occur since the formation of arene oxide intermediates is the most
general phenomenon in the monooxygenase-catalyzed metabolism of aromatic com-
pounds. The best evidence for the epoxidation of capsaicin will be the actual isola-
tion of the presumed arene oxide, but the chemical reactivity of such species pre-
cludes its direct isolation from incubation mixtures or from the biological fluids or
tissues of treated animals. The advances in the development of novel mild oxidating
agents such as dimethyl dioxirane have made it possible to prepare the extremely
reactive epoxides of certain chemical carcinogens including aflatoxin B1-8-9 oxide.
A similar synthetic approach could be applied to the synthesis of the benzoepoxide
derivate of capsaicin for testing its biological activity as well as chemical reactivity.
The covalent binding of tritium-labeled capsaicin to hepatic microsomal protein was
significantly inhibited by reduced glutathione, which implies the formation of a reac-
tive intermediate (epoxide) during the metabolism of capsaicin (Miller et al., 1983).
Since glutathione is relatively nonspecific in terms of interacting with reactive inter-
mediates including not only oxiranes but also capsaicin this does not necessarily
suggest that the study of the aforementioned arene oxide as a sole electrophilic
epoxide hydrolase might be a more sensitive approach in exploring the possible
involvement of an epoxi metabolite in the toxification processes induced by cap-
saicin.
The intermediacy of the phenoxy radical of capsaicin has been investigated by
using the electrochemical or chemical methods (Lawson, Gannett, 1989; Boersch et
al., 1991; Gannett et al., 1997). Furthermore, horseradish peroxidase plus the phe-
noxy radical intermediate have to be taken into account (Boersch et al., 1991;
131
Catechol
OH
CH NHCOCH CH CH CH CH=CHCH(CH )
2 2 2 2 2 3 2
Capsaicin
CH CH(NH)COOH
2
DOPA
Dopamine
CH(OH)CH NHCH 2 2
Epinephrine
OH
OH
Fig. 29. S t r u c t u r e s of s e l e c t e d c o m p o u n d s c o n t a i n i n g c a t e c h o l m o i e t i e s , i n c l u d i n g c a p s a i c i n
( C h a n d a , S., E r e x s o n , G . , R i a c h , C , I n n n e s , D., S t e v e n s o n , F., M u r l i , H . , B l e y , K., 2 0 0 4 :
C e n o t o x i c i t y studies w i t h p u r e t r a n s - c a p s a i c i n . M u t a t . R e s . 5 5 7 : 8 5 - 9 7 ) ( w i t h permission)
Gannett et al., 1997). Likewise, hepatic microsomal cytochrome P450 (particularly

CYP2E1) might generate the same reactive radical species that is capable of attack-
ing the nucleophilic sites of the enzyme or the target cell protein (Gannett et al.,
1997), which may lead to the loss of catalytic activity in other crucial biological
functions.
A quinone-type intermediate also represents a potential ultimate electrophilic
metabolite of capsaicin. The formation of such intermediate could proceed via O-
demethylation of the 3-methoxy group on the vanillyl ring with concomittant oxida-
tion to the semiquinone and ortho quinone derivates. The same O-quinone metabo-
lite could be generally obtained through O-demethylation of the aforementioned
phenoxy radical intermediate of capsaicin (Fig. 29). This process will also lead to
the formation of an extremely readable group of CH , which is well known to alkyl- 3
ate cellular nucleic acids and proteins. The above-mentioned reactions are likely to
132
occur in view of the presence of microsomal O-demethylase activity and relatively
high reactivity of catechol or antineoplastic agent, epoxiside, has been known to
exert its cytotoxic effect by enzymic O-demethylation of one of its methoxy groups
to the ortho-quinone derivative capable covalently binding to cellular macromole-
cules (Haim et al., 1987a,b; Mans et al., 1991). Similarly, initial ring epoxidation of
capsaicin and subsequent NIH shift in the resulting benzoepoxide derivate would
generate a catechol intermediate without O-demethylation.
9.1.5. Effects of capsaicin on xenobiotic metabolism

and chemically induced mutagenesis and carcinogenesis
Capsaicin has been suggested to exert chemopreventive effects through the modula-
tion of metabolism of carcinogens and their interactions with target cell DNA. It has
been reported that capsaicin displays a dose-related inhibition of the activity of rat
epithelial aryl hydrocarbon hydroxylase (Modly et al., 1986), a marker enzyme for
metabolism of polycyclic aromatic hydrocarbons such as benzol(a)pyrene. Fur-
thermore, capsaicin suppressed the metabolism and covalent DNA binding of ben-
zol(a)pyrene in human and murine keratocytes (Modly et al., 1986). The results of
different studies suggested the interaction of capsaicinoids with microsomal mixed-
function oxidases. Thus, the pretreatment of rats with subcutaneous dosages of cap-
saicin resulted in pronounced prolongation of phenobarbital or hexobarbital sleep-
ing time (Miller et al., 1983; Damhoeri et al., 1985; Rauf et al., 1985; Surh et al.,
1995). Capsaicin also competitively inhibited the ethylmorphine demethylase activi-
ty in rat liver microsomes and produced a type I spectral change (Miller et al., 1983).
Oral administration of capsaicin (50 mg/kg) together with 10% ethanol in drinking
water increases xenobiotic metabolizing enzyme activity to a much greater extent
that induced by ethanol alone (Iwama et al., 1990). Capsaicin pretreatment also
induced 4-hydroxylation of biphenyl groups in the rat liver (Rauf et al., 1985).
Yagi (1990) reported that capsaicin and dihydrocapsaicin repressed the energy-
transducing NADH-quinone oxidoreductase activity. This finding confirms an earli-
er observation by investigations of inhibitory effects of capsaicin on rat hepatic mito-
chondrial energy metabolism through supression of energy flow from NADH to
coenzyme Q (Chudapongse, Jathanasoot, 1981). Capsaicin was shown to inhibit
calmodulin-mediated oxidative burst in the rat macrophages as determined by the
2+
attenuation of Ca ionophore-triggered production of superoxide anion and hydro-
gen peroxide (Savitha et al., 1990). Joe and Lokesh (1994) have also shown that cap-
saicin can strongly block the generation of reactive oxygen species by rat peritoneal
macrophages in vitro. Capsaicin fed to animals was also inhibitory in these
macrophages (Joe, Lokesh, 1994). Pretreatment of rats with capsaicin (1.68 mg/kg,
intraperitoneally) for three consecutive days resulted in enhancement of activities of
pulmonary anti-oxidant enzymes such as superoxide dismutase, catalase and peroxi-
dase while long-term treatment caused an opposite effect on the latter two enzymes
(De, Ghosh, 1990). Since reactive oxygen species are known to play an important
role in phorbol-12-myristate-13-acetate (PMA)-mediated tumor promotion as well
133
as in inflammation (Kensler et al., 1986), it would be worth determinining if cap-
saicin with potential anti-inflammatory activity (Flynn, Rafferty, 1986; Joe, Lokesh,
1994) could act as an anti-tumor promoter. It is noteworthy that the inhibition of
prostaglandin synthesis by curcumin, the complex of tumeric, correlates well with
its protective activity; it was reported for curcumin to inactivate xanthine dehydro-
genase/oxygenase, which may account for its anti-promotion activity.
Capsaicinoids have also been found to retain inhibitory effects on liver microsom-
al CYP2E1 activity (Gannett el al., 1997; Lee et al., 1997; Satyanarayana 2006).
Capsaicin inhibits the metabolism and mutagenicity (Guengerich et al., 1991; Koop,
1992; Espinosa-Aguirre et al., 1993), which is known to be activated by CYP2E1.
CYP2E1-mediated mutagenicity and tumorigenicity of vinyl carbamate have also
been shown to be reduced by capsaicin (Lee et al., 1997).
The initiation of papillomas in mouse skin by benzol(a)pyrene was also signifi-
cantly lessened by topical application of capsaicin prior to the carcinogen (Lee et al.,
1997). This finding is in agreement with in vitro inhibition of rodent epidermal aryl-
hydrocarbon hydroxylase activity by capsaicin as previously reported by Modly et
al. (1986). Chili extracts have been found to modulate the mutagenic activity of par-
ticulate organic matter in urban air samples (Espinosa-Aquuirre et al., 1993).
In a series of observations, it has been observed that capsaicin has a protective
effect on the metabolism, DNA binding, and/or mutagenicity of some carcinogens
[aflatoxin Bl, tobacco-specific nitrosoamine and 4-(methylnitroso)-l-(3-pyridyl)-l-
butanone)]. These findings suggest that capsaicin might act as a chemoprotective
agent by modulating the activities of microsomal mixed-function oxidases which
play key roles in metabolic activation as well as detoxication of a wide array of
chemical carcinogens and mutagens (Surh, Lee, 1995).
9.1.6. Hepatoprotection of capsaicin in rats

Recently Abdel-Salam et al. (2006) observed in detail the dose-dependent hepato-

protective effect of capsacin in rats treated with carbon tetrachloride (CC1 ), which
4
was given orally (2 ml/kg followed by 1 ml/kg after one week). Capsaicin at three
dose levels (10, 100 and 1000 pg/kg) or silymarin (22 mg/kg) was administered oral-
ly for 10 days, starting at the time of administration of CC1 .
4
The daily administration of capsaicin conferred significant protection against the

hepatotoxic effects of CC1 in rats. It decreased the increase of serum alanine amino-
4
transferase (ALT) and aspartate aminotransferase (AST) and also prevented the
development of histological hepatic necrosis caused by CC1 as determined 10 days
4
after drug administration. Thus, compared with CC1 control group, serum ALT
4
decreased by 39.3, 59.3 and 71.1%, while AST decreased by 14.3, 21.5 and 23.3%,
after the capsaicin administration (given in doses mentioned above), respectively.
Serum bilirubin was decreased by 10 and 100 pg/kg (46.4 and 66.5% reduction,
respectively), but an increased bilirubin and ALP were observed after the highest
dose of capsaicin. Meanwhile, silymarin reduced serum ALT by 65.3%, AST by
18.9%, ALP by 22% and bilirubin by 13.4%, compared to CC1 control. Serum pro-
4
teins were significantly increased by 16.9 to 22.9% after treatment with capsaicin,
134
whilst a marked, 66.9%, increase in serum glucose was observed after the highest
dose of capsaicin compared with vehicle-treated group.
Quantitative analysis of the area of damage by image analysis technique showed a
reduced area of damage from 13.6% to 7.5, 4.3 and 2.8% by application of capsaicin
(used in the above-mentioned doses), respectively (Abdel-Salam et al., 2006).
Haematoxylin-eosin staining indicated markedly less hepatic necrosis in rats treated
with capsaicin or silymarin. Histochemical alterations such as decreased nuclear
DNA, cell glycogen and protein contents caused by CC1 in hepatocytes were pre-
4
vented by capsaicin as well as by silymarin.

It has been concluded from these results that orally applied capsaicin exerts bene-
ficial effects on liver histopathologic changes and enzymatic release caused by CC1 4
in rats, but its higher doses result in cholestasis (Abdel-Salam et al., 2006).
9.1.7. Genotoxicity studies with capsaicin or trans-capsaicin
Published information on the potential genotoxicty of capsaicin is inconsistent, both

positive and negative effects have been found in classic genetic toxicology assays
(Azizan, Blevins, 1995; Surh, Lee, 1995).
Eight bacterial point mutation tests (including Ames assays) were performed
between 1981 and 1995 on capsaicin of varying origins, using various strains of S.
typhimurium. Various forms of S9 activation were provided in seven of the eight
assays. Four of these tests resulted in a positive response and four in a negative
response, respectively. Point mutation tests in Chinese hamster V79 cells were con-
ducted twice, resulting in one positive and one negative response. The in vivo
micronucleus test was conducted once in mice and it was positive (Nagabhushan,
Bhide, 1985, 1986).
Data from one micronucleus and sister chromatid exchange study in human lym-
phocytes was interpreted to show that capsaicin is genotoxic (Marques et al., 2002).
Capsaicin was also reported to induce DNA strand breaks in human neuroblastoma
cells SHHY5Y (Richeux et al., 1999).
Most of these studies were carried out with natural extracts, and they may not
exhibit the same toxicological profile with pure capsaicin.
Recently different studies were carried out to evaluate the genotoxic potential of
pure frafts-capsaicin using different genotoxic assay used by international regulatory
agencies to evaluate drug product safety (Chanda et al., 2004). These included Ames
assay, mouse lymphoma cell mutations assay, mouse in vivo bone marrow micronu-
cleus assay and chromosomal aberration assay in human peripheral blood lympho-
cytes (HPBL). All studies were conducted according to the Organization of
Economic Cooperation and Development (OECD) principles of Good Laboratory
Practice (GLP) (Table 26).
135
T a b l e 2 6 . G e n o t o x i c i t y of c h i l l i extracts a n d their m a j o r p u n g e n t constituents c a p s a i c i n
a n d d i h y d r o c a p s a i c i n u p to 1 9 9 5
Test C o m p o u n d A n i m a l / C e l l s tested H e p a t i c S 9 for Endpoint Response

metabolic activation
CAP, Chilli 5. typhimurium Aroclor His +

reversion +
1 2 5 4 - i n d u c e d rat
CAP, Chilli C h i n e s e hamster V 7 9 Aroclor Azaguanine -
1 2 5 4 - i n d u c e d rat resistance
CAP, Chilli Swiss mice In v i v o Micronuclei + (CAP
formation only)
CAP 5. typhimurium!A98 Aroclor His +
reversion +
1 2 5 4 - i n d u c e d rat
CAP, D H C , Chilli 5. typhimuriumlA98, Aroclor His +
reversion -
TA1535 1 2 5 4 - i n d u c e d rat
CAP, D H C , Chilli C h i n e s e hamster V 7 9 Azaguanine +
resistance
-
CAP, Chilli 5. typhimurium Phenobarbital- His +
reversion
i n d u c e d rat
CAP, Chilli S. typhimurium Streptomycin- +
resistance (Chilli);
- (CAP)
CAP, D H C , Chilli S. typhimuriumTA98, + (source unclear) His +
reversion - or +
TA100 (CAP)
Chilli Mouse bone marrow In v i v o Micronuclei +
formation
CAP Albino mice In v i v o Pregnancy -
frequency
CAP M o u s e epididymis In v i v o Sperm -
abnormality
CAP H u m a n lymphocytes Chromosome +
aberrations
CAP=capsaicin; DHC=dihydrocapsaicin; Chilli=plant extract. After Surh, Y.J., Lee, S.S (1995): Capsaicin
a double-edged sword: toxicity, metabolism, and chemopreventive potential. Life Sci. 56: 1845-1855
(with modification)
9.1.7.1. Ames assay
Ames assay described in the paper of Chanda et al. (2004) was based on the method
described by Ames et al. (1975). The thymidine kinase (TK) heterozygote system,
where tk+/tk- is mutated to tk-/tk-, was described by Clive et al. (1972, 1979) and it
is based upon the L5178Y mouse lymphoma cell line established by Fischer (1958).
In this assay, cells deficient tk+/tk- to tk-/tk- are resistant to cytotoxic effects of
pyrimidine analogue trifluorothymidine (TFT). Thymidine kinase proficient cells are
sensitive to TFT, which causes the inhibition of cellular metabolism and halts further
cell devison. Thus the mutant cells are able to proliferate in the presence of TFT,
whereas normal cells which contain thymidine kinase are not (Moore et al., 2003).
136
Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 were used. The
assays were performed in the presence and absence of S9, using the direct method
and preincubation method. Capsaicin did not induce mutagenic activity in any of the
bacterial strains used, in either activation condition.
9.1.7.2. Mouse lymphoma cell mutation assay
The tk+/tk-3.7.2C heterozygote of L5178Y mouse lymphoma cells were applied in

these studies (for details of the methods used, see paper of Chanda et al, (2004).
In the presence of S9 mix both assays gave weak mutagenic responses. Both
assays contained at least two treatment groups of capsaicin that tested significant
(for log mutant fraction) at the level of 5%. Both assays had a linear trend (of mutant
fraction with concentration) that was significant at P< 0.001. The increases in the
mutant fraction obtained were small, the largest being 190 mutants per million above
the control value, obtained at near the maximum acceptable level of toxicity (12%
relative total growth).
In absence of S9 mix, the 4-hour exposure assay gave a very weak mutagenic
response, while the 24-hour assay gave no significant responses at dose levels giving
acceptable levels of genotoxicy though the test for for linear trend was significant (P
= 0.022).
Colony size distribution patterns are, in instances of weak positives, difficult to
assess due to the very small samples. The same situation occurs for vehicle control
groups, which show a high level of variation between and within experiments. The
numbers of mutant colonies assessed in the capsaicin treatments giving significant
increases were very low.
9.1.7.3. Mouse in vivo micronucleus assay
For details of this methodology, see the paper of Chanda et al. (2004). At least 2000
polychromatic erythrocytes (PCE) were scored for frequency of micronucleated
cells. The numbers of micronucleated normochromic erythrocytes (NCE), which
were observed within the same microscope fields, were similarly recorded. The
PCE/NCE ratio was assessed by scoring a total of at least 500 PCE+NCE. Each
assessment was performed on coded slides.
In preliminary toxicity tests the maximum tolerated dose of capsaicin was deter-
mined to be around 800 mg/kg per day in males and around 200 mg/kg per day in
females. Three groups of male mice were dosed with 200, 400 and 800 mg/kg per day
and one group of females was dosed with 200 mg/kg per day of capsaicin at 0 and 24
hours. Five mice per sex from each test material dose group were selected to provide
the normal assessment base. Current vehicle and positive groups were included.
Treatment-related animal deaths and clinical signs were observed in the middle-
and high-dose level groups. One death occurred in the male vehicle control group,
immediately after dosing and was considered as a result of a dosing error.
137
The frequencies of micronucleated polychromatic erythrocytes (MN-PCE) in the
capsaicin-treated groups were 0.04, 0.10, 0.09% (males) and 0.05% (females). All
of these frequencies were within the historical control range for negative responses
(0.01-0.23% for a group of five mice). The frequencies of MN-PCE in the concur-
rent vehicle control groups were 0.07% (males) and 0.11% (females), whereas the
MN-PCE frequency in the positive control group was 1.57%, demonstrating the sen-
sitivity of the test system.
9.1.7.4. Chromosomal aberration in human peripheral blood

lymphocytes ( H P B L )
Human venous blood from healthy, adult donors (non-smokers without any history
of radiotherapy, chemotherapy or drug usage and lacking current viral infections)
was used. The whole blood cultures were initiated in 15 ml centrifuge tubes by
adding 0.6 ml of fresh heparinized blood, and the final volume of culture medium
and test arcticle was 10 ml. Cultures were incubated with loose caps at 372C in a
humidified atmosphere of 51.5C in air. The medium was RPMI 1640 supplement-
ed with HEPES buffer (25 mM), about 20% heat-activated fetal bovine serum (FBS),
penicillin (100 U/ml), streptomycin 100 pg/ml9, L-glutamine (2 mM) and 2% phy-
tohemagglutinin M (PGA-M). Negative (untreated controls) and vehicle controls
(cultures treated with 10 pi of DMSO/ml) were used. The positive control agents
used in the assays were mitomycin-C (MMC) for the nonactivation series and CP
(cyclophosphamide) in the metabolic activation series.
The in vitro metabolic activation system (Maron, Ames, 1983) consisted of liver
post-mitochondrial fraction (S9) and an energy-producing system (NADP at 1.5
mg/ml (1.8 mM) and isocitric acid at 2.7 mg/ml (10.5 mM). S9 was prepared 5 days
after a single dose of 500 mg/kg of Aroclor 1254.
Two trials were conducted. In the initial trial, cultures were treated for about 3
hours with and without S9 and harvested about 22 hours after initiation of treatment.
In the second trial, cultures were treated for about 22 hours without S9 and about 3
hours with S9 and harvested about 22 hours after initiation of treatment. This har-
vesting time corresponds to 1.5 times a cell cycle time (Galloway et al., 1994). The
period of cell cycle is approximately 15 h after the lymphocytes are induced to divide
by the addition of PHA-M. At harvest, cells were swollen with 75 mM KC1 hypo-
tonic solution and fixed with absolute methanol-glacial acetic acid (3:1 v/v). Cells
selected from each duplicate culture were analyzed for the different types of chro-
mosomal aberrations (Evans, 1962a,b, 1996). Mitotic index was evaluated from the
negative control, vehicle control and a range of test article concentrations and this
was used for the measurement of toxicity and selection of doses for analysis. Per-
cent polyploidy and endoreduplication were also analyzed. For control of bias, all
slides were coded prior to analysis and read blind.
In the first trial, 6.78, 9.69, 13.8, 19.8, 28. 2, 40.4, 57.6, 82.4, 118, 168, 240, 343,
490, 700 and 1000 pg/ml of capsaicin were evaluated with and without metabolic
activation by S9. The highest concentration was limited due to the presence of a pre-
cipitate dosing. In the first trial, a precipitate was observed at >240 pg/ml, and
138
hemolysis was observed prior to washing and harvesting of these cultures. Only
dead cells were present on slides prepared from cultures treated with >343 pg/ml
due to excessive toxicity (Chanda et al., 2004).
Chromosomal aberrations were analyzed from the cultures treated with 82.4,118,
168 and 240 pg/ml. The high concentration had >50% reduction in mitotic index.
No increase in structural or numerical chromosomal aberrations was observed
(Chanda et al., 2004).
Based on the results from the initial assay, the second trial was conducted at con-
centrations of 6.15, 12.3, 24.6, 49.2, 98.4, 154, 192, 240 and 320 pg/ml without
metabolic activation and 49.2, 98.4, 123, 154, 192, 240 and 320 pg/ml with meta-
bolic activation. Treatment periods were about 22 and about 3 h without and with
metabolic activation, respectively, and the cultures were harvested about 22 h after
the initiation of treatment. In the assay without metabolic activation, a precipitate
was observed after >92 pg/ml and hemolysis was observed prior to harvest of the
cultures treated with >240 g/ml. Only dead cells were present on slides prepared
from cultures treated with > 192 pg/ml, due to excessive toxicity. Severe toxicity was
observed also at 98.4 pg/ml (92% reduction in mitotic index). Chromosomal aberra-
tions were analyzed from the cultures treated with 24.6, 49.2 and 123 pg/ml. Due to
toxicity, <100 metaphases were available for analysis in the duplicate cultures treat-
ed with 123 pg/ml. The highest tested concentration (240 pg/ml) had >50% reduc-
tion in mitotic index. No increase in structural or numerical chromosomal aberra-
tions was observed.
In the assay with metabolic activation, a precipitate was observed after dosing at
>192 pg/ml and hemolysis was observed prior to harvest of the cultures treated with
320 pg/ml. Since the cultures treated with 240 pg/ml had excessive toxicity (100%
reduction in mitotic index) the slides prepared from the cultures treated with 192 pg/ml
were selected as the highest concentration for analysis. Chromosomal aberrations
were analyzed from the cultures treated with 98.4, 123, 154 and 192 pg/ml. No
increase in structural or numerical chromosomal aberrations was observed (Chanda
et a l , 2004).
Although there are a number of publications focusing on the genotoxic potential
of capsaicin or spicy pepper extracts, the test substances used in these studies were
various according to source, purity and impurity profile. Consequently, there has not
been any systemic observation of the genotoxic potential of pure trans-capsaicin.
The majority of the studies found in the literature (Fischer, 1958; Ames et al.,
1975; Richeux et al., 1999; Marques et al., 2002) performed the Ames assay. There
was one study each with the micronucleus assay, the sister chromatoid exchange
assay and the assay invetigating DNA strand breaks in human neuroblastoma cells
(Ames et al., 1975; Nagabhushan, Bhide, 1985, 1986; Azizan, Blevins, 1995; Surh,
Lee, 1995; Marques et al., 2002). None of the studies were conducted using system-
atically pure capsaicin, though it would be very important to be able to attribute the
results to capsaicin alone, not the impurities. Chanda et al. (2004) applied synthetic
capsaicin alone (purity >99%) to perform the genotoxicity studies.
In the Ames assay with pure synthetic capsaicin, no increase in mutation frequen-
cy was observed in any assays, with and without S9. It was concluded from these
studies that capsaicin is not genotoxic in the bacterial assay, with and without metata-
bolic activation (at the highest concentrations that could be tested).
139
Capsaicin was found to be weakly mutagenic in mouse lymphoma L5178Y cells,
in the presence of S9, when it was dissolved in DMSO at concentrations that exten-
ded into the toxic range. The lowest positive concentration in the presence of S9 in
any individual test was 12 pg/ml. Limited evidence of very weak activity was also
noted in the absence of S9. The lowest positive concentration in the absence of S9
was 65 pg/ml. Although criteria for the determinations for positive response in the
lymphoma assay remained controversial, these tests are used by different laborato-
ries to evaluate the toxicity (Clive et al., 1979; Moore et al., 2003). The results of the
above-mentioned studies were interpreted as weak toxicity. With longer exposures
(about 24 hours), higher capsaicin concentrations were too toxic for analysis and the
mutant fractions were not significantly different from the controls at the usable con-
centrations. Thus it can be concluded that capsaicin is non-mutagenic at concentra-
tions up to 28 pg/ml after 24 h exposure in the absence of S9.
In the in vivo micronucleus study in mice, the frequency of MN-PCEs was
0.04-0.10% in the treated mice compared to 0.07-0.11% in the vehicle treated con-
trol mice. Both were within the historical control range, which was stated to be
0.070.08%. The frequency of MN-PCE for the positive control (50 mg/kg of
cyclophosphamide) was 1.5%. However, in this study a difference was observed
between the maximum tolerated doses estimated for males (800 mg/kg) and females
(200 mg/kg). These values are higher than those of previous reports in the literature
(Glinsukon et al., 1980; Saito, Yamamoto, 1996), which probably may reflect the
quality of the current drug substance rather than the sex difference.
Capsaicin was evaluated for its ability to induce clastogenicity in cultured human
lymphocytes with and without an exogenous metabolic activation system.
Clastogenecity was evaluated at concentrations that induced severe toxicity to no
toxicity. Cultures were harvested about 22 hours from the initiation of treatment.
Capsaicin did not induce structural and numerical chromosomal aberrations.
It can be concluded from the results of genotoxicity observations with pure trans-
capsaicin that its genotoxicity potential is very limited, differing from that when
impure capsaicin or chilli extracts were used in genotoxicity assays.
These data have important implications for analysis of risks associated with
dietary or environmental capsaicin exposures. Although the majority of epidemio-
logical data suggests that dietary capsaicin consumption is not associated with
enhanced risk of cancer (Surh, Lee, 1995), it is true that a positive mathematical cor-
relation was observed between the intake of Chilli pepper and gastric cancer. In
Mexico, other factors than capsaicin should be investigated as the causal link in such
epidemiological evaluations (Lopez-Carrillo et al., 1994, 2003).
9.1.7.5. Brief summary of the main results of observations

