BACAAN

FURTHER INVESTIGATION ON
ANTIHYPERLIPIDEMIC ACTIVITY OF
AEGLE MARMELOS CORR.
BY
MADURI SHASHANKA RAO

Dissertation submitted to the
KLE Academy of Higher Education & Research – Deemed University, Belgaum,

Karnataka
in partial fulfilment of the requirements for the degree of
MASTER OF PHARMACY
IN
PHARMACOLOGY
Under the guidance of
Mr. PRASANNA GS
Department of pharmacology,
KLE University’s college of Pharmacy, Bangalore-560010
2011
KLE ACADEMY OF HIGHER EDUCATION AND RESEARCH –
DEEMED UNIVERSITY, BELGAUM, KARNATAKA
DECLARATION BY THE CANDIDATE
I hereby declare that this dissertation entitled
“FURTHER INVESTIGATION ON
AEGLE MARMELOS CORR.”
is a bonafide and genuine research work carried out by me under
the guidance of Mr. PRASANNA GS, Assistant Professor of
Pharmacology, KLE University’s College of Pharmacy,
Bangalore.
Date:
Place: MADURI SHASHANKA RAO
KLE UNIVERSITY’S COLLEGE OF PHARMACY,
BANGALORE-560010
(A constituent unit of KLE Academy of Higher Education and
Research – Deemed University)
CERTIFICATE BY THE GUIDE
This is to certify that the dissertation entitled
is a bonafide research work done by M ADURI SHASHANKA
RAO under my supervision and guidance, in partial fulfilment
of the requirement for the award of degree of MASTER OF
PHARMACY, PHARMACOLOGY
Mr. PRASANNA GS,

DATE: Assistant professor in Pharmacology,
KLE University’s college of Pharmacy,
PLACE: Bangalore
Bangalore-560010
KLE UNIVERSITY’S COLLEGE OF PHARMACY,
BANGALORE-560010
(A constituent unit of KLE Academy of Higher Education and
Research
– Deemed University)
ENDORSEMENT BY THE HEAD OF THE DEPARTMENT AND

THE PRINCIPAL/HEAD OF THE INSTITUTION
This is to certify that the dissertation entitled
is a bonafide research work done by
MADURI SHASHANKA RAO
Under the guidance of

Mr. PRASANNA GS
Dr. Purnima Ashok, Dr. B.G. Desai

Professor & Head of the Department Principal
Dept. of Pharmacology
DATE:
PLACE: Bangalore
COPYRIGHT
DECLARATION BY THE CANDIDATE
I hereby declare that the KLE Academy of Higher Education &

Research have the rights to preserve, use and disseminate this
dissertation/thesis in print or electronic format for academic /
research purpose.
Date:
Place: Bangalore MADURI SHASHANKA RAO
© KLE Academy of Higher education and Research-Deemed University

Acknowledgement
Always bear in mind that your own resolution to succeed is more
important than any one thing.
-Abraham Lincoln
16th president of US (1809 -
1865)
Starting with the words of one of the greatest presidents- Abraham Lincoln, I would
like thank from the bottom of my heart and convey my sincere and kind gratitude to
everyone who helped me in completing my project successfully.
I am indebted to my parents Shri Maduri Srinivasa Rao and Maduri

Shyamala who have been encouraging me since my childhood in doing the things of
my own interest and let me to put my step forward in my desired ways to complete
the short term and long term goals and even for their moral support in completing the
work.
I wish to express my special thanks to my brother Sunil and my granny for

providing their all time encouragement in doing this work.
I would like to convey my sincere gratitude to my guide Prasanna GS for his

meticulous encouragement in doing this project. His generous ideas and the data
management helped me a lot in doing the work with utmost feasibility. His useful
discussions “With a Cup of Cappuccino” in the evenings made me to
inculcate the analytical way of thinking in doing research oriented works.
I am especially grateful to Dr. Purnima Ashok, Head of the Department,

Pharmacology for all time faith, suggestions and advices in the classroom which laid
the foundation to improve my scientific knowledge.
I would like to convey my token of gratitude to Prof. Dr. B.G. Desai,

Principal, and Prof. Y D Satyanarayana, Vice Principal KLE University’s college of
pharmacy, Bangalore for their moral support and providing all the requirements
needed in completing my project successfully.
I am thankful to my ever caring lecturers Mrs G P Rajani, Mrs Vanitha S

and Mr. Basavaraj Hulkoti for their great support.
I thank all the non teaching staff Mr. Sathish, Librarian for providing the
required books and journals to study and in gaining good knowledge. Mr. Biradar,
Mr. Sanjay, Mr. Ajit for their help in doing the laboratory work. Even I am thankful
to:
i
My colleagues Sahithi, sowjanya, Deepak kumar, Renuka, Deepak gupta,
Rajpreet, Nirlep, Chetan and Rema for their extreme support and love towards me.
My friends of chemistry Viswa, Garvita, Gayathri, Paramesh, Ramjith and

all other batchmates for their co operation in doing my project.
My friends of pharmaceutical technology, Dhaval, Chirag and all other

fellowmates for their support during my work.
My seniors Radhakrishna, Rajesh, Vivek, Mrs Komala, kayur, Sowjanya,

Gokul, and Kushal for their patient support and ‘n’ number of advices and
suggestions in learning the core pharmacology concepts.
My beloved juniors Deepthi, Nishi, Ramakrishna, Sagar, Rajsekhar,