with capsaicin in animals
Capsaicin is a very active compound acting at the level of capsaicin sensitive afferent
nerves. It has dual action, namely in small doses it produces a biologically significant
gastrointestinal mucosal protective effect, while in larger doses it enhances the gas-
trointestinal mucosal damages to different chemical, osmotic and pressure stimuli.
140
It is important to note that the doses (10 to 100 pg/kg) producing gastrointestinal
mucosal defensive actions are significantly lower than those (100-200 mg/kg) which
produce gastrointestinal mucosal damage.
Capsaicin is absorbed well from the animal gastrointestinal tract, its metaboliza-
tion is carried out by the liver (in pathways of enzymatic and non-enzymatic oxida-
tion). The production of epoxy (arene) by the rat liver was suggested, however, it
was not proved clearly.
There is a specific and important observation that capsaicin dose-dependently
prevents the CCl -induced hepatic injury during a one-week treatment.
4
The genotoxicity studies indicated a very limited positivity, depending on the

degree of purification of capsaicin of the plants.
9.2. Human observations with capsaicin
9.2.1. Observations with capsaicin in healthy human subjects
The capsaicin studies have been carried out since 1997, by the permission of the
Regional Ethical Committee of Pecs University, Hungary. These studies were car-
ried out in a randomized, prospective manner, respecting the Helsinki Declaration,
according to the Good Clinical Practice (GCP), with the methods required for classi-
cal drug (or drug candidate) studies.
The First Department of Medicine, Medical and Health Science Centre, University
of Pecs, Hungary, is one of the Hungarian Accredited Centres for the human phase
III examinations. This Institute has been participating in drug development since
1968 up to now.
9.2.1.1. Dose-response curves of capsaicin in the human stomach

acute observation
The dose-response curves were determined on the gastric basal acid secretion (BAO)
in healthy human subjects, and on the measurements in gastric transmucosal poten-
tial difference (GTPD) without and with topically (intragastrically) applied ethanol
(Mozsik et al., 2004 2005a, 2007b). We tested the effects of 100, 200, 400 and 800 pg/
100 ml saline solutions given intragastrically via a nasogastric tube on the gastric
BAO values, meanwhile the same doses of capsaicin given intragastrically (via
endoscopic channel of gastrofiberoscope) were tested on GTPD.
In other series of observations, the gastric microbleeding was produced by oral
(3x25 mg, plus 25 mg given at the start of examinations) indomethacin in healthy
human subjects. Indomethacin was given alone, or in combination with capsaicin
(200, 400 and 800 pg). The results were compared with the results obtained without
application of Indomethacin (baseline).
Capsaicin given in a dose smaller than 100 pg had no effect. The E D value5 0
was obtained at the 400 pg dose on the gastric basal acid output (BAO), gastric
transmucosal potential difference (GTPD) (without and with topically applied
141
ethanol), and Indomethacin-induced gastric microbleedings (Mozsik et al., 2005a,
2007b).
It was also observed that gastric microbleeding produced by inhibition of both
COX-1 and COX-2 was completely prevented by the application of 400 pg capsaicin
(Mozsik et al., 2007b; Sarlos et al., 2003).
When capsaicin was given in a dose of E D intragastrically, the "parietal compo-
50
nent" of the gastric acid secretion in healthy human subjects decreased (P<0.001),
meanwhile its "non-parietal components" (buffering secretion) increased signifi-
cantly (P<0.001) (Mozsik et al., 2004; 2005a; 2007b); gastric emptying was
increased (Debreceni et al., 1999). Recently it was observed that capsaicin (in a dose
of 400 pg orally) enhanced glucose absorption and glucagon release during the regu-
lar glucose loading test in healthy human subjects (Domotor et al., 2006).
9.2.1.2 Changes in laboratory parameters and complaints

of healthy human subjects during the study with capsaicin
No systemic laboratory changes were noted in the biochemical parameters, with the
exception of special observations with gastric juice (Mozsik et al., 2004; 2005a),
glucose loading test (Domotor et al., 2007) where specific changes were obtained in
the examined biochemical parameters.
No subjective complaints (pain, diarrhoea, vomiting) were observed.
9.2.2. Subchronic observations with capsaicin

in healthy human subjects
9.2.2.1. Two weeks treatment with capsaicin
..
The group of healthy human subjects received capsaicin treatment for two weeks
(3x400 pg given orally) in a prospective, randomized study. Gastric microbleeding
was produced by Indomethacin application before and two weeks after capsaicin
treatment. The baseline of blood loss, Indomethacin-induced gastric microbleedings
(without and with different doses of capsaicin) were measured before and two weeks
after the capsaicin treatment.
No changes were obtained in the baseline, Indomethacin-induced gastric micro-
bleeding, and on the other hand, the gastric mucosal protective effects of capsaicin
remained the same after the two-week capsaicin treatment as those at the beginning
of the two-weeks capsaicin treatment (Mozsik et al., 2005a, 2007b).
9.2.2.2. Biochemical measurements and complaints in healthy human

subjects during two weeks capsaicin treatment
No changes were noted in the biochemical parameters and no complaints were
reported in the healthy human subjects.
142
9.2.3. Human chronic observations with capsaicinoids
In a case-control study in Mexico City which included 220 cases of gastric cancer
and 752 controls randomly selected from the general population, chilli pepper con-
sumers were at a 5.5-fold greater risk for gastric cancer than non-smokers. Persons
who held themselves as heavy consumers of chilli peppers were at a 17-fold greater
risk. However, when chilli consumption was measured as frequency per day, a sig-
nificant dose-response relationship was not observed (Lopez-Carrillo et al., 1994,
2003).
In another case-control study in India, red chilli powder was found to be a risk
factor for cancers of the oral cavity, pharynx, esophagus and larynx compared with
population controls, but not with hospital controls (Notani, Jayant, 1987).
In an Italian case-control study, chilli was briefly mentioned as being protective
against stomach cancer (Buiatti et al., 1989). Chilli pepper, however, is not heavily
consumed in Northern Italy, where this study was conducted, and it is possible that
chilli consumption was correlated with other protecting spices such as onions and
garlic that are consumed in large quantities in Italy.
The Committee of Experts on Flavoring Substances of the Council of Europe con-
cluded that the available data not allow it to establish a safe exposure level of capsai-
cinoids in foods (Opinion of the Scientific Committee on Food on Capsaicin, adopt-
ed on 26 February, 2002).
It was also observed that the non-selective COX-1 and COX-2 inhibiting non-
steroidal anti-inflammatory drugs-induced gastric microbleeding can be completely
prevented by 400 pg capsaicin administrations in healthy human subjects (Mozsik et
al., 2007b).
9.2.4. Preventive effects of capsaicin against the selective

and non-selective inhibitory actions produced
by nonsteroidal anti-inflammatory drugs on COX-1
and COX-2 enzymes
Indomethacin (as a non-selective COX-1 and COX-2 inhibitor) was used to provoke
gastric microbleeding in healthy human subjects, and capsaicin itself, as a specific
stimulator at the capsaicin (TRVP1) receptor (given in small doses) was applied in
healthy human subjects. The capsaicin treatment was carried out for two weeks,
when it was given in 3x400 pg orally (400 pg capsaicin dose was found to be equal
to the E D value in different human observations) (Mozsik, 2006; Mozsik et al.,
50
1999; 2004; 2005a, 2007b) (Tables 27-28).
143
T a b l e 2 7 . C o m p a r i s o n o f i n h i b i t o r y effects ( I C ) of C O X - 1 a n d of v a r i o u s N S A I D s using h u m a n
5 0
platelet C O X - 1 a n d s y n o v i a l c e l l C O X - 2 *
NSAID Ratio COX-1 : COX-2
Aspirin 0.12
Diclofenac 38.00
Etodolac 179.00
Ibuprofen 0.86
Indomethacin 0.30
Loxoprofen-SRS 3.20
NS-398 1263.00
Oxaprozm 0.061
Zaltoprofen 3.80
* Kawai, S., Nishida, S., Kato, M., Furumaya, Y., Okomoto, T, Mizushima, Y. (1998): Comparison of cyclooxige-
nase-1 and-2 inhibitory activities of nonsteroidal anti-inflammatory drugs using humai'n platelets and synovial
cells. Eur. J . Pharamcol. 347: 87-94 (with permission)
T a b l e 2 8 . C o r r e l a t i o n b e t w e e n t h e c a p s a i c i n a c t i o n s , C O X - 1 a n d C O X - 2 systems a n d gastric
m i c r o b l e e d i n g s p r o d u c e d b y i n d o m e t h a c i n in h u m a n h e a l t h y subjects b e f o r e a n d after
2 w e e k s c a p s a i c i n (3x400 pg orally) treatment*
IC 5 0 v a l u e of i n d o m e t h a c i n t o ratio of C O X - 1 / C O X - 2 = 0.30
(1:3.25)
M i c r o b l e e d i n g in t h e s t o m a c h
- 2 w e e k s c a p s a i c i n treatment >
Before After
Baseline 2.1 0 . 1 ml/day 2.0 0.1 ml/day

After I N D 8.25 0 . 2 5 ml/day 7.8 0.3 ml/day
A I N D - i n d u c e d 6 . 1 5 0.2 ml/day 5.8 0.3 ml/day
( = inhibition on C O X - 1 + COX-2) ( = 1 0 0 % )
COX-1: 1.4470.1 ml/day 1.3640.1 ml/day
COX-2: 4 . 7 0 0 . 2 ml/day 4.440.2 ml/day
4 0 0 p g c a p s a i c i n ( I G g i v e n ) i n d u c e d d e c r e a s e of I N D - i n d u c e d gastric m i c r o b l e e d i n g
60.2 m L / d a y 5.90.2 mL/day
meansSEM in 14 healthy human subjects.

* Mozsik Gy, Szolcsanyi, J . , Domotor, A. (2007b): Capsaicin research as a new tool to approach of the human
gastrointestinal physiology, pathology and pharmacology (review). Inflammopharmacology 15: 232-245 (with
permission)
144
9.2.5. Summary of the observations with capsaicin alone or in
combination with selective and non-selective inhibition
of COX-1 and COX-2 enzymes by nonsteroidal
anti-inflammatory compounds (drugs) in animal
experiments and in human observations
Capsaicin does not represent only one chemically well identified compound, but
covers the same biological, physiological, and pharmacological actions as capsaici-
noids (capsaicin, dihydrocapsaicin, norcapsaicin, nordihydrocapsaicin). Capsaicin
(capsaicinoids) has (have) been widely used in the nutrition of the population of dif-
ferent countries in the last 7500 years.
It was an important internationally accepted discovery that capsaicin (capsaici-
noids) significantly stimulates (stimulate) a subgroup of the afferent nerves (named
as "capsaicin-sensitive afferent nerves") to different chemical agents, heat, pH gra-
dients in animal experiments and human observations.
The doses of capsaicin (capsaicinoids) are significant in the biological actions,
because when given in small doses it causes tissue protection (including the gas-
trointestinal tract), however, in larger doses enhances the organ's damage (including
the gastrointestinal tract). Capsaicin (capsaicinoids) in larger doses produces organ
damaging effects as a consequence of a capsaicin-induced desensitization process.
The existence of these principal observations was scientifically proved in animal
experiments and human observations.
The application of capsaicin has been carried out as a tool to approach the differ-
ent physiological and pharmacological regulations of different diseases and their
prevention.
The study of the afferent nerves has been carried out since 1970. The results of
this research clearly proved the principal and important role of the afferent nerves in
the physiological regulation of different organs as well as in the development of
damage and its prevention in these organs.
The human observations with capsaicin were carried out in randomized, prospec-
tive studies by the permission of Regional Ethical Committee of the University of
Pecs, Hungary, in accordance with the Good Clinical Practice (GCP) respecting the
Helsinki Declaration.
The results of the animal experiments and human observations clearly proved the
gastrointestinal protection by application of small doses of capsaicin.
The anti-inflammatory drugs (acting as selective and non-selective inhibitors of
COX-1 and COX-2 enzymes) are widely used in the prevention of thromboembolic
episodes, in the prevention of reinfarction in patients who underwent myocardial
infaction, in the treatment of patients with acute and chronic pains with degenerative
chronic joint diseases, malignant diseases and in healthy persons for the prevention
of gastrointestinal cancers, etc.
Capsaicinoids can be absorbed well from the gastrointestinal tract. They are
metabolized in the pathways of enzymatic as well as non-enzymatic oxidation of the
liver. It is very important that capsaicin alone dose-dependently prevents the hepatic
damage produced by carbon tetrachloride (in a significantly higher level than sily-
145
marin). The studies with genotoxicity clearly indicated that pure capsaicin has a
very limited toxicity (this value is higher if the capsaicin preparation is not chemi-
cally clear and is contaminated with different toxicological agents of plants) (afla-
toxin, pesticides, etc.).
The human observations also clearly indicated that capsaicin actions can be repro-
duced well in healthy human subjects. Capsaicin application produces a dose-depen-
dent increase in gastric transmucosal potential difference (without and with com-
bined ethanol), decreases the gastric acid basal acid output (BAO), and completely
prevents the Indomethacin-induced gastric mucosal microbleedings (in a range of
100 to 800 pg given intragastrically). Furthermore, two-weeks treatment with cap-
saicin (3x400 pg given intragastrically) did not modify the sensitivity of the cap-
saicin-sensitive afferent nerves in the gastric mucosa, and the gastric mucosal pro-
tective effects against Indomethacin remained as dose-dependent as those before
and after 2-weeks capsaicin treatment (Mozsik et al., 2007b).
The conclusions of these animal and human observations clearly proved that the
application of capsaicin given in small doses is able to completely prevent the nons-
teroidal anti-inflammatory drugs-induced gastrointestinal side effects. In other
words, capsaicin is able to inhibit the functions of the selective and non-selective
COX-1 and COX-2 enzymes in animal experiments and in human observations.
Further scientific research may offer an absolutely new pathway(s) for the devel-
opment of drug(s) by the stimulation of capsaicin-sensitive afferent nerves with
small doses of capsaicinoids for patients, who have to be treated with nonsteroidal
antiinflammatory drugs and for those with diseases in which the COX-1 and COX-2
enzymes are in over- (inflammations, tumors) or under- (after application of the dif-
ferent nonsteroidal anti-inflammatory drags) expressed.
146
10. Nature and characteristics
of the innovative drug research
10.1. Characterization of the innovative drug research
Our attention was focused on producing new drug combinations [aspirin, naproxen,
and diclofenac with capsaicin (capsaicinoids)] and capsaicin (capsaicinoids) alone
for the treatment of patients with different diseases.
It has been proven earlier that the natural capsaicin (capsaicinoids) - given in
small doses (nano and microgram/kg bw in animals) - prevents (prevent) the aspirin,
acidified aspirin (and other chemical agents, such as 96% ethanol 0.2 M NaOH, 25%
NaCl)-induced gastric mucosal injury, indomethacin (when it was given in basal and
in betanechol, pentagastrin and histamine-stimulated gastric secretory conditions),
acidified indomethacin-produced gastric mucosal damage in animal experiments
(Mozsik et al., 1997a; Mozsik 2006), and that the capsaicin (capsaicinoids) given in
hundred micrograms orally decreases (decrease) the orally applied indomethacin-
induced gastric microbleedings in healthy human subjects (Mozsik et al., 2005a,
2007b; Sarlos et al., 2003).
It is also known that aspirin as a selective cyclooxygenase-1 (COX-1) inhibitor
has to be administered for a long time or for the whole life time in patients with
myocardial infarction and later on for the prevention of their reinfarction (see guide-
lines: Fox et al., 2006). The beneficial effect of aspirin is based on the inhibition of
platelet aggregation. Myocardial infarction and reinfarction is a leading cause of the
mortality in the Hungarian patients.
It is also known that many people suffer from the different degenerative diseases
of the locomotor system, who have to be treated with NSAIDs also for a long time.
The number of these patients is also very high.
There is a great medical problem in the treatment that the very wide application of
NSAIDs is absolutely indicated from the points of cardiology, rheumatology, neu-
rology (stroke prevention), however, the application of these compound(s) is (are)
contraindicated from the view point of gastroenterology (based on the laws of evi-
dence based medicine, EBM). There is a general dilemma (and failure) to apply the
evidence based medicine in the patients' treatment plan. Furthermore, these diseases
occur frequently together in one patient.
The H2RA and proton pump inhibitors are widely used in the prevention of
NSAIDs-induced gastrointestinal side effects (GI mucosal bleeding and ulceration,
liver injury etc.).
Platelet aggregation can be produced by selective and non-selective COX-1 and
COX-2 inhibitors, but not by selective COX-2 inhibitors. These facts give the phar-
147
macological basis of the primary and secondary prevention of myocardial and brain
infarction.
The discovery of capsaicin-sensitive afferent nerves opened an absolutely new
gate in the physiology, pharmacology, pathology, when Szolcsanyi and Bartho
(1982) had clearly proved that the stimulation of capsaicin-sensitive afferent nerves
by application of small doses (pg/body weight) of capsaicin prevented the gastroin-
testinal mucosal damage produced by different chemical and noxious agents (for the
details see Chapter 8).
Capsaicinoids are essential compounds from the spices, which have been widely
used in the cuisine from the old ancient time up to now.
The mechanisms of gastrointestinal mucosal protection, produced by capsaici-
noids, have been extensively studied in animal experiments (Mozsik et al., 1997,
2007b; Domotor et al., 2005, 2007). The application of capsaicin (orally given in
micrograms doses) prevented the indomethacin- and ethanol-induced gastric mucos-
al damage in healthy human subjects (Mozsik et al., 2005a). The results of studies
with capsaicin in animal models and healthy human subjects offered a possibility to
conclude that the beneficial effect of application of small doses of capsaicin can be
probably used (as therapeutic tool) against the different noxious agents-induced gas-
trointestinal mucosal damage in healthy humans beings and patients. Our aim(s) was
(were) to produce new pharmaceutical preparations (capsaicin alone) or drug com-
bination^) (capsaicin plus aspirin, diclofenac and Naproxen).
The physiological and pharmacological regulatory mechanisms of actions became
more clear for medical research people, however, we had not enough knowledge in
the fields of pharmaceutical technology [industrial production of drugs; necessary
circumstances around the new drug (or drug combinations)] research approach of
the necessity the new drug (or drug combinations) composition; production of the
preclinical dossier; production of drug master file (DMF); organization and carrying
out of stability tests of a new component (or drug combinations); preparation of doc-
umentation to receive the permission of the National Institute of Pharmacy of Hun-
gary and National Clinical Pharmacological and Ethical Committee to start with the
human Phase III studies]. We had a special challenge in this field, because cap-
saicin (as mentioned earlier, in the chapter on research) is not uniform chemically.
The successful solution of these scientific, technical, industrial questions requires
very complicated and administrative work. This type of work suggests the a priori
establishment of a multidisciplinary research team. None from our research team
described below could have surveyed alone all the aspects of the above-mentioned
scientific, drug industrial laws, many national and international regulatory laws and
administrative fields.
21 researchers have participated in this "innovative drug research" from 2005;
they are the members of the Department of Pharmaceutical Chemistry, First De-
partment of Medicine, Department of Ophthalmology, Medical and Health Centre,
University of Pecs, Hungary and PannonPharma Ltd., Co., Pecsvarad, Hungary,
respectively. The university people represent the basis of research work, while the
participants from the industrial partners (PannonPharma Ltd., Co, Pecsvarad) give
the basis of technological and industrial research, including the final production of
new drug or drug compositions and their marketing.
148
The ages of researchers are between 23 to 69 years (in average 36 years). There
are 11 women and 10 men among them. They are physicians (internist, ophthalmol-
ogist), chemists, chemical engineers, biologist, bioengineers, pharmacologist, and
horticultural engineer. Some of them have different scientific qualifications [Ph.D.,
Sc.D. (med), Ph.D. (chemists, physicians, pharmaceutists)], and two Ph.D. students
joined our work team.
This "multidisciplinary team" has enough practice in clinical pharmacology, med-
icine, ophthalmology, drug research and industrial research.
The participants of this team never worked together before receiving this grant.
The time for the execution a successful study in this "innovative drug research"
covers only a four-year period. Consequently we had a very short time to acquire the
necessary knowledge from the medical, chemical, industrial, pharmaceutical chemi-
cal fields and from their experts
We had no other possibility as:
1. To identify the scientific research organization, the main steps of necessary
studies to be carried out by our "innovative drug research" (from the basic
research to reach to the drug production for human beings) during the four-year
period;
2. To receive correct information on the depth of the special knowledge from the
different members of participants;
3. To create a "functional map" on the knowledge of the participants, which is used
to give information for the others;
4. To pick up the basic and related knowledge from all the fields involved in the
study;
5. To establish a well functioning equilibrium between the researchers working in
the different university institutes and their industrial partners;
6. To follow permanently the new results of the international research;
7. To study the national (Hungarian) and international (mainly European Union)
laws existing in the field of new drug production;
8. To read many scientific papers;
9. To organize regular (monthly) meetings for the evaluation of the scientific prob-
lems together with all the participants, who are involved in the study;
10. To meet the main research people every week (before and after the above men-
tioned meetings), to discuss the further necessary steps;
11. To collect the necessary knowledge from the neighboring fields for our "innova-
tive drug research";
12. To learn the main lines and the main results of the national and international
research connected to our fields;
13. To find the different pathways, which are suitable to stimulate the work of the
participants;
14. To give absolutely new information from the different fields to all the members
of the study participants;
15. To prepare ourselves to solve creatively problems of earlier unsuccessful scien-
tific (and perhaps other) activities to receive results for innovative research.
The "innovative drug research" is an extremely hard interdisciplinary work, which
cannot be done successfully without the acceptance of the national and international
149
trends and laws. We had to recognize our real possibilities (including the spiritual,
technical and economical aspects).
No clear internationally accepted definition exists for the "innovative research".
The main point is probably to summarize the possible pathways in the research. Our
aim was to find the pathways for solving the different (interdisciplinary) problems
successfully in a relative short time, and the participants of innovative research who
are able to help the works of each others.
We never worked together, consequently we had no common experience on how
we can solve these types of current scientific, technical, industrial, marketing chal-
lenges. So, we had to pick up the necessary knowledge from the different fields, and
to learn from each other permanently, from day to day.
The author of this chapter (Gy. M.) participated in many national and internation-
al research programs (in the establishment of clinical pharmacology, clinical nutri-
tion, peptic ulcer research, drug research, medicine, producing new foods, molecular
and biochemical pharmacology) and gained significant experience in the fields of
experimental and clinical research. Nevertheless, he learned more than he did earlier
during the "innovative drug research" (furthermore in a very short time) (pharma-
ceutical chemistry, general chemistry, plant physiology, technological problems,
questions of publication, industrial research, research organization, harmonization
between the academic and industrial research, the national and international new
results and laws, etc.).
There was a special feeling in the organizer(s) and participants during our innova-
tive drug research. We learned the different pathways of problem- and success-ori-
ented works (under the different, and in some instances difficult, national and inter-
national conditions). We had about half a year to learn and accept this line of the
"innovative drug research".
The following main steps were carried out in this "innovative drug research" from
2005 to 2008:
1 .Preparation of experts' opinion;
2. Introduction of the necessary chemical, pharmaceutical, drug industrial methods
for the quantitative measurements of the chemical components;
3. Plant origin capsaicin (capsaicinoids) is (are) used as drug compound(s), so prepa-
ration of Drug Master File (DMF) is basically necessary, and it has to be used in
the preparation of the preclinical documentation(s) of the planned drug (or drug
combinations);
4. We read many original printed papers or written information;
5. We studied very carefully the new scientific results in our field, which were pub-
lished in other parts of the World;
6. We had many consultations with the National Institute of Pharmacy (Hungary) on
the chemical purity of the capsaicin (capsaicinoids), on the possibility of its use as
an effective material to produce a new orally applicable drug (or drug combina-
tion) in humans;
7. We became acquainted with a lot of pharmaceutical laws in the production of new
drugs (or drug combinations), which finally will be used during the industrial pro-
duction of the "ready drug (or drug combinations)";
150
8. We had to learn the national and international difficulties of the production of
Drug Master File (DMF) (including the problems existing in the USA, Europe,
and India);
9. The patent research was carried out by our industrial partner in this study, the
Technological and Transfer Office of the Consortium.
10. We prepared three Hungarian (P0700779, P0800602 for itraconazole and
P0700755 for capsaicin) and two PCT-safe (Patent Cooperation Treaty)
(PCT/HU 2008/000144 for intraconazole and PCT/HU 2008/000136) patents
from our work, which were accepted by the Technological and Transfer Office
of our University, while the final acceptance of these three patents by Patent's
Office is in progress;
11. The Clinical Pharmacological Unit of our Department (First Department of
Medicine, Medical Faculty, Pecs University, Hungary) was qualified by the
National Institute of Pharmacy (Hungary);
12. We permanently wrote our appraisement of new results, documentation, notes
on the meetings or other new important information. We noticed the actual state
of the problems, and we indicated continuously the forthcoming steps and their
responsible persons in a register;
13. We prepared the overview of the annual works in Hungarian and English lan-
guages;
14. We had permanent contact to the National Patent' Office in order to obtain the
final acceptance of our three patents;
15. We carried out many consultations with the different experts and national (or
international) authorities on our actual problems;
16. We were open to all new problems and all of the possible solutions;
17. We considered the possibilities of our survival after the closing of this grant (we
planned to establish different spin-off firms);
18. We published the results of the new experimental and clinical research;
19. We produced a plan to the end of 2010 for the participation in the international
science (by the organization of different international symposia, writing books
upon international invitation).
In the forthcoming part, we will emphasize some interesting and important prob-
lems, which appeared during our "innovative drug research":
1. We had many problems with learning and understanding the details of the scien-
tific knowledge of the individuals participating in this project, who received sig-
nificantly different scientific education and participated in different practices (at
different levels) on their fields. A period of nearly half a year was necessary for
understanding each other. After this period, our correspondences and personal
communication represented the main sources of our new knowledge. These events
offered a new pathway for the continuous learning for all of us;
2. We recognized that the international use of "capsaicin" in the research does not
represent a chemically uniform entity (because it means a mixture of a few com-
pounds). The "capsaicin" preparations, which were mainly used in the research,
contained at least three components: capsaicin, dihydrocapsaicin and nordihydro-
capsaicin.
151
The references in the different scientific papers mention that the "capsaicin" was
obtained from Sigma, Aldrich or Sigma-Aldrich. Interestingly the scientific papers
(except for a few ones) never mentioned that the researchers use a chemically not
uniform "capsaicin" preparation in their experiments.
Capsaicin transforms into dihydrocapsaicin chemically, which produces the same
physiological responses as capsaicin in the different experimental conditions (Burk,
Burks, 1986).
We registered this interesting event, and we initiated special consultations with
the experts of the National Institute of Pharmacy (Hungary) on the official chemical
composition of "capsaicin"(capsaicinoids) from the aspects of "innovative drug
research".
Since capsaicin (capsaicinoids) is (are) prepared from the plants, we asked the
"official authorities" how we can use it (them) as pharmaceutical sources for the
production of orally applicable drug(s) in the human medical therapy.
The constant composition of "capsaicin" is a basic requirement independently
from sources of "capsaicin preparation". That was the answer of the experts of the
National Institute of Pharmacy (Hungary). We respected the opinion of the
Hungarian authorities (as national) and international laws regarding the chemical
purity of "capsaicin preparation".
3. Capsaicin can be obtained from the plants of Capsicum (see Chapter 2) over the
World. The main producers of capsaicin preparations are India, China, Central and
SouthAmerica, and in some extent the European Countries (including Hungary).
The cultivation of domestic Capsicum and the wild Capsicum is done in signifi-
cantly different ways in the different parts of the World. The application of different
chemical compounds for increasing the quantity of capsaicin from Capsicum plants
is widely and internationally accepted.
We obtained Internet information about the pesticide contamination of sweet bell
pepper (see on the website www.ewg.org the "Report Card: Pesticides in Sweet Bell
Peppers") (Table 29):
Table 2 9 . S w e e t Bell Peppers w i t h the most pesticide residues (Report card) (www.ewg.org)
S w e e t B e l l Peppers w i t h t h e most pesticide residues
Sample 1 Sample 2 Sample 3
Acephate A z i n p h o s methyl T Acephate T
Dicofol Carbaryl T Carbaryl
Dimethoate T Endosulfans T Dimethoate T
Diphenylamine (DPA) T Malathion Endosulfans T
Fenvalerate Methamidophos T Methamidophos T
Metalaxyl Methomyl O-Phenylphenol T
Methamidophos Oxamyl Oxamyl
Methomyl
P e r m e t h r i n Total
- Animal Carcinogen; - Causes Birth Defects in Animals; V- Damages Reproductive System;