Mani, Shreya, Renuka shukla and Ramadevi for their co operation and support in
doing my work.
Also I would like to thank all companions, well wishers- Praveen, Harsha,
Navya, Veenasa, Vamshi....... Finally I would be definitely indebted to all the
animals I sacrificed for a scientific reason with a good motto of widening the horizons
of Pharmacology.
ii
LIST OF ABBREVIATIONS
AHD Atherosclerotic heart disease
ANOVA Analysis of variance
CPSCEA Committee for the purpose of control and supervision of experimental animals
EDTA Ethylene diamine tetra acetic acid
EEAM Ethanolic extract of Aegle marmelos type of cholesterol
g/dl Gram per deciliter
HDL-C High density lipoprotein type of cholesterol
HMG CoA Hydroxyl methyl glutaruyl coenzyme A
Kcl Potassium chloride
LDL-C Low density lipoprotein type of cholesterol
LPO Lipid peroxidation
MDA Malondialdehyde
mg/dL Milligram per decilitre
NADPH Reduced nicotinamide adenine dinucleotide phosphate
NaOH Sodium hydroxide
nmol Nano mole
P.O Per oral
PPARα Peroxyzome proliferator receptor activator α
PUFA Poly unsaturated fatty acids
RBC Red blood corpuscles
SEM Standard error mean
STZ Streptozotocin
Sq mm Square millimeter
TBA Thiobarbituric acid
iii
TBARS Thiobarbituric acid reactive substances
TC Total cholesterol
TCA Trichloro acetic acid
TG Triglyceride
TNF Tumour necrosis factor
VCAM Vascular cell adhesion molecule
VLDL Very low density lipoprotein
WBC White blood corpuscles
iv
ABSTRACT
Objective:
The current study was carried to further investigate the reported anti hyperlipidemic
activity of ethanolic extract of Aegle marmelos in hyperlipidemic albino rats.
Materials and methods:
Test animals were rendered hyperlipidemic by oral administration of 500mg/kg

cholesterol, confirmed hyperlipidemia. Serum triglyceride levels, total cholesterol
levels, fecal cholesterol excretion levels, HMG CoA reductase levels, MDA levels,
hematological profile, effect on weights of visceral organs, body weight, feed intake
were assessed in extract treated animals.
Results:
In hyperlipidemic animals, ethanolic extract of leaf of Aegle marmelos produced

significant antihyperlipidemic activity- reducing the triglyceride and total cholesterol
levels (p<0.01, p<0.001) and increasing the fecal cholesterol excretion. The decrease
in levels of HMG CoA reductase (p<0.001) and lipid peroxidation was also noticed.
The extract treated animals showed lighter livers and reduced weights of other
visceral organs like heart, lungs and kidneys, compared to untreated animals and a
relatively lower level WBC levels was observed in extract treated animals, compared
to untreated hyperlipidemic animals. An insignificant change in body weight and feed
intake at the end of the study were recorded by extract treated animals. Some changes
were found to be dose-dependent.
Conclusion:
Changes are suggestive of antihyperlipidemic effect of ethanolic extract of Aegle

marmelos, with significant influence on lipid peroxidation – likely to be due to
chemical constituents.
Keywords
Aegle marmelos, hyperlipidemia, fecal cholesterol excretion, lipid peroxidation,

Malondialdehyde, HMG CoA reductase.
v
TABLE OF CONTENTS
Sl. No CONTENTS PAGES
1 Introduction 1-4
2 Aim and objectives 5
3 Review of literature 6-23
4 Materials and methods 24-32
5 Results 33-53
6 Discussion 54-58
7 Conclusion 59
8 Summary 60-61
9 References 62-66
vi
LIST OF TABLES
Sl. Title of the table Page

No. No.
1 Change in the levels of serum triglycerides in 34

NC, PC, extracts and Lovastatin treated groups.
2 Change in the levels of total cholesterol in NC, 36

PC, extracts and Lovastatin treated groups.
3 Effect of extract treatment on the levels HMG 39

CoA reductase enzyme in NC, PC, extracts and
Lovastatin treated groups.
4 Effect of extract treatment on the levels of fecal 41

cholesterol excretion in NC, PC, extract and
5 Effect on extract on the levels of MDA (lipid 43

peroxidation) in NC, PC, extracts and
6 Change in body weights of animals in NC, PC, 45

extracts and Lovastatin treated groups.
7 Effect of extract on weights of visceral organs 48

in NC, PC, extracts and Lovastatin treated
groups.
8 Effect of Extract on RBC and WBC levels in 52

NC, PC, extracts and Lovastatin treated groups.
vii
LIST OF FIGURES
Sl. No Title of the figure Page no.
1 Change in serum triglyceride levels in NC, 34

PC, Extract and Lovastatin treated groups
2 Change in total cholesterol levels in NC, PC, 36
Extract and Lovastatin treated groups
3 Effect on levels of HMG CoA reductase in 39
NC, PC, Extract and Lovastatin treated groups
4 Effect on levels of fecal cholesterol excretion 41
in NC, PC, Extract and Lovastatin treated
groups
5 Effect on levels of MDA in NC, PC, Extract 43
and Lovastatin treated groups
6 Change in body weights in NC, PC, Extract 45
7 Effect on weight of liver in NC, PC, Extract 49
8 Effect on weights of lungs in NC, PC, Extract 49
9 Effect on weight of heart in NC, PC, Extract 50
10 Effect on weight on kidneys in NC, PC, 50
Extract and Lovastatin treated groups
11 Effect on levels of RBC in NC, PC, Extract 53
12 Effect on levels of WBC in NC, PC, Extract 54
viii
LIST OF PLATES
Sl. No. TITLE OF PLATE PAGE NO.
1 Leaves of Aegle marmelos 8
2 Dreid fruit of Aegle 9

marmelos
3 Fruits of Aegle marmelos 10
4 Synthesis of different 11
types of lipoproteins
ix
Introduction
1. INTRODUCTION
A
therosclerotic diseases are a leading cause of disability and death
worldwide and two thirds of these cases are associated with dyslipidemia.
Approximately 10% of the world population is affected by dyslipidemia.
In developed countries (US, Europe and Japan) there are more than 240 million
people with the abnormal lipoprotein levels. Of these more than 55 million have low
levels of HDL-C and/or high triglyceride levels and could be candidates of HDL-C
raising therapies.1
With urbanization and westernization of the society, stress is increasing day by

day. The psychological stress induces chronic inflammatory process due to an
atherosclerotic lipid profile with the oxidation of lipids. This in turn plays a
significant role in the development of atherosclerotic heart disease.2
Atherosclerosis is referred as “silent killer.” American Heart association has