- Interferes with Hormones; T- Damages Brain and Nervous System; - Damages Immune System
Pesticides were found on 68% of the sweet bell peppers tested.
There were 39 pesticides found in sweet bell peppers: Acephate, Azinphos methyl,
Benomyl, Bifenthrin, Captan, Carbaryl, Carbofuran, Chlorothalonil, Chlorpro-
pham, Cyfluthrin, DDT, Diazinon, Dichlorvos (DDVP), Dicloran, Dicofol, Dime-
thoate, Diphenylamine (DPA), Disulfoton, Endosulfans, Ethoprop, Fenvalerate,
Folpet, Iprodione, Lambda cyhalothrin total, Lindane (BHC gamma), Malathion,
Metalaxyl, Methamidophos, Methomyl, Metribuzin, Mevinphos Total, Myclo-
butanil, O-Phenylphenol, Oxamyl, Oxydemeton methyl, Permethrin Total, Pipe-
ronyl butoxide, Pronamide, Thiabendazole.
The three pesticides found most often in sweet bell peppers were Methamidophos,
Acephate, and Endosulfans (Fig. 30).
Number of Percent of Samples

pesticides
per sample
22%
Fig. 30. M o s t S w e e t B e l l P e p p e r s a r e c o n t a m i n a t e d w i t h m o r e t h a n o n e p e s t i c i d e .
Foodnews Enviromental W o r k i n g Group//foodnews.org
http://www.drgreene.org/body.cfm?xyzpdqabc=0&id=21 &action=detail&ref=l 9 2 0 "
Furthermore, the national and international controls of the applicable chemical

components are also significantly different. For example, the use of organic phos-
phate components was prohibited in the European countries, but not in India or
China.
So, the exact elucidation of the use of different components during the cultivation
of Capsicum plants is a key question.
The Department of National Chemical Safety (Bordas, 2006) published a book
on the classification of dangerous abatement means and their dose limits. Further-
more, this book summarized the chemical compounds used in the cultivation of the
plants.
153
Looking at this information concerning the toxicological conditions in the cultiva-
tion of plants, we had a new challenge to understand the conditions of cultivation of
Capsicum plants in the different countries (India, China, U.S.A., Hungary, etc.).
We received correct information on the official requirements of the use of the dif-
ferent chemicals (including the national and international recommendations) during
plant cultivation. However, we had no correct information on the usually applied
chemicals in the plant cultivation abroad.
The National Institute of Pharmacy (Orszagos Gyogyszereszeti Intezet, OGYI)
asked us to repeat the toxicological examinations (see toxicological chapter) with
our capsaicin preparation (including the chronic toxicological studies in rats and
beagle dogs). This request of the National Institute of Pharmacy surprised us,
because this meant extra work from the point of both using capsaicin for drug pro-
duction and repeating the of toxicological examinations carried out earlier. We per-
formed, of course, theses studies in the last year successfully.
How did we control these problems? The control of our activities is a very com-
plicated and hard job.
One of the most important pathways is to follow the different steps of plant culti-
vation, and thereafter the steps used in the processing of the plants. The collection
and qualification of the main steps is an essential requirement, and these factors are
the key-points in the Good Manufactorial Practice (GMP). The collection of this
information in these questions seems to be theoretically very simple, but in the prac-
tice we have no real possibility to follow and to collect all the written documentation
given by nationally (or internationally) accredited authorities.
How did we meet with these problems in our "innovative drug research"?
Earlier we tried to collect these data from the international trade firm "Sigma-
Aldrich". We have no correct and sure information on the capsaicin supplying coun-
tries for this trade firm. The mentioned data were absolutely necessary to prepare
Drug Master File (DMF). The DMF is absolutely necessary for the preparation of
the preclinical dossier before the preparation of the protocols for carrying out the
human phase III clinical pharmacological studies.
We realized with surprise that Sigma-Aldrich had no DMF for capsaicin. We
received the same information on these questions from the other trade firms con-
cerning capsaicin supplying.
We studied thoroughly the circumstances of the cultivation of the Capsicum in
Hungary. The exclusion of organic phosphate compounds from the agricultural and
horticultural practices was absolutely necessary. Concerning this, we could not obtain
correct data from the circumstances of cultivation of Capsicum. We had the same
problem with the capsaicin coming from India. The DMF of capsaicin originated
from India is signed in the documentation of Food and Drug Administration (FDA) in
the United States ("17856 A II26.10.2004 Asian Herbex Ltd: Capsaicin USP as man-
ufactured in Andhra Pradesh, India") as existing registration, which can be applied to
the use of capsaicin as basic source for the capsaicin-containing drug. We could not
understand that the results of capsaicin research increased extremely in the last years,
while only one registration of capsaicin DMF was signed by the date 26.10.2004.
over the World, registration number: 17856 A II. We followed closely the different
pharmaceutical firms over the World in the literature, however, we could not find an
154
answer as to why these firms did not use this source DMF from the Food and Drug
Administration of the USA.
It has been concluded that we need to find another pathway of the capsaicin source
for basic drug material for the production of the capsaicin containing drug (or drug
combinations). Thereafter, we returned to receiving the opinion from the experts of
the National Institute of Pharmacy (Hungary) on the possibility to surmount these
problems.
4. New consultation was done with the experts of the National Institute of Pharmacy
(Hungary) on the acceptance of capsaicin compound as basic drug material with-
out existing DMF of the capsaicin preparation obtained from Hungary.
We followed the application of the agricultural chemistry during the cultivation
of Capsicum plants (e.g. the application of organic phosphorus compounds, as pes-
ticide agents, was prohibited from the last decades). Other chemicals are also used
in plant cultivation and processing. The list of the chemicals, permitted by the
Institute of National Chemical Safety was collected and published (Bordas, 2006).
Up to this moment we could not receive a reliable DMF from the workers of
Capsicum cultivation.
We mentioned that the ordering of the basic drug compound is the task of the
industrial partner. We tried together to discover different firms supplying us with the
basic drug compound for our research work. We found a firm from India for supplying
us with capsaicin, but we did not receive from them the correct DMF. Of course, the
registration of DMF was electronically found in the collection of DMF by the Food
and Drug Administration in the USA. We received a new possibility to obtain cap-
saicin from this Indian firm.
An extremely important law was in the "innovative drug research" as follows: we
had carry out all the observations (chemical analysis, stable observation, toxicologi-
cal studies, drug formulation, human phase III clinical pharmacological studies)
with the same capsaicin preparation (charge).
In the meantime, we obtained 3 to 4 kg capsaicin (without DMF) from a Hungarian
firm. This amount of capsaicin was obtained from different charges of Hungarian pap-
rika (Capsicum), however, it was supplemented with oleoresin imported from India.
The chemical composition of this amount of capsaicin (as basic drug component)
can be chemically and toxicologically studied by the different accredited University
Institutes. We received permission from the experts of the National Pharmaceutical
Institute for preparation of DMF of the Hungarian capsaicin by the pathways above,
however, we were not able to follow the different steps of Capsaicin cultivation.
Why did we start this way? In the original agreement it was finalized that the
Hungarian cultivated Capsicum will be used by our research team in March of 2006,
and at the end of 2006 we had no capsaicin drug basic component.
In the meantime, the capsaicin supplying basic drug compound (capsaicin or cap-
saicinoids) changed from the Hungarian firm to the Indian firm in October, 2006.
On the other hand, we lost two years' time from our grant.
We had only two further years, and nearly one year was necessary for doing the
chronic toxicological examinations in animals, moreover, we could not write the
protocols for human phase III studies because of the absence of international DMF
for capsaicin.
155
The official permission from the National Institute of Pharmacy (Hungary) and of
the National Clinical Pharmacological and Ethical Committee of Hungary is basical-
ly necessary before starting the human phase III clinical pharmacological studies.
The mentioned events clearly demonstrated that we would be having many differ-
ent works in the same time period.
5. We met new events practically every day. On the other hand, all of us gave new
information to the others orally and in writing. There was a very effective regular
consultation between the university researchers and industrial researchers. Written
notes were prepared on all of consultations. We informed all of the members of
our research team permanently. This type of research is very exciting, multidisci-
plinary, responsible work. Consequently, we had to help all the participants in this
research work independently of our primary place and education. Because the
researchers are 23 to 69 years old, our common meetings represent a special edu-
cation form for the younger generation.
6. We learned at an early working stage of this grant that we have to ask help from
other experts.
7. There was a special occasion when we performed so-called "research of the pa-
tent" from the original Hungarian and foreign languages. It was a specially exci-
ting work, because our idea on the preparation of new drug (or drug combination)
is a really original discovery.
8. How can we characterize the behaviors of participants, who were involved the
innovative drug research"?
We try to collect the most important characteristic steps of our "innovative drug
research":
- The research is very quick and timed;
- The participants are intellectually at a high level;
- The participants are able to pick up a significant amount of knowledge from the
other experts of the participants, as well as from other experts;
- The participants are able to accept the opinions of others;
- They are able work quickly and use foreign language(s);
- They have good personal contacts with the people working at different levels of
research, national scientific societies, national offices or institutes which are
responsible for making decisions;
- T h e y have good contacts with foreign experts;
- They have enough practice to stimulate the work of all of us;
- They have a responsibility to accept the results of the work of others;
- They want to receive good results together;
- They are able to win acceptance for the ideas necessary to achieve our goals;
- They are able to keep their perseverance in accepting the opinion and ideas of each
other;
- They are able to learn from each other from day to day;
- They have a good dynamism in their personal contacts and in discussions;
- They have to be suitable persons for doing creative and innovative research works
(including the establishment of new ideas, problems orientated field, realization of
the research, organization, realization of the animal observations in the human
studies).
156
11. Pharmaceutical industrial research
and development
Different special terminologies are widely used in the everyday industrial pharma-
ceutical research and development (Table 30).
T a b l e 3 0 . A b b r e v i a t i o n s u s e d in t h e e v e r y d a y p h a r m a c e u t i c a l industrial r e s e a r c h
and development
API A c t i v e P h a r m a c e u t i c a l Ingredient
BMR Batch Manufacturing Record
BSE/TSE B o v i n e S e r u m Encephalitis/Transmissible S e r u m Encephalitis
CAS C h e m i c a l Abstracts S e r v i c e
CU Content Unformity
FP Finished Product
CLP G o o d Laboratory Practice
cGMP current G o o d Manufacturing Practice
ICH International C o n f e r e n c e o n Harmonisation
IHS In H o u s e S t a n d a r d
MBD Master Batch D o c u m e n t
MQD Master Quality D o c u m e n t
NMT Not M o r e Than
NSAID N o n Steroid Antiinflammatory D r u g
PH EUR European Pharmacopoeia
PMF P r o d u c t M a s t e r File
QA Quality Assurance
RET R e g i o n a l C e n t r e of S c i e n c e
RS Relative Substances
USP U n i t e d States P h a r m a c o p e i a
Preamble
In the RET cooperation Pannonpharma's role is the industrial partnership, joining in

at the start of the project and being active continuously to the end. We assume that
scientific basic research ends when its results become common practice. We think
157
that the result will be a registered medicinal product for the cure of patients who
expect their recovery from new medicines.
Although this partnership is quite new and even seemed to be a historical step in
the region, all members thought of the project as an innovative drug-research - thus,
a useful and path-finder job.
Pannonpharma Ltd. (Pecsvarad, Hungary) has realized its importance quite
enough, taking into account especially the company's young background. Not
only the business, but the challenge of the project impressed all of us, while we
believed in the chance of our first regional cooperation.
1 1 . 1 . Product design and development
As the references and partners' scientific results certified the pharmacological activ-
ity of capsaicin, confirmed in several promising and convincing animal tests (Ref.
former sections), a detailed evaluation was decided by the team in order to start a
new product development.
The main aspects of the decision were clarified by pharmacologists and clinicians
aiming at a clear pharmacological intention, while the details were worked out
together.
11.1.1. General goals
Principle of the research and development:

- QUALITY - should cover conformance of all chemical and pharmaceutical data
and requirements (reference in this section).
- SAFETY - should cover conformance of all non-clinical data and requirements.
- EFFICACY- should cover conformance of all clinical data and requirements.
All activities should be done in conformance of QA requirements (cGMP),
Legal protection of the product should be required,
Oral and written publications should be taken into account.
11.2. Chemical-pharmaceutical aspects of the product

development
The selected formulation (e.g. pharmaceutical form) is summarized in Table 31.

Having the form, questions have been grouped for clarification as follows:
158
11.2.1 Pharmaceutical form
Details are presented in Table 31.
T a b l e 3 1 . S u m m a r y of d e t a i l s of p h a r m a c e u t i c a l industrial data
yes no comment
1 . a c o m m o n tablet n e e d e d , or retard b y a c t i o n common
2 . t h e tablet s h o u l d b e c o a t e d or u n c o a t e d coated
3. if c o a t i n g is n e e d e d , w h a t t y p e of c o a t i n g :
sugarcoated
filmcoated V
4. w h a t is targeted by c o a t i n g :
aesthetic f i l m
colored film red
other f u n c t i o n a l role, s u c h as:
m a s k i n g taste/odor
p r o t e c t i o n against s t o m a c h d e c o m p o s i t i o n
i n c r e a s e stability
5. Tablet size medium

average weight appr. 3 0 0 ^ 0 0 m g
diameter 10-11 m m
shape round, biconvex
In consultation with clinicians a tablet form has been agreed, which disintegrates in
the stomach, and acts in place. It was made clear that the disintegration time should
be not more than 30 minutes. On the other hand, film-coating had to be selected
because of capsaicin's pungent taste. The color of the film-coating tablet could be
selected practically freely, and the coating film was expected even to serve for
enhancing the stability.
The size of the tablet size was determined not only simply by its active content,
but also taking into account combination variations of the NSAIDs' active pharma-
ceutical ingredients.
11.2.2 Composition and quality of starting materials
Details of these questions are presented in Table 32.

The quality of the starting materials was required to meet pharmacopoeial stan-
dards of the European Pharmacopoeia (2007): Fifth Edition, Council of Europe
Strassbourg (PH EUR 5.7) and The United States Pharmacopeial Convention, INC.
(2003): 12601 Twinbrook Parkway, Rockwille (USP 27-NF 22).
159
T a b l e 3 2 . C o m p o s i t i o n a n d q u a l i t y of starting m a t e r i a l s ( p r o d u c t s ) *
yes no quality
1 API USP27-NF22
2 Excipients PH EUR 5
Binder PH EUR 5
Disintegrant PH EUR 5
Lubricant PH EUR 5
Clidant PH EUR 5
3 C o a t i n g m a t e r i a l s (mixture) IHS
* see Table 30, for additional explanation
11.2.2.1. Active Pharmaceutical Ingredient (API) of the product
The API of the product, capsaicin, is not a homogenous substance, thus it is better to
call it capsaicinoids.
By chemical structure it is often called as pseudo alkaloid or proto alkaloid too.
The basic part of the 5 components' chemical structure is vanillyl-amide, and the dif-
ferences of the capsaicinoid components' structure are in the substituents. {Chemical
coterminus of capsaicin: 6-Nonenamide, (> Af-[(4-Hydroxy-3-methoxy-phenyl)
methyl]-8-methyl. ()-8-Methyl- Af-vanillyl-6-nonenamide, (Ref. USP, Ref. 6.2.3).}
In the formulation we used USP quality of capsaicin ("capsaicin natural", CAS
Number: 404-86-4), since PH EUR does not involve capsaicin paragraph (USP 27-
NF 22, THE UNITED STATES PHARMACOPEIAL Convention, INC. 12601
Twinbrook Parkway, Rockwille , 2003). API supplier was audited and they released
a copy of PMF for capsaicin.
The pharmaceutical characteristics of the substance:
Properties:
- Characteristics:
- pale brownish-yellow, amorphous powder
- intensive and irritating odor even in very low air-volume concentration
- Morphology:
- particle size is not homogeneous, it is varying in a wide range between 0.05-2 mm
(like a usual vacuum-dried extract)
- Polymorphism:
- no polymorphism of the substance is known
- Solubility:
- good in methanol, ethanol and acetone, practically insoluble in water
Chemical properties: testing and specification is upon USP 83/2001 EC Guide
(Directive of the European Parliament and of the Council on the Community code
relating to medicinal products for human use) (Handbook of Pharmaceutical
Excipients 5th Edition, edited by Raymond Rowe, Paul Sheskey and Paul Weller,
USA, October 2005).
160
11.2.2.2. Excipients' quality of P H EUR requirements
Excipients' quality was meeting PH EUR requirements, involving the BSE/TSE

aspect, when relevant, as well. (Directive of the European Parliament and of the
Council on the Community code relating to medicinal products for human use (2001)
(83/2001 EC Guide).
Excipients used in formulation - as above -were expected to be chemically
- indifferent,
- to serve the solid dosage form requirement (Handbook of Pharmaceutical Exci-
pients 5th Edition (2005): Eds: Rowe, R., Sheskey, P. and Weller, P., USA, October
2005)
- not to involve lactose in order of protection of lactose-sensitive patients
As from view of formulation, the excipients must have proper physical-, physical-
chemical characteristics for powder rheology in order to assure good homogenity,
fluidity and lubrication for compression.
11.2.2.3. Coating powder mixture quality
Coating powder mixture quality is by IHS, formulated for serving the above-men-
tioned purposes.
Its red color was aiming at some association with the Hungarian paprika.
Additionally, for suspension phase or solvent of capsaicin purified water was used
exclusively in order to decrease the risk of organic residual.
11.2.3. Packaging
Details of the packaging are shown in Table 33.
T a b l e 3 3 . S u m m a r y of a s p e c t s of p a c k a g i n g
yes no
1. Primary
blister V
PVC/ALU
PVC/PVdC/ALU
C o l d blister
vial
2. Secondary
carton box V
p a c k a g e insert
booklet
3. D e s i g n
design i n v o l v i n g b l i n d - c o d i n g
* abbreviations: PVC, Polyvinyl Chloride

PVdC, Polyvinylidene Chloride
ALU, Aluminium
161
A good packaging retains the products' quality, is easy to handle by patients and
externally serves some aesthetic aspects.
Blister packaging has been selected, because it was an individual packaging by
dosage form, and the indicated packaging materials were resistant against humidity,
thus supporting the stability of the tablet.
11.3. Formulation
11.3.1. Formulation steps
In the first step a lab-scale batch (appr. 3 kg) was planned to be produced, in order to
confirm the planned composition, then a pilot batch (100 000 tablets) was targeted.
As we have worked out the process in detail, FP Specification has been set up after
the complex testing of the tablets.
11.3.2. FP Specification
The details of the FP are listed in Table 34.
T a b l e 3 4 . F i n i s h e d p r o d u c t ( F P ) of t h e s p e c i f i c a t i o n s a r e s u m m a r i z e d in this T a b l e . *
Aspect Requirement
Description Characteristics R e d c o l o r e d , b i c o n v e x f i l m tablet w i t h s m o o t h surface
Dosage form - Dimensions:

- height Practical average 1 0 % (mm)
- diameter 10 3 % (mm)
- shape biconvex
- average weight T h e o r e t i c a l a v e r a g e 1 0 % (g)
- individual weight P r a c t i c a l a v e r a g e 1 0 % (g)
- disintegration time 30 min.
- friability NMT 1 %
-CU 85-115%
Composition - Identity:
-API positive
- binder positive
- lubricant positive
- glidant positive
- assay:
- capsaicin 85-115%
I m p u r i t y test - RS Total R S N M T 2%
- Microbiology III.A. - P H EUR
* see Table 30, for other additional explanation
162
11.3.3. Samples for clinical trial
After manufacturing and testing the samples taken from the pilot batch and evalua-
tion of the BMR, Master Batch Documents and Master Quality Documents have
been created for approval of QA. All the other SOPs, safety rules involving any other
aspect of producing a clinical sample have been created formerly and authorized to
achieve quality assurance requirements as well.
Then, a new pilot batch was planned to be produced for two purposes:
- t o take samples for clinical trial (11.3.3.1)
- to take samples from the same batch for stability testing of the formulation
(11.3.3.2)
11.3.3.1. Preparation of samples for clinical trial
Samples for clinical trial were planned to be taken after the release of the batch. The
number of the samples was calculated on the basis of the Clinical Protocol, their
packaging and coding were compliant with GMP regulation (Eudralex Volume 4
(2007): Good Manufacturing Practice, European Comission).
11.3.3.2. Exposing, stability and packaging of the planned final product
Samples for exposing and stability testing were packed in the same packaging as the
planned final packaging.
The product's stability is a main quality requirement. It means that the product
packed in its final form retains chemical, pharmaceutical stability and pharmacolo-
gical activity within a certain range which was set up in the finished product specifi-
cation. (If there is any reasonable reason, some requirements of the release and
expiry specification may be different within a certain range.)
A complex stability project has been created for exposition and testing by ICH
Guides in order to determine expiry specification and expiry date of the product as
well (International Conference on Harmonization (ICH) (2003) (Ql, Q8), Brus-
sels).
11.4. Plant batch manufacturing and process validation
Scaling up for plant batch is almost the final step of a development project. It was
planned and achieved by the collected information and data of the former steps. On
the other hand, planning of the process involves the site manufacturing machinery
and technical supplying system too. Taking into account all of the above, batch size
could be determined easily.
After plant batch manufacturing and quality control of the taken samples a final
report evaluates the conclusions of scaling up. Process validation and cleaning vali-
dation must be achieved upon written plans, then evaluated in a written report for
163
justification of the process and its parameters. Validation batches are also involved
in stability project.
Cleaning validation is the last important plant activity belonging to the validation
procedures, while certifying the proper method of cleaning after operation. Approved
validation reports usually confirm not only the processes, but the MBDs and MQDs
belonging to the as well.
1 1 . 5 . Summary of the Chemical-Pharmaceutical

development
The chemical-pharmaceutical part of the products' dossier is one of the main 3

detailed sections.
This is for certification and documentation of all activities of the product
development from pre-formulation to plant batch production on the basic prin-
ciple of QUALITY.
For certification of the above, we assume the next points to be relevant:
- written plans are required (involving formulation and processes) for the dif-
ferent steps of development,
- standard quality and test methods of the used starting materials are required,
- standard methods are required for quality control of products at different
levels of development,
- development activity must follow QA rules, especially in documentation:
- basic documents must have stability control,
- plans must be performed, and evaluated in correct reports,
- consecutive steps must follow an approved report and conclusion of the for-
mer step,
- records must be controlled and approved,
- the plant batch size production and the process belonging to it must be vali-
dated,
- a required number of batches must be subject to stability testing,
- in the final evaluation, i.e. in the Chemical and pharmaceutical expert report,
expiry date, storing condition and other notes must be clarified and declared
unambiguously.
The planned appearance of capsaicinoids containing pills is seen in Fig. 31.
Fig. 31. T h e v i s u a l c h a r a c t e r i z a t i o n of c a p s a i c i n o i d s c o n t a i n i n g pills

(see t h e p i c t u r e in c o l o r s o n p. 2 5 5 )
164
12. Clinical pharmacological studies
with capsaicinoids alone and with
combination of capsaicinoids with
nonsteroidal anti-inflammatory drugs
12.1. Main aims of clinical pharmacology and its relation

to the evidence-based medicine
The evidence-based medicine (EBM) was established as the basis of the activity of
medical treatment, in the everyday medical practice. The general aims of this EBM
are to give exact and clinically well proved evidences for making correct diagnoses
and for the treatment of patients suffering from different diseases.
In the last 3-4 decades (since 1970) only the "problem orientated medicine
(POM)" has been emphasized in the graduate and postgraduate medical education.
This internationally well accepted trend has been applied in the medical education
and in the diagnostics of diseases in patients.
The final aims of our medical activity are to give scientifically well proven med-
ical treatments (including the surgical treatment, treatment with different drugs, etc.)
for the patients. The drugs have a key role in the medical treatment.
It was earlier suggested that the medical treatment can be based on the:
1. classical "old" medical experiments (e.g. Chinese medicine) and
2. classical (theoretically well planned) pharmacological research.
The pharmacological studies were (and are) based on the results obtained from:
1. various animal experiments;
2. results obtained on the human isolated cell cultures [especially from that time,
when the Leagu(ag)e for Animal Defense (LAD) emphasized the defense of ani-
mals to be involved into the research activity]; and
3. human pharmacological studies.
The animal observations played essential roles in the discoveries of different phys-
iological and pharmacological facts and events. The researchers used mice, rats,
guinea pigs, dogs, chicken, etc. in their pharmacological research. Probably mice and
rats are the most accepted animals for physiological and pharmacological studies.
The different human cell cultures were also involved in medical research to clear
up the exact details of different physiological and pharmacological mechanisms of
different compounds. There is, however, a significant problem with the exclusive
use of the isolated human cell lines for the "human medical research", because these
cell lines have no innervation, no correct hormonal regulation, etc. Of course, the
results of these types of observations are basically necessary to establish the human
pharmacotherapy. Without denying the importance of these types of research on the
animal observations and on isolated human cell line, the most important physiologi-
165
cal and pharmacological regulatory mechanisms remain to be unclear in the human
body. The results of the research on cell cultures cannot be adapted directly in the
production of antibodies against the suggested mediators, in patients with inflamma-
tory bowel diseases.
The human pharmacotherapy has undergone significant changes in the last
decades. Earlier the efficacy of drug therapy was based only on the observation made
by the physicians. These observations can be done retrospectively and prospectively
(e.g. the efficacy of antituberculotic treatment was easily acceptable in Hungary,
because earlier we had no good antituberculotic drugs, such as Streptomycin; isonic-
otinic hydrazine, INH; rifampicin, Tubocin, etc). The Hungarian people died before
the discoveries of these drugs, however, the Hungarian patients were healed (practi-
cally in their whole number) after the introduction of these drugs in the everyday
medical treatment. Consequently the "Hungarian Disease" ("Morbus Hungaricus")
disappeared in the last decades. This process was a clear medical proof for the efficacy
of medical treatment in patients.
Penicillin was accidentally discovered by Fleming (1922). Its efficacy could be
proved clearly in patients with different bacterial infections (the fever disappeared
and the patients got better in a short time after the introduction of penicillin). A.
Fleming received Nobel prize in 1945.
When the different drugs are used in the treatment the patients with different dis-
eases, the efficacies of different drugs can be evaluated by parametric (measureable)
and nonparametric (subjective) parameters, which change from time to time.
Clinical pharmacology was established in the years of 1960 over the World (at the
same time in Hungary). Professor Tibor Javor (Second Department of Medicine,
Medical University of Debrecen, Hungary) and his research group (the writer of this
chapter personally participated in the establishment of clinical pharmacology of
anticholinergic drugs) were pioneers in the establishment of human clinical pharma-
cology in Hungary. The results of the human clinical pharmacology offered the most
principal research argument for the "classical human pharmacology", e.g. medical
treatments.
The main aims of the human clinical pharmacology are:
1. To give objective data on drug absorption from the gastrointestinal tract (in case
of orally applicable drugs), metabolism in the human body (dominantly by the
liver) and excretion of drugs (or their metabolites) by the urine or by the stool;
2. To observe the main pharmacodynamic actions of drugs;
3. To measure the drug (or metabolites) in the serum, urine, stool, after the applica-
tion of the drug given in different doses;
4. To identify the oral and parenteral dose rate of the drug;
5. To identify the correlation between the pharmacodynamic actions and pharmaco-
kinetic parameters in humans;
6. To identify the similarities (and differences) of these parameters obtained in
healthy human persons (volunteers) and patients;
7. To examine these parameters before and after chronic treatment with drug(s);
8. To find a correlation between the results of the clinical pharmacological studies
and those of classical human pharmacological treatment (which finally represent
the everyday medical treatments).
166
Clinical pharmacology represents a classical field in the medical sciences. It
involves a classical multidisciplinary knowledge, which can be given only by experts
working in the different fields of medical sciences (chemists, mathematicians, physi-
cians, drug technologists, drug industrial experts, clinical pharmacologists, etc.). Of
course, the special laboratories and medical wards (clinical pharmacological units)
are basically necessary for carrying out these studies.
There is no doubt about that the human clinical pharmacology has developed
extremely in the last decades in the World (including Hungary).
The classical clinical pharmacology plays an essential role in the innovative drug
research. Furthermore, the innovative drug research and clinical pharmacology can
be evaluated by the internationally accepted parameters (it was true already before
Hungary joined the European Union, 2004).
12.2. Our special scientific problems in the human clinical

pharmacology of capsaicinoids alone and together with
the application of aspirin, diclofenac and Naproxen
We mentioned earlier that "capsaicin" (which name is only used in the classical
physiological and pharmacological research) does not represent only one chemical
structure. The capsaicin of plant origin consists of capsaicin, dihydrocapsaicin, nor-
capsaicin and nordihydrocapsaicin as main components. Capsaicin is transformed
by the metabolism in human and animals into dihydrocapsaicin. Physiological and
pharmacological studies indicated clearly that there is no difference between the
chemically very similar capsaicinoids in animal experiments (Buck, Burks, 1986).
We had the following main scientific problems:
1. There was as important question as to how many components can be put into the
planned drug products (capsaicinoids + aspirin; capsaicinoids + diclofenac and
capsaicinoids + Naproxen). The measured parameters (chemical measurements of
the chemical compound in human biological samples) in the human pharmaco-
logical studies inform us on the efficacy of the drugs or drug combinations from
the point of clinical pharmacology. Capsaicin and dihydrocapsaicin give the most
important part of the chemical components of "classical capsaicin preparate".
According to special consultations with the experts of the Hungarian Institute of
Pharmacy, capsaicin and dihydrocapsaicin can be measured as main components
in the human clinical pharmacological studies. Furthermore, dihydrocapsaicin is
the main metabolite of capsaicin. Consequently the measurements of dihydrocap-
saicin can be used as one of the main chemical components for the pharmacoki-
netic approach to capsaicinoids in the different human clinical pharmacological
studies;
2. When we tried to establish new drug combinations (capsaicinoids + aspirin; cap-
saicinoids + diclofenac and capsaicinoids + Naproxen) we had to measure two
chemical compounds from the two main components of capsaicinoids (capsaicin
and dihydrocapsaicin) during the clinical pharmacological studies (together with
aspirin, diclofenac and Naproxen ) from the human sera;
167
3. The acceptance of this standpoint resulted very important consequences for our
clinical pharmacological studies:
- the pharmacokinetic measurements of capsaicinoids during the pharmacokinetic
studies in healthy human subjects and in patients with different disorders
(absorption rate, metabolisation, excretion of capsaicinoids) can be characterized
by the results of measurements of capsaicin and dihydrocapsacin,
- i f we do not have to measure all capsaicinoid components (having the same
physiological mechanisms) the carrying out of these studies becomes consider-
ably cheaper;
4. We have to perform "classical pharmacodynamic" and "pharmacokinetic" studies
with capsaicinoids alone in healthy human subjects, because there have been no
similar observations published in the world literature up to now (either in healthy
human subjects or in patients with different disorders);
5. Since we will use different combinations of capsaicinoids plus NSAIDs (aspirin,
diclofenac, Naproxen), consequently we have to carry out the human Phase I study
with capsaicinoids alone and together with aspirin, diclofenac and Naproxen.
12.3. Principal schedules for the human Phase l-ll studies

with capsaicinoids alone and together with aspirin,
diclofenac and Naproxen
12.3.1. Preparation of protocols for the human clinical

pharmacological studies (including Phase I to IV)
12.3.1.1. Medical points of the preparation of the study protocols
Very carefully carried out preparation of the medical protocols is the basis for
prospective, randomized and multicentric studies (including one or more countries
of the World).
These protocols describe the medical points of clinical pharmacological studies
[clear identification of the aim(s) of the study(studies) in the healthy human subjects
or patients with different diseases involved in the study, their exact inclusion and
exclusion criteria, schedule of the well-planned examinations, clinical controlling
examinations and their time, drop-out, determination of end points of the studies].
The protocols prepared for the clinical study (studies) have to be accepted by
nationally or internationally well known experts only from the medical points of
views.
The place and the names of person(s) responsible for performing these clinical
pharmacological studies should be given.
Details of the whole documentation of the clinical pharmacological studies
(informed consent, health insurance, case report sheet, circumstances of the data,
preparation for the chemical pharmacological studies, storage, transfer of biological
samples, the registration and archivation of the obtained results and finally to write a
168
final summary of the whole clinical pharmacological study) are clearly indicated
and regulated.
The final medical control is given by the National Institute of Pharmacy, and this
Department gives the final permission (from the medical points of views) for carry-
ing out the studies.
12.3.2. Control of the protocols by the National or Regional

Clinical Pharmacological and Ethical Committees
After the acceptance of different protocols by the National Institute of Pharmacy

(including the necessary preliminary decisions by the national and internationally
well known experts), the protocols will be controlled by the National Clinical
Pharmacological and Ethical Committee of Hungary (in case of Phase I to III, and in
some extent in case of Phase IV studies) from the ethical points of views, looking
for the health and juristic defense of participants involved in the different studies.
These Committees include medical experts from the different fields of medical sci-
ences, lawyers, ethical experts, and nurses. If the National Clinical Pharmacological
and Ethical Committee of Hungary will accept the correct circumstances (including
the medical and ethical aspects of the study planned to be carried out) of the planned
clinical pharmacological study (studies), then the examinations can be commenced.
12.3.3. Pharmacokinetic and pharmacodynamic effects

of capsaicinoids only
12.3.3.1. Human Phase I clinical pharmacological study
The results of different animal experiments clearly demonstrated that the pharmaco-
dynamic effects of capsaicinoids depend on the applied doses. Four different doses
of capsaicinoids are able to produce four different pharmacological effects (Szol-
csanyi, 1996; Mozsik et al., 2000):
1. small doses of capsaicinoids stimulate the capsaicin-sensitive afferent nerves
(these actions are reversible ones);
2. small doses of capsaicinoids, but in a little higher than those mentioned above,
produce the inhibition of the same capsaicin sensitive afferent nerve fibres (which
is also a reversible process);
3. higher doses of capsaicinoids (than those mentioned under point 2) produce an
injury of the capsaicin sensitive afferent nerves (which is probably a reversible
one);
4. the extremely high doses of capsaicinoids produce an irreversible injury of these
nerves.
These pharmacological studies were carried out in animal experiments. In healthy
human subjects we applied the very small doses of capsaicinoids (Mozsik et al.,
169
2005a), which stimulate the capsaicin-sensitive afferent nerves. The capsaicinoids-
induced side effects (diarrhoea, development of malignant diseases) were observed
and published in the world literature, in connection with the administration of
extremely high doses of capsaicinoids. We have to emphasize that the capsaicinoids
were applied in different capsaicin extracts in these studies (and not in a chemically
pure form).
The pharmacokinetic and pharmacodynamic Phase I observations were planned
to be carried out only with small doses of capsaicinoids (up to 1.0 to 1.2 mg/per-
son/day). The primary aims of these studies are to identify the tolerability, safety
and pharmacodynamic aspects of capsaicinoids in healthy human subjects and in
patients with different diseases. The capsaicinoids are orally given in doses 400 to
800 pg/person/day.
12.3.3.2. Human clinical pharmacological Phase I study

with capsaicinoids plus nonsteroidal antiinflammatory
drugs in healthy human subjects
12.3.3.2.1. Human clinical pharmacological phase I study

with capsaicinoids plus aspirin
The dose of aspirin is 100 mg/day/person, in case of aspirin resistance 300

mg/day/person, respectively, estimated by many international studies, and these
doses have been accepted recently by the European and American Societies of
Cardiology (2006) in patients with myocardial infarction. Consequently the doses of
aspirin are internationally well established.
We planned to carry out human Phase I studies with three arms:
1. E D dose of capsaicinoids (400 pg in oral dose);
50
2. aspirin (100 mg and 300 mg), given orally;

3. aspirin (in doses mentioned above) plus capsaicinoids (ED ), given orally.
50
The observations will be carried out in healthy human subjects (volunteers). Every
subject will undergo the different observations, however, the actually applied dose
will be selected according to randomization.
12.3.3.2.2. Human clinical pharmacological Phase I study

with capsaicinoids plus diclofenac
We planned also a human clinical pharmacological phase I study with capsaicinoids

plus diclofenac with three arms:
1. capsaicinoids (in dose of E D ) , (400 pg oral dose);
500
2. diclofenac (25, 50 and 75 mg), given orally;

3. diclofenac (25, 50 and 75 mg) plus capsaicinoids (in dose of ED ), given orally.
50
The clinical pharmacological observations will be done in healthy persons (volun-

teers).
170
12.3.3.2.3. Human clinical pharmacological Phase I study
with capsaicinoids plus Naproxen
Healthy human subjects (volunteers) are included in these studies. The persons
receive all the treatments mentioned below:
1. capsaicinoids in dose of E D (400 pg oral dose);
50
2. Naproxen (250, 375 and 500 mg), given orally alone;

3. capsaicinoids (in dose of ED ) plus Naproxen (in the above-mentioned doses)
50
given together orally.
12.3.3.3. Human clinical pharmacological Phase II studies in patients
12.3.3.3.1. Human clinical pharmacological Phase II study in patients

with thromboembolic diseases (myocardial infarction, stroke,
thromboembolic events)
The patients selected for the study were treated with aspirin (in doses of 100 or 300
mg given orally) without and with co-administration of capsaicinoids (orally given
ED ). The general laboratory parameters, ECC, platelet aggregation were registered
50
dayly, together with the subjective complaints of patients.

Of course, the pharmacodynamic examinations were also carried out in the pa-
tients (treated with capsaicinoids alone, aspirin alone and capsaicinoids plus aspirin).
12.3.3.3.2. Human clinical pharmacological Phase II studies in patients

with different degenerative locomotor diseases
Similar types of the human Phase II examinations are carried out in these patients as
those mentioned in Section 12.3.3.2.2.
These laboratory examinations (plus others related to the degenerative locomotor
system) and semi-quantitative parameters (complaints of patients) were registered
according to the protocols.
The patients were treated with diclofenac (25, 50 and 75 mg given orally), capsai-
cinoids (given orally in doses of E D ) and diclofenac (in the above-mentioned
50
doses) plus capsaicinoids (in dose of ED ) given orally.

50
Another group of patients received Naproxen (in doses of 250, 375 and 500 mg
orally) instead of diclofenac. The further details of this study were the same as in the
case of diclofenac.
The pharmacodynamic examinations were carried out during the clinical observa-
tion.
171
Note
We received absolutely new information from the chronic toxicological studies in

Beagle dogs (2008). These animals were treated with different doses (0.1, 0.3 and
0.9 mg/kg bw/day orally given) of capsaicin(s) for one month. No toxicological side
effects were observed in these dogs during the whole treatment periods. We noticed
surprisingly that capsaicin (capsaicin and dihydrocapsaicin) could not be detected in
the sera of Beagle dogs, either by High Pressure Chromatography (HPLC)* or by
Liquid Chromatography-Mass Spectrometry (LC-MS)** at any time after the oral
application of capsaicin (in doses of 0.1; 0.3; 0.9 mg/kg bw/day). The limit of detec-
tion by HPLC is 20 nanogram/ml serum and its value is 26 fg for capsaicin and 20 fg
for dihydrocapsaicin by LC-MS.
These results suggest that we will not be able to produce a classical pharmacoki-
netic study for capsaicin and dihydrocapsaicin in healthy human subjects and in
patients with different diseases because the dose range of capsaicin is 0.4-1.2
(400-1200 microgram)/ adult persons/ day.
* Mozsik, Gy., Past, T , Perjesi, P., Szolcsanyi, J.: Determination of capsaicin and dihy-
drocapsaicin content of dog's plasma by HPLC-FLD method. In: Mozsik, Gy., Past, T.,
Perjesi, P., Szolcsanyi, J.: Original Reports on Toxicology of Capsaicin VII. 8-Day Oral
Toxicity Study of Test Item Capsaicin Natural USP 27 in Beagle Dogs (Final Report). LAB
International Research Centre Hungary Ltd. Veszprem by the date of final report 13 June
2008. Study Code: 07/496-100K pp. 1-35 in text and 190 pages in Appendices. (Appendix
2.11) pp. 1-37 (2008)
** Boros, B., Dornyei, A., Felinger, A.: Determination of capsaicin and dihydrocapsaicin
in dog plasma by Liquid Chromatography-Mass Spectrometry (Analytical method report)
PTE TTK Analitikai Kemiai Tanszek, Pecs, Hungary (2008)
172
Acknowledgements
The study was supported by the grant of the National Office for Research and
Technology, "Pazmany Peter program" (RET-II 08/2005).
The authors express their sincere thanks to Mrs. Judit Szabo for her excellent
help in the preparation of this monograph.
They are also very grateful to Akademiai Kiado (Budapest, Hungary), especially
to Mrs. Judit Kerpel-Fronius for her precious corrections during the copy editing
and proofreading process.
173
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206
Appendix
1. European Commission
(Health and Consumer Protection Directorate General
Directorate-C Scientific Opinion): C2 Management of
Scientific Committees II Scientific Cooperation and Networks
(http://ec.europa.eu/food/fs/sc/scf/out120_en.pdf)
1.1 Opinion of the Scientific Committee on food on capsaicin
Terms of reference
The Committee is asked to advise the Commission on substances used as flavoring
substances or present in flavorings or present in other food ingredients with flavor-
ing properties for which existing toxicological data indicate that restrictions of use
or presence might be necessary to ensure safety for human health.
In particular, the Committee is asked to advise the Commission on the implica-
tions for human health of capsaicin in the diet.
Introduction
Previous evaluations The Committee of Experts on Flavouring Substances of the
Council of Europe evaluated the capsaicinoids in Capsicum preparations used as
flavourings. A TDI of 0-0.2 mg/kg bw, expressed as total capsaicinoids, was numeri-
cally derived from the results of a population based case-control study conducted in
Mexico City, where chilli pepper consumers were at high risk for gastric cancer
compared with non-consumers. The daily intake of the chilli pepper consumers was
estimated to be 4 mg capsaicinoids/kg bw and a safety factor of 20 was applied. In
addition, general limits of 5 ppm for foods and beverages, 10 ppm for hot foods and
beverages, 20 ppm for hot ketchup and 50 ppm for tabasco, harissa, hot pimento oils
and similar preparations expressed as total capsaicinoids were suggested (Council of
Europe, 2001).
Current regulatory status Capsaicin is listed in the register of chemically defined
flavouring substances laid down in Commission Decision 1999/217/EC (EC, 1999),
as last amended by Commission Decision 2002/113/EC (EC, 2002).
207
Chemical characterisation
Name: Capsaicin (N-(4-hydroxy-3-methoxybenzyl)-8-methyl-trans-6-nonenamide)
Capsaicin in Capsicum preparations is always accompanied by other capsaicinoids:
mainly dihydrocapsaicin, but also small amounts of nordihydro-, homo-, homodihy-
dro-, nor-, and nornorcapsaicin. The capsaicinoids present in the Capsicum fruit are
predominantly capsaicin and dihydrocapsaicin, making up 80 to 90%. The ratio of
capsaicin to dihydrocapsaicin is generally around 1:1 and 2:1 (Govindarajan,
Sathyanarayana, 1991).
Synonyms: 8-Methylnon-6-enoyl-4-hydroxy-3-methoxybenzylamide; trans-8
methyl-N-vanillyl-6-nonenamide; Isodecenoic acid vanillylamide (Fig. 32)
FL No: 16.014
CAS No: 404-86-4
FEMANo: 3404
CoENo: 2299
EINECS: 206-969-8
Structure:
Fig. 32. 8-Methylnon-6-enoyl-4-hydroxy-3-methoxybenzylamide;