identified the primary factor associated with atherosclerosis as elevated levels of
cholesterol and triglycerides in the blood. So treatment of hyperlipidemia to be one of
the major approaches towards decelerating the atherogenic process.3
In early stage of atherosclerosis, monocyte derived macrophages invade the

vascular wall and take up cholesterol resulting in formation of foam cells. Cytokine –
induced adhesion molecules such as vascular cell adhesion molecule – I(VCAM-I)
and chemokines in vascular endothelial cells play important roles in the invasion by
monocytes and macrophages into vascular wall. At the molecular level, oxidized low
density lipoproteins play a critical role in development and progression of
atherosclerosis enhancing VCAM-I expression in endothelial cells.4
High dietary cholesterol intake induces hypercholesterolemia which is

associated with impairment of endothelial functions and elevated blood pressure
which finally lead to atherosclerosis.5
Healthy endothelium produces NO and other mediators that protect against

atheroma, so it is likely that the adverse effects on endothelium of many metabolic
(disorders) risk factors represent a common pathway by which they promote
formation of atheromatous lesions.
1
Introduction
Atherosclerosis involve
 Endothelial dysfunction and its injury encouraging attachment of monocytes

to the endothelial cells.
 Endothelial cells later bind to LDL. The already bound
monocytes/macrophages will oxidize LDL by generating free radicals,
resulting in lipid peroxidation and destruction of receptor needed for normal
receptor mediated clearance of LDL.
 Platelets, macrophages and endothelial cells release cytokines and growth
factors causing proliferation at smooth muscle and deposition of connective
tissue. Ultimately leading to structure of dense fibrous cap of connective tissue
overlying a core lipid and necrotic debris which we will usually call
“Atheromatous plaque.”
 The lipoproteins are usually transported in blood. They are LDL, VLDL,
HDL and chylomicrons. In atheroma, LDL and VLDL levels increase and
HDL levels which are necessary for heart functioning are decreased.
So the predisposing factors that usually include along with the high cholesterol diet
and food habits include hypertension, cigarette smoking, diabetes, family history and
coagulopathy.6
The treatment concentrates on different aspects like7
 In predominant hypercholesteremia, where the first line therapy include

statins which act on the enzyme HMG CoA reductase. Alternate therapy
includes Ezetimibe (for statin intolerants) that inhibits drug intestine
absorption.
 In Hypertriglyceridemia, drugs like Gemfibrozil, Clofibrate, Clopidogrel are
usually used which act on PPARα receptor. This inhibits the synthesis of
triglyceride rich lipoproteins and fatty acids and increase synthesis of HDL-C.
Alternate therapy includes fish oil n-3 fatty acids which inhibit platelet
aggregation.
 If the modest increase is seen in triglyceride and LDL-C, monotherapy is not
sufficient and combined therapy of statin and fish oil holds good.
2
Introduction
 The above mentioned Ezetimibe is also prescribed for all the individuals who
are intolerant to other lipid lowering drugs besides statins.8
Beside the synthetic medicine, a wide range of herbal drugs show the
antihyperlipidemic activity with the lesser side effects.
These usually include the herbal extracts like seed extract of Erythina varigata
(Fabaceae),9 leaves extract of Hibiscus sabdariffa,10 Telfairia occidentalis
(cucurbitaceae),11 Ananus comosus leaves,12 leaves of Trichilia conaroids13 etc.
Aegle marmelos (Linn.) corr. Ex Roxb, a plant of Indian origin having tremendous
therapeutic potential. It belongs to family Rutaceae, family of citrus fruits. The
chemical constituents of this herb include a wide range of composition of alkaloids
(Aegeline, Aegelenine), polysachharides(glucose, arabinose, uronic acid),seed
oil(composed of palmitic acid, stearic acid), tannins(like skimmianine in leaves) and
carotenoids. Marmelosin, skimmianine and umbelliferin are therapeutically active
principles of bael plant.14
NEED FOR THE STUDY
Review of literature reveal that Aegle marmelos possessing several pharmacological

activities that extend its use in diarrhea and dysentery, producing hypoglycemia (anti
diabetic) in diabetic animals, anti microbial (antifungal) activity, anti spermatogenic
activity, anticancer activity, cardio protective activity, antipyretic and analgesic
activities etc.
The current study envisaged further investigation of anti hyperlipidemic

activity of Aegle marmelos by assessing the influence of increasing doses of ethanolic
extract of leaves of Aegle marmelos on spectrum of activities related to lipid lowering
property.
The effect of two increasing doses (150mg/kg and 250mg/kg) of ethanolic extract of
leaves of Aegle marmelos (EEAM) in the above mentioned parameters was assessed
and compared with that of standard drug. Study is based on preliminary report of
Vijaya et.al
3
Introduction
15
Recently, Vijaya et al., have reported lipid lowering activity of ethanolic
extract of Aegle marmelos corr. in hyperlipidemic wistar albino rats. Most important
findings include
 Significant reduction in triglyceride and total cholesterol levels of extract

pretreated animals in triton induced hypercholesterolemia and similar
significant reduction in triglyceride and total cholesterol levels in high fat diet
induced hyperlipidemic animals in one week study and
 Consequent decrease in atherogenic index of extract treated animals.
 Antihyperlipidemic effect - comparable to Gemfibrozil.
The current study envisaged to further investigate the antihyperlipidemic activity and
study protocol was designed to
 Study and confirm dose dependent antihyperlipidemic activity (claimed) for

an extended duration, for 3 weeks.
 Possible influence of extract on lipid peroxidation, fecal cholesterol
(excretion), hematological features, hepatic HMG CoA reductase enzyme
levels following treatment,
 Influence of extract treatment on weight of various visceral organs.
Such a detailed investigation is likely to help understand the spectrum

of its activity and identifies a herb worth further study.
4
Aim and objectives
2. AIM AND OBJECTIVES