trans-8-methyl-N-vanillyl-6-nonenamide; Isodecenoic acid v a n i l l y l a m i d e
Exposure assessment
Capsaicinoids are mainly ingested as naturally occurring pungency-producing com-
ponents of capsicum spices (chilli, cayenne pepper, red pepper). They typically range
from 0.1 mg/g in chilli pepper to 2.5 mg/g in red pepper and 60 mg/g in oleoresin
red pepper (Parrish, 1996). Pepper varieties from Capsicum frutescens, annuum and
chinense were found to contain 0.22-20 mg total capsaicinoids/g of dry weight
(Thomas et al., 1998). Cayenne pepper samples had mean capsaicin and dihydro-
capsaicin contents of 1.32 and 0.83 mg/g dry weight, respectively (Lopez-Her-
nandez, 1996).
The consumption of capsicum spices was reported to be 2.5 g/person/day in India,
5 g/person/day in Thailand (Monsereenusorn, 1983; Monsereenusorn et al., 1982)
and 20 g/person (one chilli pepper) per day in Mexico (Lopez-Carrillo, 1994).
Assuming a content of capsaicinoids in these spices of about 1%, the daily intake of
capsaicinoids in these countries has been estimated to be 25-200 mg/person/day or
in the case of a person with 50 kg body weight 0.5-4 mg/kg bw/day (Council of
Europe, 2001).
The maximum daily intake of capsaicin in the U.S. and Europe from mild chillies
and paprika was roughly estimated to be 0.025 mg/kg bw (Govindarajan, Sa-
thyanarayana, 1991), equivalent to 1.5 mg/person/day. According to a recent estima-
208
tion, the mean and maximum intake of capsaicin from industrially prepared food
products containing the recommended general limit of 5 pm would be 0.77 and 2.64
mg/day, respectively (CREDOC/OCA, 1998).
Hazard identification/characterisation
Capsaicin and other members of the group of capsaicinoids produce a large number
of physiological and pharmacological effects such as effects on the gastrointestinal
tract, the cardiovascular and respiratory system as well as the sensory and ther-
moregulation system. These effects result principally from the specific action of
capsaicinoids on primary afferent neurons of the C-fiber type. This provides the
rationale for their use to treat some peripheral painful states, such as rheumatoid
arthritis (Surh, Lee, 1995)
In addition, capsaicinoids are powerful irritants, causing burn and pain at low con-
centrations on the skin and mucous membranes. Given orally, they induce an in-
crease of salivation and gastric secretion, a rapid change of sensation, warm to in-
tolerable burning, and gastrointestinal disorders depending on the dose (Govin-
darajan, Sathyanarayana, 1991).
Absorption, distribution, metabolism and excretion
Capsaicinoids, when administered to rats intragastrically are readily absorbed and
metabolized to a great extent in the liver before reaching the general circulation and
extrahepatic organs (Donnerer et al., 1990). In vitro and in vivo studies have shown
that capsaicinoids are metabolized by different pathways: (1) hydrolysis of the acid-
amide-bond and oxidative deamination of the formed vanillylamine, (2) hydroxyla-
tion of the vanillyl ring, possibly via epoxidation, (3) one electron oxidation of the
ring hydroxyl forming phenoxy radicals and capsaicinoid dimers, (4) oxidation at
the terminal carbon of the side chain (Surh, Lee, 1995). Within 48 hrs after oral
administration of dihydrocapsaicin to male rats, 8.7% of the dose were excreted
unchanged in urine and 10% in faeces. Metabolites found in urine were vanillyl-
amine (4.7%), vanillin (4.6%) vanillyl alcohol (37.6%) and vanillic acid (19.2%) in
free form or as glucuronides (Kawada, Iwai, 1985). Based on results of Miller et al.
(1983), who demonstrated the covalent binding of dihydrocapsaicin to hepatic
microsomal proteins, the formation of electrophilic intermediates (arene epoxides,
phenoxy radicals or quinone type derivatives formed after O-demethylation) and
subsequent covalent binding to cellular macromolecules is discussed to play a role
in the etiology of capsaicin-induced toxicity including mutagenicity and carcino-
genicity (Surh, Lee, 1995).
Acute toxicity
The acute toxicity of capsaicin shows a large variation depending on the route of
administration. In male mice, the LD 50 varies from 0.56 mg/kg bw (i.v.) to 60-75
mg/kg bw (in ethanol) and 190 (122-294) mg/kg bw (in dimethyl sulfoxide), fol-
lowing intragastric intubation. The possible cause of death was considered to be due
to respiratory paralysis (Glinsukon et al. 1980). Intraduodenal and intragastric
administration of 10% Capsicum as well as 0.014% capsaicin in 0.85% saline to
male rats produced morphological damages in the duodenal mucosa (Nopanitaya,
Nye, 1974).
209
Subacute/subchronic toxicity
A 4-week feeding study with groups of 5 male B6C3F1 mice with 0, 0.5, 1.0, 2.5,
5.0, 7.5, and 10% ground red chilli (Capsicum annuum) in the diet showed slight
glycogen depletion and anisocytosis of hepatocytes in the 10% group. Other organs
did not reveal any lesions. General health, body weight and food intake were not
adversely affected (Jang, Kim 1988; Jang et al., 1992).
Groups of 10-14 rats were fed by stomach tube with 50 mg/kg bw/day capsaicin
or 0.5 g/kg bw/day Capsicum extract for 10-60 days. There were significant reduc-
tions of growth, plasma urea, glucose, phospholipids, triglycerides, total cholesterol,
free fatty acids, glutamic pyruvic transaminase, and alkaline phosphatase in both
groups with a tendency for Capsicum treated animals to show more adverse effects.
No gross pathological changes and no differences in organ weights from control val-
ues were observed at autopsy, only a slight hyperemia in the livers and reddening
with increasing mucous materials in the gastric mucosa. The organs, however, were
not examined histopathologically (Monsereenusorn, 1983).
BALB/c mice received an alcoholic chilli extract in drinking water 5 days a week
till 16 months of age (27 males, 25 pg capsaicin/week, equivalent to about 0.125
mg/kg bw/d) or on the tongue 2 days a week for 14 months (22 males without and
19 males with 1% atropin solution prior to application, 50 pg capsaicin/week, equiv-
alent to about 0.25 mg/kg bw/d). Compared with 40 untreated mice, the treated ani-
mals showed increased mortality and histopathological changes in liver, kidneys,
stomach and tongue. The lesions in the liver observed in all treated mice were in the
form of focal necrosis with inflammatory cells around, fatty changes and fibrosis
(Agrawal, Bhide, 1987).
36 male Syrian hamsters received 20 pi alcoholic chilli extract with 50 pg cap-
saicin (equivalent to about 0.5 mg/kg bw/d) 5 days a week by cheek pouch applica-
tion for 14 months. 30 untreated animals and 17 hamsters treated with 20 pi alcohol
were used as controls. The animals treated with chilli extract had increased mortali-
ty and histopathological lesions in liver, kidneys, stomach and cheek pouch. The
main lesions were liver cirrhosis, observed in 49 % of examined livers from exposed
hamsters compared to 8 and 17 % in the control groups and glomeruli degeneration
in 50% of examined kidneys of exposed animals compared to 8 and 0 % in the con-
trol groups, respectively (Agrawal, Bhide, 1988).
Capsaicin, administered intraperitoneally to adult male mice at doses of 0.4, 0.8
or 1.6 mg/kg bw/day on 5 consecutive days, did not induce significant alterations in
epididymal weights, caudal sperm counts, testicular weights or testicular histology.
In the sperm morphology assay, sperms at 1, 3, 5 and 7 weeks did not reveal any
treatment-related increase in the incidence of sperm-head abnormalities (Muralidhara,
Narasimhamurthy, 1988).
In a 13-week study performed to determine the maximum tolerated dose, groups
of 10 male and 10 female B6C3F1 mice received a mixture of 64.5% capsaicin and
32.6% dihydrocapsaicin at those levels of 0, 0.0625, 0.125, 0.25, 0.5, and 1% in the
diet. Significant reduction of food intake and body weight gain in all dose groups,
especially in treated females, and significantly increased liver/body weight ratios of
both sexes and renal toxicity (focal tubular dilatation) in the 1 % treated males were
observed (Akagi et al., 1998).
210
Capsaicin (purum) was administered at concentrations of 0.0625, 0.125, 0.25, 0.5
and 1% in the diet of groups of 4 male and 4 female Swiss albino mice for 35 days.
When the animals died at an age of 62-126 weeks, one adenocarcinoma of the duo-
denum had developed at each dose level, except for the highest dose, while no such
tumours occurred in a historical control group of 100 males and 100 females. There
was no concurrent control group and the observed tumour incidence was not dose-
related (Toth, Gannett, 1992; Toth et al., 1984).
Chronic toxicity/carcinogenicity
15 out of 26 rats fed for seven months with 10% chillies in a semisynthetic diet con-
taining ardein, a purified protein of the ground nut, developed neoplastic changes in
the liver (hepatomas, multiple cystic cholangiomas, solid adenomas or adenocarci-
nomas of the bile duct). Although no tumour developed in rats fed the basic diet
without chillies, the authors stress, that it cannot be said whether chillies have a spe-
cific carcinogenic effect or whether a deficiency in the diet aggravated by a non-spe-
cific irritant caused the tumours (Hoch-Ligeti, 1951).
Capsaicin (capsaicin 65%, dihydro- 31%, nordihydro- 0.9%, homo- 1%, homodi-
hydro- 0.6%, nor- 0.5%, nornor- 0.3%), administered in a semisynthetic diet at
0.03125% to 50 male and 50 female Swiss albino mice for their life span from 6
weeks of age, induced benign polypoid adenomas of the caecum in 22% of females
(p <0.05) and 14% of males, compared to 8% in the untreated female and male con-
trols (incidence of historical controls not given). The survival rate was not substan-
tially altered (Toth, Gannet, 1992).
Groups of 50 male and 50 female B6C3F1 mice were given 0, 0.025, 0.083, and
0.25% capsaicinoid mixture (64% capsaicin and 32.6% dihydrocapsaicin) in the diet
for 79 weeks, equivalent to daily doses of up to 220 and 200 mg/kg bw in males and
females, respectively. In all dose groups, food intake was significantly reduced, in
females also the body weight gain and in males the liver/body weight ratio. No evi-
dence of carcinogenicity was found. Renal cell adenomas developed only in one
male mouse of the 0.025 and 0.25% groups. (Akagi et al., 1998).
A number of studies have shown that capsaicin or chilli extract can act as tumour
promoters (Surh, Lee, 1995, 1996). Thus, capsaicin (0.002% in drinking water for 6
weeks) has been reported to act as a promoter for the development of diethylni-
trosamine-initiated enzyme-altered foci in the liver of male rats (Jang, Kim, 1988).
Chilli extract has also been shown to have a promoting effect on the development of
stomach and liver tumours in BALB/c mice initiated by methyl-acetoxy methylni-
trosamine and benzene hexachloride, respectively (Agrawal et al., 1986). In another
study, rats fed diets containing hot chilli pepper showed slightly higher incidence of
N-methyl-N-nitrosoguanidine-induced gastric cancer (Kim et al., 1985).
On the other side, capsaicin has been suggested to exert chemoprotective effects
through modulation of metabolism of carcinogens and their interaction with target
cell DNA (Surh, Lee, 1995, 1996).
Genotoxicity
Capsaicin (purum) was found to be mutagenic in Salmonella typhimurium strain TA
98 in the presence of Aroclor induced rat liver S9 fraction (Toth et al., 1984), where-
as another study in which S9 from phenobarbital-induced rats was used for metabolic
211
activation was negative (Buchanan et al., 1981). Capsaicin containing 20% dihydro-
capsaicin exhibited mutagenicity in Salmonella strains TA 98, TA 100 and TA 1535
in the presence of S9 mixture from Aroclor-induced rats. An alcoholic chilli extract
was mutagenic only in strain TA 98 (Nagabhushan, Bhide, 1985, 1986). Capsicum
pepper oleoresin was reported to have mutagenic activity in Salmonella strains SD
1018 and SD 7823 without metabolic activation (Damhoeri et al., 1985). In addition,
also a modified SOS microplate assay indicated a genotoxic activity of capsaicin
(Venkat et al., 1995).
Capsaicin containing 20% dihydrocapsaicin and alcoholic chilli extract failed to
induce 8-azaguanine resistant mutants in Chinese hamster V79 cells with and without
metabolic activation by S9 from Aroclor-induced rats (Nagabhushan, Bhide, 1985).
On the other side, synthetic capsaicin, dihydrocapsaicin and a crude mixture of cap-
saicinoids from Capsicum frutescens activated with hamster hepatocytes were muta-
genic in the V 79 assay measured by resistance to ouabain and 6-thioguanine (Lawson,
Gannet, 1989).
The results of the comet assay and a DNA fragmentation assay show that cap-
saicin is able to induce DNA damage in human neuroblastoma cells (Richeux et al.,
1999). Capsaicin also induced DNA strand breakage with calf thymus and plasmid
DNA in the presence of Cu (II) (Singh et al., 2001).
Capsaicin containing 20% dihydrocapsaicin induced micronuclei in polychromat-
ic erythrocytes in the mouse-bone-marrow assay at 7.5 mg/kg i.p. (Nagabhushan,
Bhide, 1985). It also produced a significant increase of micronucleated normochro-
matic erythrocytes in the peripheral blood and SCEs in bone marrow cells of male
mice at 1.46 and 1.94 mg/kg i.p. (Diaz Barriga Arceo et al., 1995). A fraction of an
alcoholic extract from the fruits of Capsicum frutescens, containing 3-acetamido-2-
methyltetradecane as major component, has been found positive in the mouse-bone-
marrow micronucleus assay after i.p. administration (Villasenor, Ocampo, 1994,
1995). Dominant-lethal mutations in mice were not induced by capsaicin (Mu-
ralidhara, Narasimhamurthy, 1988).
Furthermore, capsaicin inhibited DNA biosynthesis in the testes of Swiss mice
injected intraperitoneally (Nagabhushan, Bhide, 1985).
Some other studies cannot be evaluated, because important experimental details
have not been published.
Reproductive and developmental toxicity
No data available
Human data
In a case-control study in Mexico City which included 220 cases of gastric cancer
and 752 controls randomly selected from the general population, chilli pepper con-
sumers were at a 5.5-fold greater risk for gastric cancer than non-consumers. Persons
who rated themselves as heavy consumers of chilli peppers were even at a 17-fold
greater risk. However, when chilli pepper consumption was measured as frequency
per day, a significant dose response relationship was not observed (Lopez-Carrillo et
al., 1994).
In another case-control study in India, red chilli powder was found to be a risk
factor for cancer of the oral cavity, pharynx, esophagus, and larynx (2- to 3-fold risk
212
with a dose-response relationship) compared with population controls, but not with
hospital controls (Notani, Jayant, 1987).
In an Italian case-control study, chilli was briefly mentioned as being protective
against stomach cancer (Buiatti et al., 1989). Chilli peppers, however, are not heavi-
ly consumed in Northern Italy, where this study was conducted, and it is possible
that chilli consumption was correlated with other protecting spices such as onions
and garlic that are more heavily consumed in Italy (comment from Lopez-Carrillo et
al., 1994).
Summary of hazard identification/characterisation
Capsaicin, capsaicinoid mixtures, chillies and chilli extracts have been tested toxico-
logically by oral administration to mice, rats and hamsters. Some of these studies
indicated a carcinogenic potential of capsaicin. These studies are regarded, however,
as limited. A more recent carcinogenicity study did not show carcinogenic effects in
mice. In humans, however, high consumption of chillies has been reported to be a
risk factor for cancer of the upper gastrointestinal tract, possibly due to the irritating
effect of capsaicinoids.
Genotoxic effects of capsaicin and capsaicinoid mixtures have been shown in vitro
and in vivo.
Risk characterisation
The Committee concluded that the available data did not allow it to establish a safe
exposure level for capsaicinoids in food.
The human intake of capsaicinoids in India, Thailand and Mexico, where cap-
sicum spices are heavily consumed, has been estimated to be 25-200 mg/day. The
high consumption of chillies in Mexico and India was reported to be associated with
cancer of the upper digestive tract. In contrast, the maximum daily intake from mild
chillies and paprika in Europe was roughly estimated to be 1.5 mg/day. In the one
study conducted in Europe, no increase in the incidence of gastric cancer was found
in association with occasional and lower intakes of chillies.
1.2. European Herbal Practitioners Association
Response to EMEA Consultation Document CPMP/QWP/2819/00 REV 1 AKA

EMEA/CVMP/814/00 REV 1: Guideline on Quality of Herbal Medicinal Pro-
ducts/Traditional Herbal Medicinal Products (Released 21 July 2001/Con-
th
sultation Date 3 0 September 2005)
1. The European Herbal Practitioners Association (EHPA) was founded in 1993.

It represents the interests of professional herbal practitioners and their patients across
the EU. The EHPA is designated an "interested party" by the EMEA Herbal
Medicinal Products Committee, attending its open meetings.
2. The EHPA notes that Footnote 1 of the document states, "Throughout the guide-
line and unless otherwise specified, the term 'herbal medicinal products' includes
'traditional herbal medicinal product'". It is thus clear that so far as quality assur-
ance is concerned, the Guideline on the Quality of Herbal Medicinal Products
213
regards herbal medicinal products as synonymous with traditional herbal medicinal
products. The EHPA contends that quality guidelines for traditional herbal medici-
nal products should be specifically designed to recognise the characteristics and
meet the needs of traditional herbal medicinal products. As explained below, these
should not be taken to be exactly the same as those of herbal medicinal products.
3. The Guidelines on Specifications CPMP/QWP/2819 (hereinafter referred to as
"the Guidelines") were originally developed by the Herbal Medicinal Products
Working Party (HMPWP) in 1999. These guidelines were published to provide guid-
ance in support of a full marketing authorisation for licensed herbal products and
were adopted by the Committee for Proprietary Medicinal Products (CPMP) and the
Committee for Veterinary Medicinal Products (CVMP) in July 2001. Thus these
guidelines were developed and agreed a considerable time before the passage into
law of the Traditional Herbal Medicinal Products Directive (THMPD) in April 2004.
This Guideline and CPMP/QWP/2820/00 Rev 1, both part of the present consulta-
tion process, seek to ensure the appropriateness of the Guidelines for the THMPD.
4. Although the EHPA concurs with the general principle proposed in the
Guidelines that "the quality of a medicinal product is independent of its traditional
use", nevertheless, it should be recognised that the Guidelines were developed for
assuring the quality of products licensed under Directive 2001/83/EC (e.g. the "Well-
Established Use" category), not Directive 2004/24/EC (THMPD). The EHPA sub-
mits that these current Guidelines now out for consultation should be redrafted to
suit the new class of herbal products destined to be licensed under Directive
2004/24/EC (THMPD). The adoption of specific Guidelines appropriate to this new
class of herbal products will enable the THMPD to achieve its objective of bringing
traditionally-used herbal medicinal products within the EU medicines licensing sys-
tem. We submit that this goal will not be achieved if the currently proposed
Guidelines are adopted without such revision and that this will lead to an increase
by the public of unregulated herbal products available via the Internet.
5. It is a fact that many traditional herbal medical products currently on sale in the
EU are multi-herb preparations. It is not unusual for such traditional formulae to
comprise 5 or more herbs. In the case of traditional Chinese, Tibetan and Ayurvedic
medicines, the number of herbs in a complex herbal formula may frequently number
10 or more.
6. The EHPA submits that the relatively rigid quality standards being implement-
ed by these Guidelines and those also under review in the consultation Guideline
entitled Guideline On Quality of Herbal Medicinal Products (CPMP/QWP/2820/00
Rev 1) are proving technically very difficult and in many cases unworkable in prac-
tice. The Guideline (2819/00 Rev 1) calls for tests to demonstrate that the known
constituents of any herbal medicines in the product are present in the finished prod-
uct. The Guideline states (vide page 7 "Control Tests on the Finished Product") that
if a herbal medicinal product contains a combination of several herbs, "the determi-
nation may be carried jointly for several active substances." The Guidance note
advises (vide page 6 "control of herbal preparations") that such identification tests
should be carried out using "appropriate chromatographic methods ". The problem
here is that demonstrating exactly what is present in the finished product by chro-
matographic means is easier said than done. These quality control measures are rela-
214
tively easy to carry out for an orthodox drug which contains a single chemical entity
or for a single herbal compound for which the Guidelines were originally designed
but often impossible to demonstrate when evaluating a complex herbal mixture of
several herbs each one containing a multiplicity of chemical signatures. The EHPA
contends that the proposed Guidelines must be adapted so that they can apply to
complex herbal mixtures.
7. The EHPA submits that quality and safety of multiherb products should be
assured by the identification and control of the raw materials of these products rather
that by the quantitative testing of the end product.
8. Similarly, the EHPA submits that stability testing of the final product should
focus on microbial, physical and finger-print chromatographic testing and only
require the quantification of claimed active(s) or marker(s) solely for single-herb
products where appropriate.
9. The rationale of the THMPD is that herbal products must demonstrate 30 years
of safe traditional use, with 15 years of this usage being within the EU. In this way,
by definition, herbal products licensed under the THMPD must have demonstrated a
good safety profile over many years so that the quantification of actives is not neces-
sary for establishing a safety profile. Moreover the quantification of actives is unnec-
essary for the establishment of efficacy since the basis of efficacy for the THMPD is
traditional use.
The EHPA proposes that this proposed quality and safety model is supported by
required pharmacovigilance, providing a clear method of evaluating the safety of
traditional herbal medicinal products on the market.
This response sent to QWP@emea.eu.int 30/09/2005 should be read in con-

junction with the EHPA response to CPMP/QWP/2820/00 Rev 1 sent previously.
Contact Information: European Herbal Practitioners Association 8 Lion Yard
Tremadoc Road London SW4 7NQ Tel: +44 (0) 20 7627 2680 ENDS
1.3. Commmission Decision of 1 8 M a y 2005 amending

Decision 1999/21 7/EC as regards the register
of flavouring substances used in or on foodstuffs
(notified under document number C(2005) 1437)
(Text with EEA relevance) (2005/389/EC)
THE COMMISSION OF THE EUROPEAN COMMUNITIES,

Having regard to the Treaty establishing the European Community,
Having regard to Regulation (EC) No 2232/96 of the European Parliament and of
the Council of 28 October 1996 laying down a Community procedure for flavouring
substances used or intended for use in or on foodstuffs (OJ L 299, 23.11.1996, p. 1.
Regulation as amended by Regulation (EC) No 1882/2003 (OJ L 284, 31.10.2003,
p.l)), and in particular Article 3(2) and Article 4(3) thereof,
215
Whereas:
1. Regulation (EC) No 2232/96 lays down the procedure for the establishment of
rules in respect of flavouring substances used or intended to be used in foodstuffs.
That Regulation provides for the adoption of a register of flavouring substances (the
register) following notification by the Member States of a list of the flavouring sub-
stances which may be used in or on foodstuffs marketed in their territory and on the
basis of scrutiny by the Commission of that notification. That register was adopted
by Commission Decision 1999/217/EC (OJ L 84, 27.3.1999, p. 1. Decision as last
amended by Decision 2004/357/EC (OJ L 113, 20.4.2004, p. 28).).
2. In addition, Regulation (EC) No 2232/96 provides for a programme for the
evaluation of flavouring substances in order to check whether they comply with the
general criteria for the use of flavouring substances set out in the Annex to that
Regulation.
3. The European Food Safety Authority (the Authority) concluded in its opinion
of 13 July 2004 on para-hydroxybenzoates, that propyl 4-hydroxybenzoate (FL
09.915) had effects on sex hormones and the male reproductive organs in juvenile
rats. The Authority was unable to recommend an acceptable daily intake (ADI) for
this substance because of the lack of clear no-observed adverse-effect-level
(NOAEL). The use of propyl 4-hydroxybenzoate as a flavouring substance in food is
not acceptable, as it does not comply with the general criteria for the use of flavour-
ing substances set out in the Annex to Regulation (EC) No 2232/96. As a conse-
quence, propyl 4-hydroxybenzoate should be deleted from the register.
4. The Authority concluded in its opinion of 7 December 2004 on aliphatic dial-
cohols, diketones and hydroxy-ketones, that pentane-2,4 dione (FL07.191) is geno-
toxic in vitro and in vivo. Accordingly, its use as a flavouring substance is not accept-
able, because it does not comply with the general criteria for the use of flavouring
substances set out in the Annex to Regulation (EC) No2232/96. As a consequence,
pentane-2,4-dione should be deleted from the register.
5. In application of Regulation (EC) No 2232/96 and Commission Recom-
mendation 98/282/EC of 21 April 1998 on the ways in which the Member States and
the signatory States to the Agreement on the European Economic Area should pro-
tect intellectual property in connection with the development and manufacture of
flavouring substances referred to in Regulation (EC) No 2232/96 of the European
Parliament and of the Council (OJ L 127, 29.4.1998, p. 32. has adopted this decision),
for a number of substances, the notifying Member States requested that they should
be registered in such a way as to protect the intellectual property rights of the manu-
facturer.
6. Protection for these substances, listed in Part B of the register, is limited to a
maximum period of five years following the date of receipt of the notification. That
period has now expired for 28 substances which should consequently be transferred
to Part A of the register.
7. Decision 1999/217/EC should therefore be amended accordingly.
8. The measures provided for in this Decision are in accordance with the opinion
of the Standing Committee on the Food Chain and Animal Health,
216
Article 1
The Annex to Decision 1999/217/EC is amended in accordance with the Annex to
this Decision.
Article 2
This Decision is addressed to the Member States.
Done at Brussels, 18 May 2005.

For the Commission
Markos KYPRIANOU
Member of the Commission
ANNEX
The Annex to Decision 1999/217/EC is amended as follows:
1. Part A is amended as follows:

(a) The rows set out in the table for the substances attributed with FL-numbers
07.191 (pentane-2,4-dione) and 09.915 (propyl 4-hydroxybenzoate) are delet-
ed
(b) The following rows are inserted in the Table 35:
2. The table of part B is replaced by the following
Flavouring substances notified in application of Article 3(2) of Regulation (EC)
No 2232/96, for which the protection of the intellectual property rights of the manu-
facturer has been requested (Table 36)
T a b l e 3 5 . C o m m i s s i o n D e c i s i o n of 18 M a y 2 0 0 5 a m e n d i n g 1999/21 7/EC as regards to register of

f l a v o u r i n g s u b s t a n c e s u s e d in or o n foodstaffs part A *
FL. N o . Chemical CAS Name Einecs Syno- Comments

group nyms
01.070 31 111-66-0 1 -Octene 203-893-7

01.071 31 111-67-1 2-Octene 203-894-2
01.072 31 544-76-3 Hexadecane 280-878-9
07.251 21 577-16-2 Metylaceto- CAS N o
phenone corresponds
to 2-methyl-
acetopenone
01.073 31 592-99-4 4-Octene
01.074 31 593-45-3 Octadecane 209-790-3
16.084 30 627-67-8 3-Methyl- 211-008-0
1-nitrobutane
01.075 31 629-78-7 Heptadecane 211-108-4
12.260 20 4131-76-4 Methyl-2-methyl-
3-mercapto
propionate 223-949-4
217
T a b l e 3 5 . C o m m i s s i o n D e c i s i o n of 18 M a y 2 0 0 5 a m e n d i n g 1999/21 7/EC as regards to register of
f l a v o u r i n g s u b s t a n c e s u s e d in o r o n foodstaffs part A *
FL. N o . Chemical CAS Name Einecs Syno- Comments

group nyms
12.261 20 6725-64-0 Methaneditiol

01.076 31 2099635-4 3,7-Decadiene
16.085 20 27959-66-6 4,4-Dimethyl-
1,3-oxathiane
12.262 20 29414-47-9 (Methylthio)
methanethiol
05.210 04 30390-51-3 4-Dodecenal 250-174-9
05.211 02 30689-75-9 6-Methyloctanal
14.166 30 32536-43-9 Indole acetic acid
07.252 05 33655-27-9 4-Octen-2-one
02.244 04 54393-36-1 4-Octen-1-ol
10.070 09 57681-53-5 4-Hydroxy-
2-heptenoic acid
lactone 260-902-7
05.212 04 76261-02-04 6-Dodecenal
05.213 04 90645-87-7 5-Nonenal
15.124 29 103527-75-9 3-Methyl-2-butenyl- Rose
thiophene thiophene
05.214 04 121052-28-6 8-Dodecenal
05.215 03 134998-59-7 2,3-Decadienal (c,c)
05.216 03 134998-60-0 2,6-Decadienal (t,t)
12.263 20 3-Mercapto-
3-methylbutanal
12.264 20 92585-08-5 4,2-Thiopentanone
03.021 16 142-96-1 Dibutyl ether 205-575-3
The rows set out in the table for the substances attributed with FL-numbers 07.191 (pentane-2,4-dione) and
09.915 (propyl 4-hydroxybenzoate) are deleted
T a b l e 3 6 . C o m m i s s i o n D e c i s i o n of 18 M a y 2 0 0 5 a m e n d i n g 1999/21 7/EC as regards t o register of

f l a v o u r i n g s u b s t a n c e s u s e d in o r o n foodstaffs part B*
Code D a t e of receipt of the notification by the Commission Comments
CN065 26.1.2001
CN074 18.4.2003 6
CN075 18.4.2003 6
CN076 18.4.2003 6
* Flavouring substances notified in application of Article 3(2) of Regulation (EC) No 2232/96, for which the pro-
tection of the intellectual property rights of the manufacturer has been requested
218
2. Medical products for human use:
Common Technical Document (CTD)
2 . 1 . Foreword
This Notice to Applicants (NTA) has been prepared by the European Commission in
consultation with the competent authorities of the Member States, the European
Medicines Agency and interested parties in order to fulfil the Commission's obligations
with respect to article 6 of Regulation (EC) No. 726/2004, and with respect to the Annex
I to Directive 2001/83/EC as amended (Directive 2003/63/EC, OJ L 159 27.6.2003 p.46
NTA, Vol. 2B-CTD, foreword & introduction, edition June 2006).
The first edition of the Notice to Applicants (Volume 2 in the series "The Rules
Governing Medicinal Products in the European Union") was published in 1986. A
revised and completed version, the second edition, was issued in January 1989. In
1993, the procedures for applications for marketing authorisations were amended,
and the centralised and mutual recognition procedures became applicable from 1995.
It was decided to separate the procedural and presentational parts of this guidance as
Volumes 2A and 2B respectively. In 2000, a need for additional specific regulatory
guidelines was recognised and a Volume 2C was prepared. The NTA is now pub-
lished in the following volumes:
Volume 2A dealing with procedures for marketing authorisation
Volume 2B dealing with the presentation and format of the application dossier
Volume 2C dealing with regulatory guidelines.
The latest updates of all of the above-mentioned volumes can be found on the
European Commission's pharmaceutical unit's web-site at the following address:
http://ec.europa.eu/enterprise/pharmaceuticals/eudralex/homev2.htm.
2.2. Introduction
Volume 2B is concerned with the presentation of the application dossier and was
first published as a separate volume in 1998. It provides guidance for the compilation
of dossiers for applications for European marketing authorisations and is applicable
for the centralised procedure and national procedures, including mutual recognition
and decentralised procedures. The update takes account of the international agree-
ments on the structure and format of the Common Technical Document (CTD) which
were agreed in November 2000 within the International Conference on Harmonisation
(ICH) framework and further documents and revised guidelines agreed upon since that
time. This introduction should be read together with other published documents (e.g.
219
documents published on the ICH-website: http://www.ich.org, and "questions and
answers" published on the Website of the European Commission).
The update of the NTA, Vol. 2B: EU-CTD also reflects the revised ICH CTD-
guidelines (see http://www.ich.org) for Quality, Safety and Efficacy. The revised
ICH-guidelines were signed off at the ICH meeting in Washington, September 02.
The reasons for revision were minor changes in the numbering and the headings of
the CTD, which have been incorporated in the updated Modules 2, 3, 4 and 5 of the
EU CTD NTA.
Module 1 was updated in April 2006 taking into account the requirements of the
new pharmaceutical legislation.
To each Module a list of relevant CHMP /ICH-guidelines is annexed, which have
to be taken into consideration when preparing an EU Marketing authorisation
dossier. These will be updated at regular intervals.
The CTD is an internationally agreed format for the preparation of applications to
be submitted to regulatory authorities in the three ICH regions of Europe, USA and
Japan. It is intended to save time and resources and to facilitate regulatory review
and communication.
The CTD gives no information about the content of a dossier and does not indi-
cate which studies and data are required for a successful approval. Regional require-
ments may affect the content of the dossier submitted in each region, therefore the
dossier will not necessarily be identical for all regions.
The CTD indicates an appropriate format for the data that have been required in
an application. Applicants should not modify the overall organisation of the Com-
mon Technical Document as outlined in the guideline. However, in the Non-clinical
and Clinical Summaries, applicants can modify individual formats if needed to pro-
vide the best possible tabulated presentation of the technical information, in order to
facilitate the understanding and evaluation of the results.
The new EU-CTD-presentation will be applicable for all types of marketing autho-
risation applications irrespective of the procedure (CR MRR DCP or national) and of
type of application (stand alone, generics etc). The CTD-format will be applicable for
all types of products (new chemical entities, radiopharmaceuticals, vaccines, herbals
etc.) To determine the applicability of this format for a particular type of product,
applicants should consult with the appropriate regulatory authorities.
2.3. Terminology
The Common Technical Document was developed as an international document,

and therefore specific European legal terms such as "active substance", "medicinal
product", and "marketing authorisation" were not used in its development.
Applicants are reminded that the term "medicinal product" covers both pharmaceu-
tical and biological medicinal products. Unless otherwise indicated, it should be
considered to be synonymous with the term "drug product". Similarly, the term
"active substance" should be considered as synonymous with "drug substance".
The terms used in the ICH documents may be used in the CTD part of the appli-
cation.
220
2.4. Presentation of European Marketing Authorisation
Applications
The current requirements for the content of the European application dossier are set
out in Annex I to Directive 2001/83/EC as amended, as stated in Article 8.3 "the
application shall be accompanied by the following particulars and documents, sub-
mitted in accordance with Annex I".
Annex I of Directive 2001/83/EC sets out the legal provision for implementation
of the CTD-format.
The provision of this update of Volume 2B (EU CTD), which take into account
the ICH agreements, replaces the previous structure of the European marketing
authorisation dossier described in the 1998 edition of Volume 2B.
st
From 1 July 2003, all applications should be made entirely in accordance with
the EU-CTD presentation outlined in the July 2003 edition of NTA, Vol. 2B or its
subsequent updates.
In order to take into account experience with CTD structure and changes of a tech-
nical or scientific nature, it is anticipated that NTA, Volume 2B will be updated reg-
ularly and additional guidance will be provided in the form of Question & Answer
documents as experience is gained (Question & Answer document: Presentation and
content of the dossier Common Technical Document (CTD) Volume 2B. Applicants
Notice to NTA, Vol. 2B-CTD, foreword & introduction, edition June 2006.
Applicants are advised to consult the Commission web-site: http://ec.europa.eu/
enterprise/pharmaceuticals/eudralex/homev2.htm to verify the latest updated infor-
mation).
2.5. Presentation of Applications in the Mutual Recognition

Procedure or Decentralised Procedure
All new applications have to be submitted in accordance with the CTD format. For
Mutual Recognition Procedure based on marketing authorisation approved to the old
format it is an obligation to reformat the Quality data of the dossiers before starting
a new Mutual Recognition Procedure or a Repeat use procedure after 1.5.2005. For
Repeat use procedures and duplicate applications/multiple applications there is no
need to reformat the non-clinical and clinical data of dossiers for medicinal products
for human use, authorised before 1 July 2003. Furthers guidance is given in section
"presentation of the application" and the corresponding Annex to the Question &
Answer document.
If the original Part II contained data on bioequivalence, then this data should be
extracted from the Part II and reformatted into the new CTD structure, and annexed
in a separate binder as a separate section 5.3.1.2.
The applicants are strongly reminded and encouraged to submit the Quality part
of a dossier in the EU-CTD format as soon as possible.
If a MAH wants to reformat the dossier into the CTD-format, it must first be sub-
mitted to the RMS, who has to take this reformat of the dossier into account. For
221
updating the Assessment Report it will be sufficient for the RMS to attach one page
to the Assessment Report explaining that the format of the relevant dossier has been
changed to the CTD-format but not the content of the dossier.
2.6. Presentation of Follow-up Measures,

Specific Obligations and P S U R s
Also for the submission of Follow-up Measures, a Specific Obligation dossier or a

dossier including post-marketing experience, the CTD structure needs to be used.
The CTD structure should always be utilised whereby documents are assigned to
the most appropriate sections in Modules 1-5. They should be structured according
to the granularity defined in the ICH guidance on CTD Organisation, Annex:
Granularity Document (CHM/ICH/2887/99 Rev 2 Correction Organisation CTD)
with Tables of Content and tab dividers for paper submissions or as separate files
according to eCTD guidance.
When submitting via the Centralised Procedure the Post-authorisation Guidance
provided on the EMEA website (http://www.emea.eu.int/htms/human/postguid-
ance/list.htm) should also be followed.
The PSUR should be located in Module 5.3.6, Reports of Post-Marketing
Experience. If a Summary Bridging Report is also to be provided then this should be
included in Module 5.3.6 as well.
2.7. Reformatting of dossiers of already authorised products
There is no obligation to reformat the dossier of already authorised medicinal prod-

ucts into the new EU-CTD format. If a marketing authorisation holder (MAH) wish-
es to reformat the documentation, such reformatting will be allowed, although it is
not recommended for the Non-clinical and Clinical parts of the documentation.
However, for the Quality part of the documentation, companies are encouraged to
voluntarily reformat into the CTD-format, especially to facilitate the handling of
st
variations and line extensions after 1 July 2003. Such reformatting must however
involve the complete Quality parts, including any Drug Master Files (if applicable)
and also including and integrating all approved variations. A signed declaration from
the MAH, must also be submitted stating that the content/data of the Quality Module
is identical to the currently approved Quality part and that there has been no changes
to the dossier as a result of the reformatting.
Reformatted Quality documentation submitted in the CTD-format must consist of
a new Module 3 in CTD format, but need not necessarily contain the Quality Overall
Summary together with the signed template for the Quality Expert.
A Module 1 need not be submitted. If the original Part II contained data on bio-
equivalence, then this data should be extracted from the Part II and reformatted into
the new CTD structure, and annexed in a separate binder.
222
The submission of reformatted documentation should preferably occur simultane-
ously but separately with the submission of a variation, extension or renewal. A clear
distinction between the reformatted (unchanged) information and the documentation
supporting the simultaneously submitted variation / line extension or renewal should
be made. Any reformatted documentation should also be submitted in electronic for-
mat (e-CTD) if available. Reformatting of a dossier does not fall under the legal def-
inition of a variation, because there is no amendment of the dossier's content. For
dossiers of products which are already NTA, Vol. 2B-CTD, foreword & introduc-
tion, edition June 2006 approved via Mutual Recognition Procedure in more than one
Member State, any reformatting has to be made simultaneously in all the Member
States concerned. Where products are approved via national procedures in different
Member States it is also highly recommended to reformat the dossiers at the same
time.
The relevant competent authority has to decide whether a fee would be charged
or not.
In case of Mutual Recognition Procedures the reformatted dossier (new CTD)
format of an already approved medicinal product cannot be submitted directly to the
Concerned Member States. It must first be submitted to the Reference Member State,
who has to take this reformat into account. For the RMS it will be sufficient to attach
one page to the Assessment Report explaining that the format has been changed and
not the content.
2.8. Presentation of the application
The Common Technical Document is organized into five modules. The content of
Module 1 is defined by the European Commission in consultation with the compe-
tent authorities of the Member States, the European Agency for the Evaluation of
Medicinal Products and interested parties. Concerning the structure of Modules 2, 3,
4, and 5 they are common for all ICH regions.
2.9. Administrative, regional or national information

is provided in different Modules
Module 1
This module contains the specific EU-requirements for the administrative data (e.g.
the application form, the proposed summary of product characteristics, labelling and
package leaflet, etc.).
Module 2
The module 2 contains high level summaries (the Quality Overall Summary, the
Non-clinical Overview / Summaries, and the Clinical Overview / Summaries), which
must be prepared by suitably qualified and experienced persons (experts). Although
the term "Expert Report" must be maintained for legal reasons, the content is expected
223
to be given in the Quality Overall Summary, the Non-clinical Overview / Summaries,
and the Clinical Overview / Summaries documents. Old Expert Reports are now
replaced by Module 2. The experts have to sign and add brief information on their
educational background and specific expertise in a special section in Module 1.4.
Module 3
Chemical, Pharmaceutical and Biological documentation is provided by module 3.
This information should be structured as described in Guideline M4Q (M4Q (Rl):
QUALITY Module 2 :Quality Overall Summary (QOS) Module 3 : Quality The
section of the application covering chemical and pharmaceutical data including data
for biological/ biotechnological products).
Module 4
The documentation on the Toxicological and Pharmacological Tests performed on
drug/active substance and a drug/medicinal product is provided in the Non-clinical
Written Summaries (from Module 2) and by the Non-clinical Study Reports. These
reports should be presented in the order described in Guideline M4S (M4S (R2):
SAFETY Nonclinical Summaries and Organisation of Module 4 The non-clinical
section of the application).
Module 5
The documentation on the Clinical Trials performed on the drug/medicinal product
is provided in the Clinical Written Summaries (from Module 2) and in the Clinical
Study Reports. These reports should be presented in the order described in Guideline
M4E (M4E (Rl): EFFICACY Module 2: Clinical Overview and Clinical Summary
Module 5 Clinical Study Reports The clinical section of the Application) (Fig 33).
http://www.ich.org/cache/compo/276-254-1 .html
2.10. Preparing and Organizing the C T D
Throughout the CTD, the display of information should be unambiguous and trans-
parent, to facilitate the review of the basic data and to help a reviewer become quick-
ly oriented to the application contents. Text and tables should be prepared using
margins that allow the document to be printed on A4 paper. The left-hand margin
should be sufficiently large that information is not obscured through binding. Font
sizes for text and tables should be of a style and size that are large enough to be eas-
ily legible, even after photocopying. Times New Roman, 12-point font is recom-
mended for narrative text. Acronyms and abbreviations should be defined the first
time they are used in each module.
However when preparing the product information for applications in the cen-
tralised procedure (ref. Module 1.3.) it is mandatory to use the "QRD (Quality
Review of Documents) convention".
224
Fig. 33. D i a g r a m m a t i c R e p r e s e n t a t i o n of t h e O r g a n i z a t i o n of t h e C T D
2.11. Pagination and Segregation
Every document should be prepared in line with the CHMP /ICH/2887/99 Revision
1 Organisation CTD recommendation.
225
2.12. Information about national administrative
requirements
Information about the addresses of the national authorities, the numbers of copies of
dossier-modules required, and further information are published by the EC in the
NTA, Vol. 2A, Chapter 7
(http://ec.europa.eu/enterprise/pharmaceuticals/eudralex/vol-2/a/ctd-
chap7_200603.pdf).
2.13. Special guidance for different kinds of applications
This international format is intended to apply to all categories of drug products /

medicinal products (incl. NCE's, radiopharmaceuticals, vaccines, herbals, etc.) and
all types of applications (stand alone, generics, biosimilar etc.), although some adap-
tations may be necessary for specific application/product types.
It is not designed to indicate what studies are required for successful approval, but
to indicate an appropriate organization for the information included in the applica-
tion. If no information is available or required under a specific heading, that section
of the application should be marked "not applicable" or "not relevant" whilst retain-
ing the section title and numbering, and, if necessary, a justification for the absence
of a study should be provided in the Quality Overall Summary, the Non-clinical
Overview and the Clinical Overview.
Applicants are reminded that for bibliographical, generic and biosimilar,
"hybrid" and extensions the non-clinical/clinical overviews/summaries should
focus on particular issues concerning the basis for the application. Applicants should
also consult Chapter 1 of the NTA, Vol. 2A - Marketing authorisation.
For generic, biosimilar and "hybrid" applications and extensions cross-references
to previous applications in the "old" EU-format will be accepted. No reformatting of
already assessed and authorised "old" documentation into the CTD-format is neces-
sary.
1. Bibliographical applications For applications based upon Article 10a of
Directive 2001/83/EC, non-clinical/clinical overviews/summaries should demon-
strate that the constituent(s) of the medicinal product have a well-established use,
with an acceptable level of safety and/or efficacy, as outlined in Annex I to Di-
rective 2001/83/EC.
Tabulated clinical and non-clinical summaries in Module 2 shall be provided.
Tables may not be necessary for very old, well known substances, but a proper justi-
fication will be required. Overviews always have to be provided.
2. Informed consent, Generic, "Hybrid" or Bio-similar applications
2 a) Consent from the marketing authorization holder For applications based
upon Article 10c of Directive 2001/83/EC, the original expert reports or quali-
ty/non-clinical/clinical overviews/summaries of the original marketing autho-
rization holder may be referred to.
2 b) Applications relating to generic medicinal products and biosimilar medici-
nal products; For applications according to Article 10 (1), (3) and (4)
226
Directive 2001/83/EC, Module 2 must include the Quality Overall Summary,
Non-clinical Overview and Clinical Overview. Non-clinical and Clinical
Summaries can be provided, but they are only mandatory if new additional stud-
ies have been provided within the documentation.
3. Variation Applications in accordance with Regulation 1084/2003/EC and
Regulation 1085/2003/EC
st
After 1 July 2003, all variation applications must be submitted using the EU-
CTD format. However, cross-references to "old" EU-format documentation will be
accepted, because the content is identical. Clear references to any "old" format do-
cumentation are essential. Examples: Any new (either additional or revised) data in
support of the variation must be submitted using the CTD format.
If any data need to be submitted which are unchanged, for example, the Type I varia-
tion Guideline might specify the need for submission of a copy of the approved specifi-
ations, then the marketing authorisation holder should update the specifications into the
new CTD-format. The marketing authorisation holder must also provide a declaration
that the content of any reformatted documents is unchanged. Any future variation appli-
cations would then be able to use these "updated" (CTD) specifications.
Where only a cross-reference to already authorised data is required, such cross-
references can still be made to the relevant "old" format dossier (Part and section).
However, if the marketing authorisation holder prefers to take the opportunity to
present the (unchanged) data in the new CTD-format instead, this would be equally
acceptable as it would facilitate the handling of future variations. The marketing
authorisation holder must also provide a declaration stating that the content of any
reformatted documents is unchanged.
Type IA/IB Variation Applications and their supportive documentation - where
appropriate - should be presented as follows
Where a cover letter is provided it should be placed in Module 1, 1.0 Cover Letter.
The checklist of conditions to be fulfilled and documentation to be supplied, i.e.
the extract of the relevant page from the Guideline on Dossier Requirements for
Type IA and Type IB notifications, should be placed in 1.2 Application Form after
the Application Form itself.
http://ec.europa.eu/enterprise/pharmaceuticals/eudralex/homev2.htm
Documents should be assigned, wherever possible, to the relevant CTD section,
primarily within Module 3 Quality and 1.3.1 Summary of Product Characteristics,
Labelling and Package Leaflet. These would include replacement sections and addi-
tional information.
Where documents cannot be assigned to specific CTD-defined locations then they
should be included in 1.2 Application Form. These might include declarations, cer-
tificates, justifications etc. Where possible, they should be organised according to
the Annexes for the Application Form for new products (Annexes 1 to 22). Where
documents cannot be assigned to one of the annexes then it is appropriate to place
them after the annexes. The 'Additional Data' section of Module 1 should not be
used except for country-specific information defined in Table 3.2 of Chapter 7,
General Information, of the Notice to Applicants.
Documents should be presented with tab dividers for paper submissions or as sep-
arate files according to eCTD guidances.
227
Type II Variation Applications and their supportive documentation - where
appropriate - should be presented as follows (non-exhaustive list depending on the
scope of the variation and supportive data):
Module 1 :
1.0 Cover Letter
1.1 Comprehensive Table of Contents
1.2 Application Form
1.3 Product Information
1.3.1 SPC, Labelling and Package Leaflet - where appropriate
1.3.4 Consultation with Target Patient Population (e.g. in case of significant changes)
1.3.6 Braille (when Braille is implemented for an already authorised medicinal prod-
uct as part of a variation)
1.4 Information about the Experts: The relevant expert declaration(s) and signa-
ture^) must be provided, corresponding to the Overview/Summary submitted in
Module 2. In cases where MAHs wish to distinguish this declaration from any
previous declarations, the Variation Procedure Number of the RMS/EMEA may
be included on top.
1.5 Specific Requirements for Different Types of Applications
1.5.3 (Extended) Data/Market Exclusivity In case the applicant wants to claim a one-
year data exclusivity at the time of the application for a new therapeutic indica-
tion, a document, justifying that the application concerns "a new therapeutic
indication which is claimed to bring a significant clinical benefit" or that sig-
nificant preclinical or clinical studies have been carried out, has to be provid-
ed. Related study reports and supporting literature references shall be placed in
the relevant Modules of the dossier and thus cross-referred to accordingly.
1.6 Environmental Risk Assessment (e.g. in case of a new indication with significant
increase of the extent of use)
1.7. Orphan Market Exclusivity (in case the indication applied for is the same as an
already authorised orphan medicinal product)
1.8.1 Pharmacovigilance system (e.g. in case of changes), where appropriate
1.8.2 Risk-Management System (e.g. in case of a significant change in indication)
1.9 Information relating to clinical trials (in case clinical trials supporting the varia-
tion application were carried out outside the EU)
Module 2:
As mentioned in the Variation Regulation any Type II variation should be accompa-
nied by the relevant Overviews/Summaries updates or addenda (even if a variation is
submitted at the request of the Competent Authority/CHMP). Expert details and sig-
nature are to be provided in Module 1.4 separated from the actual Overview/
Summary.
Modules 3, 4, 5:
Supportive data are to be included in Modules 3, 4 and/or 5 as appropriate and in
accordance with the EU-CTD structure.
4. New Applications as referred to in Annex II of Regulation 1084/2003/EC and
Regulation 1085/2003/EC ("Extensions").
The non-clinical/clinical overviews/summaries should particularly focus on the
following elements: an evaluation of the results of the additional studies. The results
228
should be discussed in the perspective of what is known from published literature
and previous submissions. Additional studies should also be submitted in tabular
formats provided in this Notice to Applicants:
- if applicable an update of published literature relevant to the substance and the
present application. The documentation may include annotated articles published
in "peer review" journals, which may be acceptable for this purpose;
- every claim in the Summary of Product Characteristics (SPC) not known from or
inferred from the properties of the medicinal product and/or its therapeutic group
should be discussed in the non-clinical/clinical overviews/summaries and substan-
tiated by published literature and/or additional studies.
st
After 1 July 2003, all applications for extensions must also be submitted using
the new EU-CTD format. However, references can be made to already assessed and
authorised "old" Parts of the dossier, but only if no new additional data are submit-
ted in these parts. In such cases, it is not necessary to reformat already assessed and
authorised "old" documentation.
Modules 1 and 2 always have to be provided. Where no new clinical and/or non-
clinical data are submitted the respective overviews/summaries can be replaced by
expert-statements. It is however necessary to provide new CTD-format summaries
and overviews to cover any new information or data provided in support of the
application. In these CTD-format summaries/overviews, all the headings (numbers
and titles) must be included, but where cross-references can be made, as the data
has not changed, it is sufficient to include a statement such as "Not changed" (or
similar).
Marketing authorisation holders are also encouraged, in addition to presenting the
new extension quality data in the CTD-format, to reformat the entire assessed and
authorised "old" format Part II into the new CTD-format, in order to obtain a com-
plete CTD Module 3, covering all strengths/pharmaceutical forms. Exceptionally, in
those cases where there are multiple strengths and/or pharmaceutical forms, the
Quality module of the extension may include only the data for the new strength/phar-
maceutical form and cross-refer to the relevant "old" quality data. At the occasion of
the next variation affecting the "old" part, marketing authorisation holders should
"reformat" (at least) that part into the CTD format.
5. Renewal Applications:
st
Since 1 July 2003 all applications for renewals must be submitted using the EU-
CTD format. The guidance stated in the relevant guidelines/recommendations on the
processing of renewals, have to be taken into account. See Vol. 2C,
http://ec.europa.eu/enterprise/pharmaceuticals/eudralex/homev2.htm
2.14. Special guidance on herbal medicinal products
For the purposes of this document the terms "herbal substances and preparations"
shall be considered equivalent to the terms "herbal drugs and herbal drug prepara-
tions", as defined in the European Pharmacopoeia.
For ease of reference the section titles of Modules 2 and 3 have been copied. The
text following the section titles is intended to be explanatory and illustrative to herbal
229
medicinal products only. The content of these sections should include relevant infor-
mation described in Guidelines published by the Agency.
Click here to obtain the specific information on Modules 2 and 3 for herbal medi-
cinal products: http://www.ich.org.
Information relevant to the Active Substance Master File (ASMF). According to
the current EU guideline on the ASMF procedure (Active Substance Master File
guideline (CHMP QWP/227/02) (Former European Drug Master File (EDMF)
CPMP/QWP/227/02 Guideline on Active Substance Master File procedure;
http://www.emea.eu.int/htms/human/qwp/qwpdraft.htm. NTA, Vol. 2B-CTD, fore-
word & introduction, edition June 2006) it is the responsibility of the applicant for a
marketing authorisation for a medicinal product to ensure that the complete ASMF,
that is both the applicant's ("open") part and the active substance manufacturer's
restricted ("closed") part with original signed Letter of Access is supplied to the
authorities directly by the active substance manufacturer in the CTD format, syn-
chronised to arrive at around the same time as the marketing authorisation applica-
tion. A copy of the "Letter of Access" shall be included in Annex 6.10 of the appli-
cation form, in Module 1 and addressed to the regulatory authority to where the
application is made. The applicant's ("open") part of the ASMF should be included
in section 3.2.S of the Quality documentation presented in the CTD-format.
The active substance manufacturer's restricted ("closed") part of the ASMF should
follow the structure of Module 3.2.S of the CTD. A separate Quality Overall
Summary for the information included in the active substance manufacturer's
restricted ("closed") part should also be provided, as part of the ASMF.
When an ASMF is provided as part of a new application for which the Quality
data are submitted in the EU-CTD format, the complete ASMF (open and closed
part and the Quality Overall Summary on the ASMF) must also be presented in the
EU-CTD format.
2.15. Variation of an A S M F
If a change concerns a section of an ASMF, the documentation for this change must
be submitted in the CTD-format. The ASMF-holder should be strongly encouraged
to reformat the complete ASMF at this occasion. This will facilitate the handling of
changes/variations which affect data in the ASMF concerned.
The ASMF-holder should make a clear distinction between the:
- reformatting of the ASMF data, already assessed by the competent authorities and
- the new documentation supporting the change to ASMF-data.
If an ASMF is reformatted into the new EU-CTD-format without any change in
data, a signed declaration must be provided by the ASMF-holder certifying that the
content of that ASMF is identical to the currently held version of the ASMF.
After reformatting of the ASMF, the new "open part" of the ASMF in CTD-for-
mat has to be sent by the ASMF-holder to the MAHs concerned, in order that the
MAH is able to update all marketing authorisations where that ASMF has been used.
It will be acceptable to have the ASMF in the CTD format, without reformatting of
the corresponding Quality data in the dossier of the medicinal product.
230
If there is a change to the ASMF-data, the corresponding variation has to be sub-
mitted to the authorities by the MAH. For extension applications to MAs where an
ASMF is used, the ASMF Holder should reformat the complete ASMF into the EU-
CTD-format so that the "new" format ASMF can be included in the extension appli-
cation, rather than any cross reference being made to an "old" format ASMF.
2.16. European Certificate of Suitability of monographs