Aim: To further investigate antihyperlipidemic activity of leaf extract of Aegle
marmelos in hyperlipidemic animals.
Objective:
 To render normal albino rats into hyperlipidemic and confirming the same.
 To determine and compare serum triglyceride and serum total cholesterol of
vehicle treated normal animals, untreated hyperlipidemic, extract treated (two
doses) hyperlipidemic and standard treated hyperlipidemic albino rats.
 To assess and compare complete hematological profile that includes RBC and
WBC levels among the different drug treated standard and normal groups.
 To determine and compare fecal cholesterol excretion among above
mentioned group of animals.
 To measure liver HMG-CoA reductase activity in the isolated liver samples of
different groups of animals and their comparison to know the significant
effects of different drugs.
 To determine the level of lipidperoxidation (MDA) and compare between
above mentioned group of animals.
 To compare relative weight of visceral organs between above mentioned
group of animals to know the effect of test and standard drugs.
5
Review of Literature
3. REVIEW OF LITERATURE
Review of literature was carried out by searching data bases Eg: (www.pubmed.org)
of abstracted, peer reviewed articles, as well print and e-journals.
Literature review pertinent to current investigation is summarized in the following

three parts.
Part I: Aegle marmelos – It’s taxonomical details and uses in folklore/traditional

system of medicine.
Part II: Animal models used in the screening/evaluation of anti hyperlipidemic

animals.
Part III: Reported biological activities of Aegle marmelos (corr.)
6
PART- I
3.1 Taxonomical details of Aegle marmelos and use in traditional/folklore system

of treatment16, 17
Aegle marmelos corr. is a small or medium sized deciduous tree armed with straight
sharp axillary thorns, 2.5-6.3cm long, terete. Leaflets are 5-10 by 2.5-6.3 cm. ovate or
ovate-lanceolate, crenate, acuminate, membranous, pellucid- punctuate the lateral
opposite, sub sessile and the terminal long petiolated. Flowers greenish white, sweet-
scented, about 2.5cm across, 2- sexual in short axillary panicles. Calyx flat, pubescent
4-lobed; lobes rounded, sometimes obscure. Petals 4, spreading, oblong, thick, gland
dotted, much exceeding the sepals, imbricate.
Stamens –numerous; anthers-elongate, apical-elongate, apiculate filaments

free or fascicled, inserted round an inconspicuous disk. Ovary ovoid, cells 10-20;
style terminal, short, deciduous; stigma capitate; Ovules numerous, 2-seriate.
Fruit 5-18cm. diam., globose, grey or yellowish, rind woody. Seeds numerous,
oblong, compressed with a wooly mucous testa, embedded in orange – coloured sweet
pulp.
Different names of Aegle marmelos corr.
English: Bael fruit tree,

Indian: Bel,
Sanskrit: Bilva,
Distribution
Wild in sub-Himalayan tract, central and south India and Burma often planted all over
India and Burma. Plant is cultivated throughout India.
Parts of plants and their use in traditional system of medicine

Roots
The taste of roots is sweet. It cures
 Fever due to trishoda (pain in abdomen),
 Palpitations of heart,
7
 Urinary troubles,
 Hypochondriasis,
 Melancholia,
 Removes “vata”; “pitta” and “kapha”.
Decoction of root of Aegle marmelos is given with sugar and fried rice for checking
diarrhoea and gastric irritability in infants.
Leaves
Astringent- Digestive, laxative and febrifuge when fresh, useful in ophthalmic,
deafness and inflammation.
Made into poultice, used in treatment of ophthalmic, fresh juice diluted is praised in
catarrhs and feverishness. Fresh juice of leaves is given with the addition of black
pepper in Anasarca with costiveness and jaundice.
In external inflammations: The juice of leaves is given internally to remove the
supposed derangement of humours.
Expressed juice of leaves is used in Opthalmia and other eye infections.
In Malabar, a decoction of leaves is valued in asthama complaints. Hot poultice to the
head is used in delirium of fevers.
Plate 3.1.a: Leaves of Aegle marmelos

Flower
 To Allay thirst and vomiting; it is useful in dysentery.
 Water, distilled from the flower, is said to be aledipharmic.
8
Fruit
 Unripe fruit – oily, bitter, acrid and sour.
 Tasty but difficult to digest, appetizer; binding: cures dysentery; removes pain.
 Oil – hot, cures “vata”.
 Good for heart
 Extract of fruits lowered blood sugar level in normal rabbits but was
insignificant in diabetic animals.
 Essential oil is antifungal.
Plate 3.1.b: Dried fruit of Aegle marmelos
9
Plate 3.1.c: fruit of Aegle marmelos

Chemical composition:
 The chemical composition of Aegle marmelos includes umbelliferin,
skimmianine, marmin, β-sitosterol, lupeol and Y- sitosterol from immature
bark and roots.
 Fruit contains psoralein and tannic acid; aegelinol, furocoumarins,
furanocoumarin, marmelosin, marmelide.
 Ripe fruit contains xanthotoxol, marmesin etc.
 The pulp contains mucilage, pectin, reducing sugars, tannin, a volatile oil,
bitter principle.
 Fresh leaves yield yellowish green oil.
10
PART – II
Plate 3.2 Synthesis of different types of lipoproteins, cholesterol and

bile acids18
Partial summary of lipoprotein metabolism in humans.
 I to VII are sites of action of hypolipidemic drugs.
 I, stimulation of bile acid and/or cholesterol fecal excretion;
 II, stimulation of lipoprotein lipase activity;
 III, inhibition of VLDL production and secretion;
 IV, inhibition of cholesterol biosynthesis;
 V, stimulation of cholesterol secretion into bile fluid;
 VI, stimulation of cholesterol conversion to bile acids;
 VII, increased plasma clearance of LDL due either to increased LDL receptor activity
or altered lipoprotein composition.
 CHOL, cholesterol; IDL, intermediate-density lipoprotein.
11
3.2.1: Animal models for screening and evaluating anti

hyperlipidemic activity (potential)19
With advances progress in understanding consequences of hyperlipidemia alone or as
a secondary consequence of Diabetis mellitus, a robust animal model to assess and
evaluate anti hyperlipidemic activity is a necessity.
Complications due to hyperlipidemia include
 Atherosclerosis, one of the risk factor/predisposing factors is also considered
while screening drugs for anti hyperlipidemic activity.
 Conventionally test animals- both rodent and non rodents can be converted to
hyperlipidemic by feeding them on high fat diet rich in cholesterol (with bile
acid) or administered cholesterol (orally) for sufficiently longer period.
 Acute elevation in lipid profile in small lab animal is another choice. Eg:
Fructose and olive oil load to induce acute hyperlipidemia, Triton induced
triglyceridemia serves as acute model.
More reliable ones include hereditary models (of hypercholesteremia) and transgenic
animals.
Following is the brief description
1. Hereditary hypercholesteremia in rats