of the European Pharmacopoeia (CEP)
Applicants may use the CEP-scheme to replace some of the information needed in
Module 3 for drug substances described in the European Pharmacopoeia.
The Drug Substance section should refer to the Certificate of Suitability in the rel-
evant sections in Module 3.2.S. The Certificates of Suitability are deemed to replace
the data of the corresponding sections and therefore no further additional informa-
tion is necessary except concerning technical characteristics of the substance where
not covered by the Certificate of Suitability (e.g. when the Certificate of Suitability
does not describe a specific technical grade).
A complete copy of the Certificates of Suitability (including any annexes) should
be provided in the annex 6.10 of the application form in Module 1 and in Module 3 R.
TSE-compliance can also be demonstrated by a CEP.
2.17. European Community Guidelines on Quality,

Safety and Efficacy
In assembling the dossier for application for marketing authorisation, applicants are
required to take into account the Community guidelines relating to the quality, safe-
ty and efficacy of drug/medicinal products published by the Commission in "The
rules governing medicinal products in the European Community", Volumes 3A, 3B,
3C: Guidelines on the quality, safety and efficacy of drug/medicinal products for
human use, and subsequent updates as adopted by the Committee for Human
Medicinal Products. The guidelines adopted within the ICH process are considered
as Community guidelines once adopted by the CHMP and published. References to
the relevant Community or ICH guidelines have been included either within the rel-
evant sections, or as annexes to each part of the dossier. For the latest updates of
Community / ICH guidelines, applicants are advised to consult the Website of the
EMEA on http://www.emea.eu.int/index/indexhl.htm (Regulatory Guidance and
Procedures - Notes for Guidance).
With respect to the quality part of the dossier, the monographs and general chap-
ters of the European Pharmacopoeia are also applicable. All materials of ruminant
origin have also to comply with the TSE requirements.
Correlation
EU-CTD (NTA, Vol. 2B, edition May 2006) vs. NTA, Vol. 2B (edition 1998)
(Table 37)
231
T a b l e 3 7 . S u m m a r y of t h e parts of C o m m o n T e c h n i c a l D o c u m e n t s ( C T D )
( E U - C T D ( N T A , V o l . 2 B , e d i t i o n M a y 2 0 0 6 ) v s . N T A , V o l . 2 B ( e d i t i o n 1998)
M O D U L E 1 -ADMINISTRATIVE I N F O R M A T I O N A N D PRESCRIBING INFORMATION
CTD E U C T D ( N T A , Vol. 2 B , Edition 2006) N T A , Vol. 2 B (Edition 1998) NTA
1.0 C o v e r Letter
1.1 C o m p r e h e n s i v e t a b l e of c o n t e n t
1.2 Application Form Administrative Data I A
1.3 Product Information S u m m a r y of P r o d u c t C h a r a c t e r i s t i c s ,

L a b e l l i n g a n d P a c k a g e Leaflet I B
1.3.1 S u m m a r y of P r o d u c t C h a r a c t e r i s t i c s , S u m m a r y of P r o d u c t C h a r a c t e r i s t i c s
L a b e l l i n g a n d P a c k a g e Leaflet I B 1
P r o p o s a l for p a c k a g i n g , l a b e l l i n g
& p a c k a g e leaflet I B 2
1.3.2 Mock-up I B 2
1.3.3 Specimen
1.3.4 C o n s u l t a t i o n w i t h Target Patient

Groups
1.3.5 Product Information already S P C s a l r e a d y a p p r o v e d in t h e

a p p r o v e d in t h e M e m b e r States M e m b e r States I B 3
1.3.6 Braille
1.4 I n f o r m a t i o n a b o u t t h e Experts Expert R e p o r t s : S i g n a t u r e of Experts I C
1.4.1 Quality
1.4.2 Non-clinical
1.4.3 Clinical
1.5 S p e c i f i c R e q u i r e m e n t s for different

t y p e s of a p p l i c a t i o n s
1.5.1 I n f o r m a t i o n for b i b l i o g r a p h i c a l
applications
1.5.2 I n f o r m a t i o n for G e n e r i c , " H y b r i d "

or B i o - s i m i l a r A p p l i c a t i o n s
1.5.3 (Extended) Data/Market Exclusivity
1.5.4 Exceptional Circumstances
1.5.5 Conditional Marketing Authorisation
1.6 E n v i r o n m e n t a l risk assessment E n v i r o n m e n t a l risk assessment
1.6.1 Non-GMO E n v i r o n m e n t a l risk assessment

/ e c o t o x i c i t y (for n o n - G M O s ) III R
1.6.2 GMO D a t a r e l a t e d to t h e e n v i r o n m e n t a l risk

assessment for p r o d u c t s c o n t a i n i n g ,
or c o n s i s t i n g of g e n e t i c a l l y m o d i f i e d
organisms ( G M O s ) II H
232
CTD E U C T D ( N T A , Vol. 2 B , Edition 2006) N T A , V o l . 2 B (Edition 1998) NTA
1.7 I n f o r m a t i o n relating to O r p h a n
Market Exclusivity
1.7.1 Similarity
1.7.2 M a r k e t Exclusivity
1.8 I n f o r m a t i o n r e l a t i n g to
Pharmacovigilance
1.8.1 Pharmacovigilance System
1.8.2 Risk-management System
1.9 I n f o r m a t i o n relating to C l i n i c a l Trials
R e s p o n s e s to Q u e s t i o n s R e s p o n s e s to Q u e s t i o n s
CTD E U C T D (NTA, Vol. 2 B , Edition 2006) N T A , Vol. 2 B (Edition 1998) NTA
Additional Data Additional Data
2.1 O v e r a l l C T D T a b l e of C o n t e n t s of T a b l e of C o n t e n t s for r e m a i n d e r
M o d u l e s 2, 3, 4, a n d 5 of t h e dossier I.A
2.2 Introduction Product profile I.C
2.3 Quality Overall Summary Expert report o n t h e c h e m i c a l ,

pharmaceutical and biological
documentation I C 1
2.4 Non-clinical O v e r v i e w Expert R e p o r t o n t h e t o x i c o -

pharmacological documentation I C 2
2.5 Clinical O v e r v i e w Expert R e p o r t o n t h e C l i n i c a l

Documentation I C 3
2.6 Non-clinical Summary A p p e n d i c e s to t h e t o x i c o -

p h a r m a c o l o g i c a l Expert R e p o r t I C 2
2.6.1 Pharmacology Written Summary Written Summary I C 2
2.6.2 Pharmacology Tabulated S u m m a r y T a b u l a r Formats I C 2
2.6.3 Pharmacokinetics Written Summary Written Summary IC2
2.6.4 Pharmacokinetics Tabulated S u m m a r y Tabular Formats I C 2
2.6.5 Toxicology Written S u m m a r y
2.6.6 Toxicology Tabulated S u m m a r y Tabular Formats I C 2
2.7 Clinical Summary A p p e n d i c e s to t h e c l i n i c a l

Expert R e p o r t I C 3
2.7.1 S u m m a r y of b i o p h a r m a c e u t i c s Written Summary

and associated analytical methods I C 3
2.7.2 S u m m a r y of c l i n i c a l p h a r m a c o l o g y studies Written Summary I C 3
2.7.3 S u m m a r y of c l i n i c a l e f f i c a c y Written Summary I C 3
2.7.4 S u m m a r y of c l i n i c a l safety Written Summary I C 3
2.7.5 S y n o p s e s of I n d i v i d u a l S t u d i e s T a b u l a r Formats I C 3
233
MODULE 3 - QUALITY
3.1 M O D U L E 3 TABLE O F CONTENTS
3.2 B O D Y O F DATA Chemical, Pharmaceutical,

Biological Documentation II
3.2.S DRUG SUBSTANCE
3.2.S.1 General Information Scientific Data II C 1 .2
3.2.S.1.1 Nomenclature Nomenclature II C 1 .2.1
3.2.S.1.2 Structure D e s c r i p t i o n : Structural f o r m u l a II C 1 .2.2
3.2.S.1.3 G e n e r a l Properties Physico-chemical

characterization II C 1 .2.5
3.2.S.2 Manufacture Manufacture II C 1 .2.3
3.2.S.2.1 Manufacturer(s) N a m e ( s ) address(es) of t h e

m a n u f a c t u r i n g source(s) II C 1 .2.3
3.2.S.2.2 D e s c r i p t i o n of m a n u f a c t u r i n g S y n t h e t i c or m a n u f a c t u r i n g
process a n d process controls route D e s c r i p t i o n of p r o c e s s II C 1 .2.3
3.2.S.2.3 C o n t r o l of m a t e r i a l s Q u a l i t y control during

manufacture II C 1 .2.4
3.2.S.2.4 C o n t r o l s of c r i t i c a l steps Quality control during

and intermediates manufacture II C 1 .2.4
3.2.S.2.5 P r o c e s s v a l i d a t i o n and/or e v a l u a t i o n
3.2.S.2.6 Manufacturing process d e v e l o p m e n t
3.2.S.3 Characterisation
3.2.S.3.1 E l u c i d a t i o n of structure D e v e l o p m e n t chemistry

a n d other characteristics II C 1 2.5
3.2.S.3.2 Impurities Impurities II C 1 2.6
3.2.S.4 C o n t r o l of d r u g s u b s t a n c e Specifications a n d routine

tests II C 1 1
3.2.S.4.1 Specification S p e c i f i c a t i o n s a n d r o u t i n e tests II C 1 1
3.2.S.4.2 Analytical Procedures S p e c i f i c a t i o n s a n d r o u t i n e tests II C 1 1
3.2.S.4.3 V a l i d a t i o n of a n a l y t i c a l p r o c e d u r e s Development Chemistry:

Analytical Validation II C 1 2.5
3.2.S.4.4 Batch analyses Batch analysis II C 1 2.7
3.2.S.4.5 J u s t i f i c a t i o n of S p e c i f i c a t i o n D e v e l o p m e n t Chemistry:
C o m m e n t s o n the c h o i c e
of r o u t i n e tests a n d standards II C 1 2.5
3.2.S.5 R e f e r e n c e S t a n d a r d s or M a t e r i a l s D e v e l o p m e n t c h e m i s t r y : Full
c h a r a c t e r i z a t i o n of t h e p r i m a r y
reference material Batch II C 1 2.5
analysis: R e f e r e n c e material II C 1 2.7
3.2.S.6 Container Closure System
3.2.S.7 Stability Stability Tests o n A c t i v e

Substance(s) II F 1
234
MODULE 3 - QUALITY
CTD E U C T D (NTA, Vol. 2 B , Edition 2001) NTA, Vol. 2 B (Edition 1998) NTA
3.2.P DRUG PRODUCT
3.2.P.1 Description and composition Composition and container

of t h e d r u g p r o d u c t (brief d e s c r i p t i o n ) II A 1 II A 2
3.2.P.2 Pharmaceutical Development Development Pharmaceutics

a n d c l i n i c a l trial f o r m u l a e II A 4 II A 3
3.2.P.2.4 C o n t r o l s a n d c r i t i c a l steps Manufacturing process

a n d intermediates ( i n c l u d i n g in-process c o n t r o l
and phamraceutical assembly
process) C o n t r o l tests
on intermediate products II B 3 II D
3.2.P.3 Manufacture M e t h o d of P r e p a r a t i o n II B
3.2.P.3.1 Manufacturer(s) Administrative Data I A
3.2.P.3.2 Batch formula Manufacturing Formula II B 1
3.2.P.3.3 D e s c r i p t i o n of M a n u f a c t u r i n g Manufacturing Process

Process and Process Controls ( i n c l u d i n g In-process C o n t r o l
and Pharmaceutical Assembly
Process) 11 B 2
3.2.P.3.4 C o n t r o l s of c r i t i c a l steps Manufacturing Process (inclu-

and intermediates d i n g In-process C o n t r o l
and Pharmaceutical Assembly
Process) II B 2
3.2.P.3.5 P r o c e s s v a l i d a t i o n a n d / or V a l i d a t i o n of t h e P r o c e s s
evaluation II B 3
3.2.P.4 C o n t r o l of e x c i p i e n t s Excipients(s) II C 2
3.2.P.4.1 Specifications S p e c i f i c a t i o n s a n d r o u t i n e tests II C 2 . 1
3.2.P.4.2 Analytical procedures S p e c i f i c a t i o n s a n d r o u t i n e tests II C 2 . 1
3.2.P.4.3 V a l i d a t i o n of a n a l y t i c a l p r o c e d u r e s S c i e n t i f i c data II C 2.2
3.2.P.4.4 Justification of s p e c i f i c a t i o n s S c i e n t i f i c data II C 2.2
3.2.P.4.5 E x c i p i e n t s of h u m a n or a n i m a l o r i g i n
3.2.P.4.6 N o v e l E x c i p i e n t s (ref to A 3) Excipient(s) not d e s c r i b e d in a II C 2.2.1

p h a r m a c o p o e i a S c i e n t i f i c data II C 2.2
3.2.P.5 C o n t r o l of d r u g p r o d u c t C o n t r o l Tests o n t h e F i n i s h e d
Product II E
3.2.P.5.1 Specification(s) Product specifications

Q u a l i t y specifications II E 1.1 II
for t h e p r o p o s e d shelf life F 2
3.2.P.5.2 Analytical Procedures Control Methods II E 1.2
3.2.P.5.3 V a l i d a t i o n of A n a l y t i c a l P r o c e d u r e s Analytical validation

of m e t h o d s II E 2 . 1
3.2.P.5.4 Batch analyses Batch analysis II E 2 . 2
3.2.P.5.5 C h a r a c t e r i s a t i o n of I m p u r i t i e s
3.2.P.5.6 Justification of s p e c i f i c a t i o n ( s ) C o m m e n t s on the choice

of r o u t i n e tests a n d s t a n d a r d s II E 2.1
235
MODULE 3 - QUALITY
CTD E U C T D (NTA, Vol. 2 B , Edition 2001) N T A , V o l . 2 B (Edition 1998) NTA
3.2.P.6 R e f e r e n c e S t a n d a r d s or M a t e r i a l s Batch analysis: Reference

material II E 2 . 2
3.2.P.7 Container Closure System Packaging Material

( I m m e d i a t e Packaging) II C 3
3.2.P.8 Stability Stability Tests o n t h e F i n i s h e d

Product II F 2
3.2.A APPENDICES
3.2.A.1 Facilities a n d E q u i p m e n t
3.2.A.2 A d v e n t i t i o u s A g e n t s Safety
Evaluation
3.2.A.3 Excipients
3.2.R REGIONAL INFORMATION V a l i d a t i o n of t h e p r o c e s s -II B 3 -
3.3 LITERATURE REFERENCES OTHER INFORMATION II Q
MODULE 4 - NONCLINICAL STUDY REPORTS
CTD E U C T D ( N T A , Vol. 2 B , Edition 2001) N T A , Vol. 2 B (Edition 1998) NTA
4.2 STUDY REPORTS TOXICO-PHARMACOLOGICAL

DOCUMENTATION III
4.2.1 PHARMACOLOGY PHARMACODYNAMICS III F
4.2.1.1 Primary pharmacodynamics P h a r m a c o d y n a m i c s effects

relating to t h e p r o p o s e d
indications III F 1
4.2.1.2 Secondary pharmacodynamics General pharmacodynamics III F 2
4.2.1.3 Safety p h a r m a c o l o g y General pharmacodynamics III F 2
4.2.1.4 P h a r m a c o d y n a m i c drug interactions D r u g interactions III F 3
4.2.2 PHARMACOKINETICS PHARMACOKINETICS III G
4.2.2.1 Analytical Methods and Validation O t h e r Information

Reports IIIQ
4.2.2.2 Absorption P h a r m a c o k i n e t i c s after a single

dose III G 1
P h a r m a c o k i n e t i c s after r e p e a t e d
administration III G 2
4.2.2.3 Distribution D i s t r i b u t i o n in n o r m a l
a n d pregnant animals III G 3
4.2.2.4 Metabolism Biotransformation III G 4
4.2.2.5 Excretion Pharmacokinetics III G 1 , 2
4.2.2.6 P h a r m a c o k i n e t i c D r u g Interactions
(nonclinical)
4.2.2.7 O t h e r Pharmacokinetic Studies
236
MODULE 4 - NONCLINICAL STUDY REPORTS
4.2.3 TOXICOLOGY TOXICITY III A
4.2.3.1 Single-dose t o x i c i t y S i n g l e d o s e t o x i c i t y studies III A 1
4.2.3.2 Repeat-dose toxicity R e p e a t e d d o s e t o x i c i t y studies III A 2
4.2.3.3 Genotoxicity M u t a g e n i c Potential III D
4.2.3.4 Carcinogenicity C a r c i n o g e n i c Potential III E
4.2.3.5 Reproductive and developmental Reproductive Function Embryo-

toxicity foetal a n d P e r i n a t a l T o x i c i t y III B III C
4.2.3.6 Local tolerance Local Tolerance III H
4.2.3.7 O t h e r t o x i c i t y studies O t h e r Information IIIQ
4.3 LITERATURE REFERENCES OTHER INFORMATION IIIQ
M O D U L E 5- C L I N I C A L S T U D Y REPORTS
CTD E U C T D (NTA, Vol. 2 B , Edition 2001) N T A , V o l . 2 B (Edition 1998) NTA
5.2 T A B U L A R LISTINGS O F ALL EXPERT REPORT O N THE

CLINICAL STUDIES CLINICAL DOCUMENTATION,
A P P E N D I X 2: W R I T T E N
SUMMARY -TABULAR OVERVIEW I C 3
5.3 CLINICAL STUDY REPORTS CLINICAL DOCUMENTATION IV
5.3.1 Reports of B i o p h a r m a c e u t i c S t u d i e s Pharmacokinetics IV A 2
5.3.2 Reports of S t u d i e s Pertinent to P h a r m a - Pharmacokinetics

cokinetics using H u m a n Biomaterials IV A 2
5.3.3 Reports of h u m a n p h a r m a c o k i n e t i c Pharmacokinetics

( P K ) studies IV A 2
5.3.4 Reports of h u m a n p h a r m a c o d y n a m i c Pharmacodynamics

( P D ) studies IV A 1
5.3.5 R e p o r t s of e f f i c a c y a n d safety studies C l i n i c a l Trials IV B 1
5.3.6 R e p o r t s of post-marketing e x p e r i e n c e Post-marketing e x p e r i e n c e

(if a v a i l a b l e ) IV B 2
5.3.7 C a s e report forms a n d i n d i v i d u a l A p p e n d i x to e a c h c l i n i c a l study

patient listings, w h e n s u b m i t t e d report, w h e n s u b m i t t e d
( A p p e n d i x 16.3) IV B 1
5.4 LITERATURE REFERENCES PUBLISHED AND UNPUB-