Hereditary hypercholesteremia is seen in genetically hyperlipidemic rats (RICO). In
contrast to zucker-rats, RICO rats are non-obese and triglyceridemic. The
hypercholesteremia of RICO rat is correlated to decreased rate of catabolism of
chylomicrons and LDL, but more specifically to an excessive production of these two
types of lipoproteins. This strain has been proposed to study the hypolipidemic drugs,
particularly those designed to decrease plasma concentrations of chylomicrons and
LDL.
Eg: Hypolipidemic effects of β- cyclodextrin were assessed using this model.
12
2. Hereditary hyperlipidemia in rabbits

Watanabe et al described strain of rabbits with hereditary hyperlipidemia. This has
been used by several scientists for studying development of atherosclerosis, as well as
histological and functional changes of the aorta.
In this strains animals at the age of 10-14 months homozygous animal exhibit an
atheromatous plaque, distributed heterogeneously over luminal surface of the aorta.
Serum cholesterol is increased up to 400-600mg/dL.
3. Transgenic animals
Several transgenic animals are used as disease models.
 Apo E knockout mouse created by Nubuyo Maeda, University of North
Carolina, Chappell Hill, NC.
 Apo E knock out mouse have spontaneously elevated plasma cholesterol
levels, and develop atherosclerosis even on irregular chow within 3-4 months.
 Time dependent progression of atherosclerosis leads to lesions similar in
histopathology to those observed in humans.
 This model is used for atherosclerosis research and validation.
Several models (as described below) can be used to assess influence of drugs
(potential) on lipid metabolism.
Influence on lipid metabolism
1. Hypolipidemic activity in rats
Hyperlipoproteinemia with increased concentrations of cholesterol and triglyceride
carrying lipoproteins is considered to be the cause of atherosclerosis with its dual
sequel of thrombosis and infarction. Increased levels of VLDL and LDL are taken up
by macrophages via scavengers mechanism.
So anti arteriosclerotic drugs should reduce VLDL and LDL levels and/ or elevate
HDL levels.
13
Procedure
 10 male Albino wistar rats weighing 180-200g are used. They are given once
daily in the morning over a period of 8 days. The test compounds or the
standard in various doses ranging from 1 to 100mg/kg via stomach in a
volume of 5 ml/kg.
 Control group is given the solvent (eg: PEG 400) only.
 Body weights of animals before beginning the experiment are registered.
 Then the basal blood cholesterol and triglyceride levels were measured 24
hours prior to the experiment.
 After 8 days, the TG and TC levels should be measured again.
 Liver is isolated and stored in liquid nitrogen at -25˚C for the lipid analysis.
Evaluation
Statistical differences between the controls and treatment groups are established by co
variance analysis.
Critical assessment
Normocholesterolemic animals faced difficulty that starting cholesterol levels are
relatively low and achieve significance for the lowering of cholesterol requires large
group of animal.
2. Hypolipidemic activity of Syrian hamsters
 Syrian hamsters are widely used to study effect of drug and diet on lipoprotein
metabolism to that of human.
 Increase in plasma cholesterol can be induced by adding small physiological
amounts of cholesterol to diet (0.05-0.01 wt %), saturating with coconut oil (5-
10 wt %) has synergistic effect.
 As stable hyperlipidemia with a human like lipoprotein pattern can be induced
in hamster within 2-3 weeks by the above mentioned diet.
 Hamster HDL cholesterol can be measured by precipitation of VLDL and
LDL with phosphotungstinic acid/ Magnesium chloride.
14
3. Triton induced hyperlipidemia

 Systemic administration of the surfactant triton to the mice or rats results in a
biphasic elevation of plasma cholesterol and triglycerides.
 Male Sprague Dawley rats (200-350g) starved for 18h are injected I.V with
200mg/kg. Triton WR 1339(iso octyl-polyoxy-ethylene phenol) .
 Serum levels increase sharply 2-3 times after 24h (phase I) and within next 24
(phase II), after which hypercholesteremia decrease nearly to control levels.
 Usually 6, 24, 48 hour analysis is made after simultaneously giving test and
standard drugs with triton.
 In phase I, mechanism of action is thought to be due to increased hepatic
synthesis.
 Drugs active in phase I are those, which effect synthesis of cholesterol.
 Drug acting on excretion are active in phase II.
 Triton induced hyperlipidemia is simple and rapid for the detection of
compounds interfering with synthesis and excretion of cholesterol.
4. Fructose induced hyperlipidemia
 Rats switched from a diet low in carbohydrates and high in protein to a high
intake fructose, develop an acute hypertriglyceridemia.
 Usually male Sprague dawley rats (200-250g) fed over a period of week with
high protein low carbohydrate meal.
 10 animals are treated for 3 days daily with test or standard drug or vehicle.
 From 2nd or 3rd day water is withheld for a 24 hour period. Immediately
afterwards, animals are offered 20% fructose solution and ad libitum for a
period of 20h.
 Then finally blood is withdrawn from the animals.
Several models are available to assess the ability of drugs to inhibit cholesterol
biosynthesis which includes assessment of invitro HMG CoA activity.
Following is the brief description of models to inhibit cholesterol absorption.
15
Inhibition of cholesterol absorption

Acyl co enzyme A: cholesterol acetyl transferase catalyses the intracellular formation
of cholesteryl esters, important role in absorption of cholesterol, foam cell within
arterial wall and VLDL production in liver
1. In vivo activity of ACAT
This is done to test in vivo anti-atherosclerotic and anti hyperlipidemic effect of
ACAT inhibitors in cholesterol fed hypercholesterolemic animals.
Procedure
 Male Sprague dawley rats (200-250g) are fed with diet of 5.5% peanut oil, 0.5
% cholic aid and 1.5% cholesterol with or without drugs for a week.
 On the last day, food is removed, in morning and isotopes are administered
after 4-5 hours.
 [3H] cholesterol is given by oral gavage (125 mg of cholesterol in 1625ml of
oil)
 [14C] cholesterol by tail vein injection.
 Oil phase is suspended by sonication in 25ml of water containing 156mg
taurocholate (Na salt), each animal receives 1ml.
 The I.V dose is prepared by drying the cholesterol and adding 300ml warm
ethanol followed by 12.5ml of saline.
 Each animal receives 0.5ml of this colloidal suspension.
 Rats are allowed to consume respective diets after an hour and sacrificed 48h
after isotope administration.
 Here, percentage of oral dose of cholesterol absorbed is calculated from
plasma isotope ratio.
2. Lymph fistula model:

In this method, direct evidence for an inhibitory effect on cholesterol absorption can
be obtained.
This method provides an indication as to the duration of inhibition and relative
selectivity of the compound on absorption of cholesterol versus triglycerides and
phospholipids.
16
 Here rats are anesthetised. Then silicon rubber cannulae are placed into main
mesenteric lymph duct and into duodenum and secured with sutures.
 After overnight period, lipid emulsion of 0.1 % cholesterol, 0.1% sodium
taurocholate, 15% intralipid, 2.4 % safflower oil, 82.6% saline is infused into
duodenal cannulae (3ml/h).
 Then for 2hours lymph collections obtained.
 Lymph samples extracted into hexane in presence of stigmasterol internal
standard.
 Total and free cholesterol are quantitated by liquid gas chromatography.
 Critical assessment found was that, the intestinal ACAT activity was
significantly reduced by ACAT inhibitors.
Another aspect of lipid metabolism- interruption of bile acid recirculation that is

likely to influence elevation in plasma lipid level is described below.
1. Interruption of bile acid recirculation
Cholesterol is converted to bile acids by acids by oxidation and then undergoes
enterohepatic circulation.
 In untreated state 95% of bile acids that are secreted, are reabsorbed and
returned to liver, while small loss is replaced by de novo synthesis from
cholesterol.
 Increased bile acid excretion increases rate of oxidation of cholesterol in liver
finally depletes cholesterol pool.
 So compensatory increase in uptake via LDL receptors result in lower serum
LDL levels. This can be achieved by addition of bile acid binding resin eg:
cholestyramine, to the food.
 For rabbits of weight (2.5 – 3 kg) are switched from standard diet to diet with
10-20% polymeric basic anion exchange resin, cholesteryramine.
 Cholesterol levels in serum are measured at the beginning and at the end of 4
weeks from the feeding period.
 Following method helps to assess inhibition of lipid peroxidation of isolated
plasma low density lipoproteins
17
1. Inhibition of lipid peroxidation of isolated plasma low density lipoprotein

Purpose
Hypercholesterolemic Watanbe rabbits are considered to be a suitable model to study
effect of anti oxidants and anti atherosclerotic agents.
 Plasma of Watanbe heritable hyperlipidemic rabbits is used to test the
inhibition of Cu+2-induced lipid peroxidation of isolated LDL.
 Animals are fed over a period of 12 weeks with rabbit chow diet with or
without 1% of test compound or standard.
 Plasma samples collected in Disodium Ethylene Diamine tetra acetic acid
(EDTA). LDL is isolated from rabbit plasma by ultracentrifugation.
 LDL dialyzed against phosphate buffer saline at 4˚C for 24 h.
 Each 100µg of LDL sample is adjusted to volume of 1.5ml with distilled water
for knowing lipid peroxidation induced by Cu+2.
 Lipid peroxidation is initiated by addition of CuSO4 to a final concentration of
5µM followed by incubation at 37˚C for 3h.
 Reaction is stopped by adding 100µl of 50mM Disodium EDTA.
50µg of LDL from reaction mixture is added to 1.5ml of 20% TCA (Trichloroacetic
acid) and vortexed. Finally 1.5ml of 0.67% thiobarbituric acid in 0.05N NaOH is
added and mixture is incubated at 90˚C for 30min. Samples centrifuged at 1500rpm
for 10min and absorbance of supernatant fractions is determined at 532nm to estimate
content of lipid peroxides (TBARS- Thio barbituric acid reactive substances).
Standard curve (0-5 nmol) of Malondialdehyde is generated using Malondialdehyde
bis (dimethyl acetyl) as reference to determine the lipid peroxidation content in Cu+2
treated LDL.
3.2.2: Animal models frequently employed to assess
antihyperlipidemic activities.
Various animal models reported to be used for screening and evaluating
antihyperlipidemic activity are as follows:
Literature review in several abstracted journals revealed different version of models
has been employed.
Following are the animal models reported for screening and evaluation of
herbs/synthetic compounds for the potential anti hyperlipidemic activity.
18
 Hasimum et al has found the newer anti hyperlipidemic model by induction of