LISHED EXPERIENCE (OTHER IV B 3
T H A N 1) O T H E R INFORMATION IV Q
237
About the auth
Professor Gyula Mozsik, MD, Sc.D.
He was born in Dancshaza, Hungary in 1938. After earning a Medical Doctorate at

the University of Debrecen in Hungary in 1962, he served as an assistant at the
Second Department of Medicine at the University Medical School of Debrecen in
Hungary, where he received the degree of specialist in internal medicine in 1967. He
was visiting scientist at the Department of Pharmacology at the University of Oslo
in Norway from 1968-1969. The following year he received a Philosophy Doctorate
(Ph.D.) in medicine at the University of Pecs. As assistant professor of the First
Department of Medicine, at the Medical University of Pecs in 1969, he worked as a
trainee in clinical pharmacology at the Medical Universities of Debrecen and Pecs
as well as a trainee in experimental and clinical biochemistry at the Medical
University of Pecs and the University of Oslo from 1962 to 1970. He received a
Science Doctorate (Sc.D.) in medicine in 1977 at the University of Pecs, Hungary.
He became a specialist in gastroenterology in 1980, and was visiting scientist in the
Chemical Pathology Laboratory of Harvard Medical University of Boston,
Massachusetts, USA in 1985. Four years later he became full professor of internal
medicine at the First Department of Medicine, Medical University Pecs, he was
named head of the department from 1993 to 2003. He was full professor of medicine
from 2003 to 2008, and from May 2008 is Professor Emeritus at the First Department
of Medicine, University of Pecs, Hungary.
Serving on the Advisory Board of the National Institute of Dietetics of the
Hungarian Ministry of Health, he is a member of the European Society for Clinical
Investigation, Hungarian Society of Physiology, Hungarian Society of Pharma-
cology, Hungarian Society of Nutrition, Hungarian Society of Gastroenterology,
International Brain-Gut Society, International Society of Internal Medicine,
International Society of Metabolic Therapy, American Dietetical Association,
American Gastroenterological Association, International Union of Pharmacology
Gastrointestinal Section's Transition Committee (1989), National Board of Gastro-
enterology and New York Academy of Sciences. Serving on the Standing Committee
of the International Conferences on Ulcer Research, General Secretary of Executive
Committee (from 2000). He is the leader of both the Gastroenterological and Clinical
Nutrition and Dietetic Sections of the Pecs Committee of the Hungarian Academy of
Sciences. He is the President of the International Union of Pharmacology Gastro-
intestinal Section (from 2002). He is the member of Leadership of the Hungarian
241
Society of Gastroenterology, Hungarian Society of Internal Medicine, Transda-
nubian Section of the Hungarian Society of the Internal Medicine and he is the
President of this Section (from 1998).
During his activity five Szechenyi Awards, three Bolyai Awards and three Bekesi
Awards were given to the colleagues of the First Department of Medicine. Twenty-
eight colleagues of the Department received Ph.D. qualification, eleven physicians
obtained the title of the habilitation and three physicians were named as Academic
Doctor (Sc.D.). Six physicians became full professors of medicine and one of
Professor of the Health Sciences from the member of the First Department of Medi-
cine, Pecs University, and two researchers became full professors of Pharmacology
at Universities of Habana (Cuba) and of Cairo (Egypt).
r d
The Department of Family Medicine and 3 Department of Medicine and De-
partment of Human Nutrition and Dietetics were established by the members of the
First Department of Medicine.
Professor Mozsik is the author of seventeen textbooks for college students in clin-
ical nutrition and dietetics, 20 textbooks for medical students. He wrote 16 mono-
graphs, edited 26 books, 191 book chapters, 350 regular papers (published in Eng-
lish), and he participated in giving 700 lectures. The Hungarian Ministry of Health
gave to Professor Mozsik the award of Excellent Worker of Health in 1977, while
the Hungarian Ministry of Education named him Excellent Worker of Education in
1983. That year, he received the Hetenyi Geza medal, the highest medal for gas-
troenterological research, from the Hungarian Society of Gastroenterology. He
received the Sos Jozsef medal from the Hungarian Society of Nutrition in 1984, the
medal of Pro Optimo Merito in Gastroenterologia from the Hungarian Society of
Gastroenterology in 1989, Diploma of the International Brain Gut Society in 1997,
the High Quality Price of Hungarian Academy of Sciences (for the book of Mozsik
Gy., Abdel Salam OME., Szolcsanyi J.: Capsaicin Sensitive Afferent Nerves in
Gastric Mucosal Damage and Protection. Akademiai Kiado, 1998) in 1998, He re-
ceived Exellent Prize of the Intellectual Work from the Faculty of the Health Science
from the Pecs, University for the book of Metabolic Ward in Human Clinical
Nutrition and Dietetics 2005 in 2006 and Excellent Prize of the Intellectual Work
from the Medical Faculty of Pecs, University for the book Discoveries in Gas-
troeterology: from Basic Research to Clinical Perspectives 1960-2005 in 2007.
Award of the Hungarian Society of Internal Medicine Transdanubian Section in
1999, and the medal of Pro Facultate Sciential Sanitatis from the University of Pecs
in 2002. From 1998 he won Szechenyi Scholarship (by the Hungarian Government).
In 2003 the Rector of University of Pecs gave him the Pro Facultate Medicinae-
Golden Medal.
His special fields for the research are: internal medicine, experimental and clini-
cal nutrition (dietetics), gastroenterology, clinical pharmacology, clinical biochem-
istry and genetics.
242
Andras Domotor, MD
Date of Birth: December 5, 1980

Place of Birth: Szekszard, Tolna County, Hungary
Nationality: Hungarian
Citizenship: Hungary
Marital Status: Single
Working place: First Department of Medicine,
Medical and Health Centre
Medical Faculty
University of Pecs, Hungary
H-7643, Ifjusag u. 13., Pecs, Hungary
Home address: Epitok utja 14. H-7030 Paks, Hungary
E-mail: domotorjandras @yahoo. com
Education:
1995-1999. Vak Bottyan High School, Paks, Hungary
1999-2005. Medical University of Pecs, Hungary.
2005- PhD student at the University of Pecs, Hungary.
Subject: The mechanisms of gastrointestinal mucosal damage and protection.
Supervisor: Prof. Dr. Gyula Mozsik
Scientific Student Association:

2000-2002. Biochemistry and Medical Chemistry, University of Pecs
Subject: Detection of GSH and GSSG by HPLC
Supervisor: Prof. Dr. Robert Ohmacht
st
2003-2005. 1 Department of Medicine, Medical and Health Centre of University
of Pecs
Subject: Capsaicin-sensitive afferent nerves in the development of human gastroin-
testinal disorders
Supervisor: Prof. Dr. Gyula Mozsik
243
Tibor S. Past, PhD
Date of Birth: January 05. 1944.

Place of Birth: Pecs, Baranya County
Citizenship: Hungarian
Marital Status: Married
Education
Graduated Eotvos Lorand University, Faculty of Sciences Subject Chemistry (1968).
Hospital and University Appointments:
st
Chem.eng. sci. research worker, 1 Department of Medicine, Med. Univ. Pecs 1969.
st
Chem.eng. senior researcher, 1 Department of Medicine, Med. Univ. Pecs 1982.
Invited lecturer of Janus Pannonius University, Faculty of Sciences Subject
Chemistry 1995.
License
Licensed Biology
Certified Med.Univ. Pecs, Hungary, 1973.
Licensed Isotopic Diagnostics and Therapy
Certified Postgraduate Medical Institute Budapest, Hungary, 1973.
Licensed Biometry
Certified Medical Post-graduate Institute Budapest, Hungary, 1978.
Licensed Candidate in Biological Scholarship
Certified Hung. Sci. Academy, 1982.
Societies
Hungarian Pharmacological Society (1970)
Hungarian Society of Nuclear Medicine (foundation member) (1972)
Hungarian Chemical Society (local secretary (1993)) (1970)
Hungarian Gastroenterological Society (1978)
Hungarian Humangenetic Society (1980)
John von Neumann Computer Society (1984)
Hungarian Diabetes Society (1982)
Member of Committee of the Hungarian Academy of Sciences on Coordination of
Research of Biologically Active Compounds (1975)
Member of Management of Polytechnic and Natural Science (local, Baranya
County) Associations (1993)
Member of Local Committee of the Hungarian Academy of Sciences (1993)
Public member of the Hungarian Academy of Sciences (1995)
244
Grants and Contracts
Pharmacokinetic, metabolization and bioavailability studies of about 55-60 drugs
which were produced by Hungarian and foreign pharmaceutical works, certified by
the archives of the National Institute of Pharmacy and the archives of different
Hungarian pharmaceutical works, Research Institute for Pharmaceutical Chemistry,
Budapest, Tiszavasvari, Debrecen (Hungary) and abroad.
Developments of new drugs [Tisacid, Cr(III)-complexes, dietary fibre preps, artifi-
cial dental cleaner, oral capsaicinoids contained compositions, antifungal enamel
and solution, eye drop], manufacturing process, new analytical instruments (H-tester,
electric alcohol-tester, electric gastrointestinal transit time tester, instrument for con-
trolled electric deposition), new analytical methods (quantitative analysis of
transuran elements in environmental and power plant samples, new electric-deposi-
tion methods, quantitative measurement of iodine micro amounts in complex phar-
maceutical samples), new therapeutic method (in vivo dissolution of pigment gall-
stones).
Certified by partly the archives of the Hungarian Patent Office, Home Office, partly
in archive of Beres articles of association and different scientific journals.
245
Viktoria Vas
She was born in 1984 at Szekszard. She finished secondary school in 2000. She
entered the Faculty of Adult Education and Human Resourches, University of Pecs,
in 2004. She successfully finished the First Degree of first teaching program of Bo-
logna in this field, and she started her studies for receiving the Master Degree. Her
special field is human manager. She joined the work of the Regional South-Danubian
Knowledge Centre, as a co-worker to the leader of one subchapter involved in this
programme (Gy.M.).
She typed the whole text, prepared figures and tables for the book. She carried out
all of the necessary corrections on the final form on the manuscript, as well as on the
proof of this book (together with Gy.M.).
She helped us in the solutions of our scientific and human problems during the
research period of 2005-2008 (in the above-mentioned programme) based on her
knowledge on Adult Education and Human Resources.
Besides this, she participated in the preparation of five books, published in
Hungarian and English.
246
Pal Perjesi, PhD
He was born in Oroshaza, Hungary in 1956. He received an M.S. degree in Pharmacy

at the University of Szeged (Hungary) in 1979. After qualifying he started work as a
graduate fellow at the Institute of Pharmaceutical Chemistry of the University
Medical School of Szeged. As a result of his work here he got a "University Doctor"
degree qualification in Pharmaceutical Chemistry in 1983. Since 1981 he has been
working at the University of Pecs (Hungary). He completed and defended his disser-
tation for the "Candidate" degree in organic/pharmaceutical chemistry at the
Hungarian Academy of Sciences in 1994. Based on his "Candidate" degree he
received a Doctor of Philosophy (PhD) degree at the University of Szeged in 1996.
He got a Specialization Certificate of preparative chemical laboratory investiga-
tions (1985) and that of toxicology (1994) from the Postgraduate Medical School
(Budapest). In 1990/1991 he worked at the Department of Medicinal Chemistry,
University of Washington, Seattle (USA) as a Fogarty International Fellow. In 2001
and 2003 he worked at the University of Florida, Gainesville (USA) as a visiting sci-
entist at the Center for Drug Discovery and the Department of Medicinal Chemistry,
respectively. In 2000 he got a Habilitation Certificate from the University of Pecs. At
present, he is working as an Associate Professor, the founder and the first head of the
Institute of Pharmaceutical Chemistry, School of Pharmacy, University of Pecs. He
also serves as the head of the GLP Laboratory of the Institute of Pharmaceutical
Chemistry and the MEDIPOLISZ (Regional University Science Center of Pecs),
which actively participates in the drug development projects sponsored by the
National Office for Research and Technology.
His scientific interest is focused on the investigation of structure-to-reactivity and
structure-to-biological activity relationship of selected natural products and their
synthetic analogs. So far he has been involved in chemical, physico-chemical and
biological investigations of chalcones, cyclic chalcone analogues, estrogens and
synthetic TRH analogues. Recently, he has started research to investigate molecular
pathways of degradation and metabolism of active pharmaceutical ingredients. In
connection with these latter studies, his former research activities have been extend-
ed by the development of analytical methods to be used during stability as well as
preclinical and clinical studies.
247
He is coauthor of more than 80 publications and 90 conference presentations. He
is a member of the Hungarian Pharmaceutical Society, the Hungarian Chemical
Society, and the Medicinal Chemistry and Pharmaceutical Technology Section of
the Hungarian Academy of Sciences. Since 2005 he has been serving as President of
the Section of Drug Research of the Hungarian Pharmaceutical Society. He is mem-
ber of the editorial advisory board of The Open Medicinal Chemistry, and Open
Medicinal Chemistry Letters. He is member of the editorial board of Gyogyszere-
szet (Pharmacy) and Acta Pharmaceutica Hungarica.
248
Monika Kuzma
She was born in Nagykanizsa, Hungary in 1982. She received an M.S. degree in
Pharmacy at the University of Pecs, Hungary in 2005. After qualifying she started
work as a graduate fellow at the Institute of Pharmaceutical Chemistry of the
University Medical School of Pecs and she is a qualified pharmacist.
At present, she is working as a research fellow of the Institute of Pharmaceutical
Chemistry, School of Pharmacy, University of Pecs and participates in the teaching
program of Pharmaceutical Chemistry. She also serves as the analytical associate of
the GLP Laboratory of the Institute of Pharmaceutical Chemistry and the MEDI-
POLISZ (Regional University Science Center of Pecs), which actively participates
in the drug development projects sponsored by the National Office for Research and
Technology.
Her scientific interest is focused on the investigation of non-enzymatic metabolism
of non-steroidal anti-inflammatory drugs, especially related to salicylic acid and its
hydroxylated derivates. Recently, she has participated in the development of analyti-
cal methods to be used during stability as well as preclinical and clinical studies.
She is a member of the Hungarian Pharmaceutical Society.
249
Gyula Blazics, PhD
H-7720 Pecsvarad, Pannonpharma ut 1., Hungary * Tel: (+36) 72 566 750

E-mail: blazics.gy@pannonpharma.hu;
Personal
Date of birth: 10 January, 1953
Nationality: Hungarian
Marital status: married
Objective
To use my educational and practical maximum performance experience in a profes
sional business, or management environment while pursuing personal and com
pany set goals
Education
Budapest Medical University - Pharmaceutical Faculty, Hungary, 1972-1977
Training for qualification of Inspector Pharmacist, 1981
Training for GMP production, 1993
Coating course at COLORCON (Orpington-UK) 1995
Profile
Eight-year experience in drug control
More than 14-year experience in pharmaceutical production
Advanced GMP knowledge (involving QC&QA fields) and application in pharma
ceutical fields
Advanced experiences in planning GMP conform plants - especially for OSD phar
maceuticals
Advanced experiences in generic development, especially for solid forms
Proven ability to initiate, maintain and develop positive business relationships
Qualifications
Pharmacist 1977
Inspector Pharmacist 1981
PhD (phytochemistry) 1982
Expert of physical, physical-chemical and chemical drug control 1985
Expert of pharmaceutical technology 1994
QP 2004
250
Experience
09.2002- Quality Assuarance Director of Pannonpharma Pharmaceutical Ltd
2002-1999 Managing Director of Pannonpharma Pharmaceutical Ltd
1999-1993 Manager of Production & Development of Pannonpharma Pharma-
ceutical Ltd
1989-1993 Manager of Pharmaceutical/Galenic Laboratory of Pannonmedicina Co.
1982-1993 Inspector Pharmacist of Baranya Regional Pharmaceutical Co.
Honors and activities
Adviser member of the Management of the Analytical Session of the Hungarian
Pharmaceutical Society
International Experience
Traveled through thirteen European and Asian countries (involving Egypt and India)
over last fifteen years, including GMP audits and some business development dis-
cussions at appr. 20 pharmaceutical companies.
251
Janos Szolcsanyi, MD
Present position:
Professor Emeritus, Department of Pharmacology and Pharmacotherapy Faculty of
Medicine, University of Pecs and head of the Neuropharmacology Research Group
of the Hungarian Academy of Sciences
H-7624 Pecs, Szigeti str. 12.
Personal
Date of Birth: 24 February, 1938
Place of Birth: Budapest
Marital Status: Married, two children
Degrees
1962 M.D. (Univ. Medical School of Szeged "summa cum laude")
1977 C.Sc. (PhD)
1987 D.Sc. (Hungary)
1995 Corresponding member of the Hungarian Academy of Sciences (HAS)
2001 Regular member of HAS
Employment
1962-1964 Research Associate: Dept. Pharmacol. Univ. Med. School of Szeged
1965-1966 Warner Research Fellowship in the Department of Pharmacology,
University of London, King's College
1966-1970 Research Associate of the Hungarian Academy of Sciences
(1966-1967), then Assistant Professor (1968-1970) in the
Department of Pharmacology, University of Szeged
1970-1977 Associate Professor in the Department of Pharmacology, University
of Pecs, Hungary
1977-1978 Visiting Associate Professor in the Department of Physiology,
University of North Carolina at Chapel Hill, USA (Professor Ed Perl)
1981-1994 Visiting scientist in the Max-Planck-Institut fiir physiologische und
klinische Forschung W.G. Kerckhoff Institut, Bad Nauheim (BRD)
for 14 months
252
1985- Visiting professor in II. Physiologisches Institut, Universitat
Heidelberg (BRD) for 7 months (Professor H.O. Handwerker)
1988-1991 Consultant of the Sandoz Institute for Medical Research, London
(Professor H.P. Rang).
1990- Professor in the Dept. of Pharmacology, University of Pecs
1994-2003 Head of the Department of Pharmacology, then Pharmacology and
Pharmacotherapy, University of Pecs
1991-1995 Pro-Rector for Research of the University Medical School of Pecs
1995- Head of the Neuropharmacology Research Group of the Hungarian
Academy of Sciences in Pecs
Professional associations
Scientific committee membership:
Hungarian Pain Society (president)
Hungarian Society for Experimental and Clinical Pharmacology (president elect)
Doctor of Science (D.Sc.) committee of HAS (chairman)
Committee of HAS on Drug Research and Pharmacotherapy
Neuroscience Research Committee of the Hungarian Ministry of Health Social and
Family Affairs (chairman)
Hungarian Neuroscience Association (council member)
IASP Committee on Research (member 1997-2000)
r d
Member of the Scientific Committee of the 3 EFIC Meeting, Prague 2003. and 4th
EFIC Meeting, Istanbul, 2006
Councillor of EFIC
Other societies
Hungarian Physiological Society
British Pharmacological Society
International Association for the Study of Pain (IASP)
Federation of European Neuroscience Societies (FENS)
European Neuropeptide Club (founding member)
Society for Neuroscience (USA)
Honours and awards

1987 Award for Excellence in Teaching (Ministry of Education, Hungary)
1990 Nonvoting member of the Hungarian Academy of Sciences
1993 Medal of the Helsinki University
1994 Szent-Gyorgyi Albert Award (Ministry of Culture and Education, Hungary)
1995 Corresponding member of the Hungarian Academy of Sciences
1996 Plaque of Appreciation (Seoul National University)
1997 Batthyany-Strattmann Award (Ministry of Welfare, Hungary)
1998 Issekutz Medal, Hungarian Pharmacological Society
1999 Ipolyi Arnold Medal of the National Science Foundation (OTKA)
1999 Award of Excellence of the Hungarian Academy of Sciences
2000 Szechenyi Professorship
2001 "Pro Facultate Medicinae" gold medal of the University of Pecs
253
2001 Medal of the Krakow University.
2003 Jancso Miklos Award, University of Szeged
2003 Szechenyi Award, Hungary
2004 Manfred Zimmermann Award (ENC)
2004 Valyi-Nagy Tibor Award
Main research field

Capsaicin and pharmacology of nociceptors, neurogenic inflammation. Preclinical
pharmacology of two new drugs (thymoxamine, setastine), patents on somatostatin
analogs.
Publications
The number of full publications is 179 (121 in international journals, 20 in
Hungarian journals in English, 37 book chapters, 1 book, 1 book edition). During
the last 5 years one European and one Hungarian Patent.
Citation
Independent SCI citation is around 5600 (full SCI citations: 6033). The most cited
paper received 838 citations and further 16 papers over 100 SCI citations (102-328).
Around 70% of the citations are on papers from his own laboratories without foreign
coworkers.
Keywords of PubMed (first appeared under his name): Thymoxamine (1965),

Capsaicin (pharmacological) receptor (1975), "Capsaicin-sensitive" afferents
(1979), "Sensory-efferent" or local efferent function of sensory receptors in differ-
ent organs (gut 1978), gastric ulcer (1981), bronchi (1982), Setastine (1990),
"sensocrine" function of sensory receptors (2003), and among the firsts "capsaicin
and pain".
254
T h e v i s u a l c h a r a c t e r i z a t i o n of c a p s a i c i n o i d s c o n t a i n i n g pills
255

Gyula Mózsik, András Dömötör, Tibor Past, Viktória Vas, Pál Perjési, Mónika Kuzma, Gyula Blazics, János Szolcsányi Capsaicinoids, From The Plant Cultivation To The Production of The Human Medical Drug

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Gyula Mózsik, András Dömötör, Tibor Past, Viktória Vas, Pál Perjési, Mónika Kuzma, Gyula Blazics, János Szolcsányi Capsaicinoids, From The Plant Cultivation To The Production of The Human Medical Drug

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168x238_paprika_cimnegyed_vegleges.qxd 2009.04.21.

ISBN 978 963 05 8694 8

Gyula Mzsik, 2009

Published by Akadmiai Kiad

2. Capsaicin (capsaicinoids) is (are) a famous family of the spices . . . . . . . . . . . . 21

4. Cultivation of Capsicum or paprika . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

5. General chemical structure and composition of Capsicums . . . . . . . . . . . . . . . 39

7. General physiology of retinoids and carotenoids. . . . . . . . . . . . . . . . . . . . . . . . . 81

9. Toxicological studies with capsaicin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

9.2.5. Summary of the observations with capsaicin alone or in combination

12.3.3.2.1. Human clinical pharmacological phase

Pcs, Hungary, September 2008

The participants of this multidisciplinary group receiving the grant (National

Pcs, Hungary, September 2008 The Authors

1.1. General introduction to the interactions of foods (or food

1.1.1. Foods and food components

Our attention is focused on the nonsteroidal anti-inflammatory drugs (NSAIDs).

1.1.3. Levels of the drug-food interactions in healthy human

becomes insufficient, however, in patients with ileitis terminalis B absorption

3. Most of the macronutrients (carbohydrates, proteins) are absorbed by an active

1.1.4. A short historic background of the interaction between the

The mechanism of action produced by capsaicin (capsaicinoids) is very wide both in

2 . 1 . Cultural background of spices

2.2. History of spices

The term "chilli" is a rather confusing terminology: "chilli", "aji", "paprika",

Language Common name

Amharic Mit'mita, Berbere

Burmese N g a yut thee, Nil thee

English C a y e n n e pepper, R e d pepper, C h i l l i , Chili

Estonian Kibe paprika

German R o t e r Pfeffer, C a y e n n e - P f e f f e r , Chili-Pfeffer, B e i f t b e e r e

Gujarati Lai m a r c h a (red), Lila m a r c h a (green)

Hebrew Pilpel adorn

Hindi Lai m i r c h (red), H a r i m i r c h (green)

Hungarian C s i l i p a p r i k a , Igen eros a p r o , C a y e n n e bors, C a y e n n i b o r s ,

Icelandic Chilipipar, Cayennepipar

Indonesian Lombok, Cabe, Cabai

Malay Lada merah

Marathi Lai m i r c h y a (red), H i r v y a m i r c h y a (green)

Russian Perets krasni

Sanskrit Marichiphala, Ujjvala

Singhalese Rathu miris, Gasmiris

Spanish C h i l e , G u i n d i l l a , C a y e n a inglesa, P i m i e n t a d e C a y e n a , P i m i e n t a p i c a n t e , Ajf

Language Common name

Thai Pisi h u i , Prik k h e e , Prik

Turkish A c i kirmizi biber

2.2.2. History of Capsicum in Hungary

Species Flower color Number flw/node Seed color Calyx constriction

C. annuum white 1 tan absent

C. pubescens purple 1-2 black absent

4 . 1 . Cultivation of Capsicums in India

4.2. Cultivation of Capsicums in other Asian countries

4.3. Cultivation of Capsicums in Africa

4.4. Cultivation of Capsicums in America

4.5. Cultivation of Capsicums in Europe and Hungary

4.5.1. Cultivar types in Hungary

4.5.1.1. Sweet Capsicum cultivar

W h i t e fruit, d e t e r m i n a t e without pungency

Pale g r e e n fruit, i n d e t e r m i n a t e without pungency

Hornshaped, indeterminate pungent, without pungency

P o i n t e d , hot, i n d e t e r m i n a t e without pungency

P o i n t e d , hot, i n d e t e r m i n a t e without pungency

Tomato-shaped without pungency