hyperlipidemia in short time period using propylthiouracil 0.01% in drinking
water followed by 400mg/kg cholesterol administration in male wistar rats.20
 Ochani et al, while investigating claimed lipid lowering activity of leaves of
Hibiscus sabdariffa in albino wistar rats, produced hyperlipidemia in test
animals by oral administration of cholesterol in the dose of 25mg/kg/day in oil
for 30 days.21
 Yuce et al has identified the anti hyperlipidemic activity of synthetic
compound 7, 8- dihydroxy -3-(4-methyl phenyl) coumarin in female Sprague
dawley rats on hyperlipidemia produced by high fat diet (1.63 % cholesterol,
0.41g cholic acid and 16.3g sunflower oil) for 17 days.22
 Adaramoye et al observed the anti hyperlipidemic activity of leaves of
Telfaira occidentalis in male wistar rats, induced hyperlipidemia by dietary
cholesterol orally at 30mg/mL per animal for 9 weeks.11
 Xie et al screened the hypolipidemic activity of Ananus comosus in male ICR
mice, in which the hyperlipidemia was induced by Triton WR 1339 at
500mg/kg besides a free access to high fat diet for hypercholesterolemia
progression test.22
 Harnafi et al confirmed the anti hyperlipidemic activity of aqueous extract of
flowers of Erica multiflora on female wistar rats on the hyperlipidemia
induced by Triton WR 1339 at 200mg/kg in normal saline solution.23
 Santhosh kumari et al has found the lipid lowering activity of ethanolic extract
of Eclipta prostate in male wistar rats which were previously rendered
hyperlipidemic by oral administration of cholesterol at 500mg/kg in groundnut
oil for 30 days.24
 Purohit et al has recognized and reported the hypolipidemic activity of
ethanolic extract of fruits and shoots of Capparis decidua in white adult male
rabbits in which hyperlipidemia was previously induced by giving high fat diet
of cholesterol at 400mg/kg in oil for 60 days.25
 Arun Samlal et al has investigated the anti hyperlipidemic activity of
suspension of Coriandrum sativum in male wistar rats which were made
19
hyperlipidemic by giving Triton WR 1339 at 200mg/kg bw in 0.05M 7.2 pH

buffer (phosphate buffer).3
 Ohmori et al wistar has screened that the novel anti hyperlipidemic agent S-2E
has shown the lipid lowering properties in Sprague Dawley rats, produced
triglyceridemic by fructose induced hypertriglyceridemia in four weeks at a
defined doses of fructose (high fructose chows i.e,70% fructose,15.2%
casein,4.8 % corn oil, 2.0% avicel cellulose, 1.0% vitamins, 7.0% minerals).26
20
PART - III
3.3: Reported biological activities of Aegle marmelos corr.
Almost all the parts of Aegle marmelos are reported to possess significant biological
activities. The following are some of the reported biological activities of the extracts
obtained from the leaves and other plant parts.
 A new anthraquinone derivative extracted from the seeds of Aegle marmelos
corr. showed significant anti fungal activity against pathogenic strains of
Aspergillus species and Candida albicans.27
 A 50% ethanolic extract of Aegle marmelos corr. leaves was fed orally to male
albino rats at the dose levels of 200 and 300mg/kg bw/day for 60 days and a
significant anti fertility effect (i.e., reduction in sperm count and motility) was
found at 300mg/kg of dose. Normal situation was reversed after 120 days.28
 A novel compound 1-hydroxy-5, 7- dimethoxy-2- naphthalene-
carboxaldehyde, showed the activation of apoptosis and also inhibited growth
of HCT 116 colon cancer tumour xenografts in vivo through the activation of
tumour necrosis factor-alpha (TNF-alpha), TNF receptor associated death
domain and capsizes. 29
 Leaves of Aegle marmelos corr. contain an alkaloidal amide, Aegeline 2,
which was isolated and found to have antihyperglycemic activity, lowering
blood glucose level by 12.9% and 16.9% at 5 and 24h in sucrose challenged
streptozotocin induced diabetic rats (STZ-S) model at a dose of 100mg/kg
body weight. It also significantly decrease plasma triglyceride levels by 55%
and total cholesterol by 24% and free fatty acids by 24%, accompanied with
increase in HDL-C by 28% in dyslipidemic hamster model at the dose of
500mg/kg body weight.30
 Isoprenaline – induced myocardial infarction in rats showed that decreased
levels of cholesterol and triglycerides and increased levels of phospholipids in
heart and aorta. All deranged biochemical parameters were restored with
200mg/kg of aqueous extract of leaves of Aegle marmelos. Similarly α-
tocopherol (60mg/kg) pretreatment to isoprenaline treated rats exhibited a
significant effect on all the parameters studied.31
21
 The comparative effects of leaf extract of Aegle marmelos corr. and alpha
tocopherol on serum lipids, lipid peroxides and cardiac enzyme levels in rats
with isoproterenol induced myocardial infarction were carried on. Treatment
with 100mg/kg and 200mg/kg bw for 35 days showed significant effect on
activities on marker enzymes lipid peroxides, lipids, lipoproteins and anti
oxidant enzymes in isoproterenol treated rats.32
 The alcoholic extract of leaves of Aegle marmelos corr. on isolated guinea pig
ileum and tracheal chain was investigated. The H1 receptors were depressed
and the contractions produced by histamine were antagonized. This even
relaxed ileal and tracheal smooth muscles was observed at 1mg/ml and
2mg/ml dose. The positive results obtained, were due to the presence of one or
more anti histaminic constituents present in the alcoholic extract of this plant
and used in treating asthama complaints.33
 The serial extracts of the leaves of Aegle marmelos corr. were investigated for
anti inflammatory property. Most of the extracts caused a significant inhibition
of the carageenan induced paw oedema and cotton pellet granuloma in rats.
Extracts also produced marked analgesic activity by reducing early and late
phases of paw licking in mice. Further, a significantly reduced hyperpyrexia in
rats was also observed. 34
 The polyherbal ayurvedic formulation of four different drugs namely Bilwa
(Aegle marmelos), Dhanyak (Coriandrum sativum), Musta (Cyperus rotundus)
and Vala (Vetiveria zinzanoids) were assessed in two inflammatory bowel
disease models – acetic acid induced colitis in mice and indomethacin induced
enterocolitis in rats. This formulation showed significant inhibitory activity
against inflammatory bowel disease. 35
 The methanolic and aqueous extracts from leaves of Aegle marmelos corr.
were and studied their toxic effects. Intraperitoneally 50mg/kg, 70mg/kg,
90mg/kg and 100mg/kg of extracts were given for 14 days in study acute and
chronic studies and LD50 studies were conducted on male and female wistar
rats and reported absence of any short term toxicity. Such investigations
established that of the extracts of leaves of Aegle marmelos have high margins
of drug safety. 36
22
 Aqueous extract of the leaves of Aegle marmelos corr. are reported to possess
the contraceptive effect on the reproductive organs of male rats. Albino rats
treated with 150, 300 and 600mg/kg bw/day for 60 days showed significant
decrease in weight of the reproductive organs, decrease in sperm motility and
sperm density of the cauda epididymis and testes. Even testosterone levels
were also significantly reduced. Biochemical analysis of reproductive tissues
for sialic acid, protein, glycogen, fructose, ascorbic acid, acid and alkaline
phosphatase showed significant decrease where as testicular cholesterol levels
increased showing alterations in biochemical mileage of genital organs.37
 Lupeol, a triterpenoid isolated from the leaves extract of Aegle marmelos

showed antihyperglycemic activity and antidyslipidemic activity in sucrose
challenged streptozotocin induced diabetic rats model at a dose of 100mg/kg
body weight.38
23
Materials and Methods
4. MATERIAL AND METHODS
4.1: Collection and Authentication of Aegle marmelos Corr. Leaves

Fresh leaves of Aegle marmelos corr. (family: Meliaceae) were collected in Bangalore
during the month of March 2010 and authenticated by the competent personnel from
Botanical Survey of India, Pune, Maharashtra state, communicated vide letter no.
BSI/WRC/Tech/2010/25 dated 13-04-2010 (Annexure).
The voucher herbarium specimen (PGSAMI) was deposited at Botanical
Survey of India, Pune, Maharashtra state Pune, Maharashtra state, as well in our
laboratory (H3/KLEUCOP, BNG/2010) for the further reference. The leaves were
shade dried and stored in an air tight container till extraction.
4.1.1 Extraction
Extraction is carried out by cold maceration technique. Shade dried leaves (around
300 grams) of Aegle marmelos was crushed into coarse powder and soaked in 95 %
ethanol for 72 hours. The contents were shaken frequently. After 72 hours, leaves
were filtered off and solvent was evaporated to get a dark green, semisolid paste like
extract. Extraction was repeated twice to get sufficient quantity for investigation.
4.1.2 Yield, Storage & Organoleptic Features

The yield was around 12 grams i.e., 4%. The resulting ethanolic extract of Aegle
marmelos (EEAM) was stored in an air tight container and preserved in cool and free
from light prior to use. EEAM - so obtained was dark green, bitter, pleasant / aromatic
and was of semisolid consistency.
4.2 Chemicals used

Following chemicals used for the investigation were procured from reliable
manufacturer/ supplier and stored in appropriate / applicable conditions. Chemicals,
wherever required were of analytical grade.
24
Name of the Chemical Manufacturer
1. Bovine Albumin Fraction Central Drug House, New Delhi.
2. Copper Sulphate Central Drug House, New Delhi.
3. Cupric Sulphate Nice Chemicals Pvt. Ltd, Cochin.
4. EDTA S.D Fine Chemicals, Mumbai.
5. Ethanol From Govt. Bonded Ware House.
6. Ferric Chloride Central Drug House, New Delhi.
7. Folin Ciocalteu Reagent Central Drug House, New Delhi.
8. Hydrochloric Acid Central Drug House, New Delhi.
9. Perchloric Acid Lobachem, Mumbai.
10. Potassium Dichromate S.D Fine Chem Ltd, Mumbai.
11. Potassium Dihydrogen O- S.D Fine Chem Ltd, Mumbai.

Phosphate
12. Potassium Sodium Tartrate S.D Fine Chem, Mumbai.
13. RBC Diluting Fluid Central Drug House, New Delhi.
14. Sodium Arsenate Lobachem, Mumbai.
15. Sodium Carbonate Nice Chemicals, Pvt, Kochi.
16. Sodium Chloride S.D Fine Chem Ltd, Mumbai.
17. Sodium Citrate S.D Fine Chem, Mumbai.
18. Sodium Hydrogen Carbonate S.D Fine Chem Ltd, Mumbai.
19. Sodium Hydroxide Nice Chemicals, Ltd, Kochi.
20. Thiobarbituric Acid Spectrochem. Pvt Ltd, Mumbai

21. Trichloroacetic Acid S.D Fine Chem Ltd. Mumbai.
22. WBC Diluting Fluid Central Drug House, New Delhi.
25
4.3: Experimental Animals
4.3.1 Source of Experimental Animals
Naive, Adult, female, healthy Albino Wistar rats in the weight range of 100-150gms
were procured from local breeder, M/s Sri Venkateshwara Traders, subramanya
Nagar, Bangalore. All the animals were housed for a week in the Institute’s Animal
house Facility.
4.3.2 Maintenance of Experimental Animals

Prior to and during the investigation, all the experimental animals were housed in
standard Animal house Facility conditions of this institution. Temperature (18-25˚c),
Relative humidity (50-70 %) and Light–Dark cycle of 12:12 hours and other
environmental conditions suggested by CPCSEA was followed.39 Naïve and test
animals were housed in a cage not more than four in number poly propylene cage (26
×19×13cms) covered with stainless steel wire mesh (28×20.5cm) and on a paddy husk
bed. Animals were maintained on pelleted commercial feed (M/s Amrut Feeds,
Bangalore) and water ad libitum. Clearance from the Institutional Animal Ethics
Committee of KLE University’s College of Pharmacy, Bangalore was taken prior to
the commencement of the experimental work. vide letter dated 19/6/2010.
4.3.3 Induction of hyperlipidemia
The animals were rendered hyperlipidemic by oral administration of 500mg/kg

cholesterol with groundnut oil for 30days (4 weeks).24The animals with 20-30%
increase in cholesterol levels (than normal) were employed in the investigation.
4.4 Experimental study protocol
Hyperlipidemic animals were randomly assigned to five groups (n=6) and

simultaneously, a group of (n=6) normal animals were employed for comparison
purpose. During the investigations, hyperlipidemic and normal control animals were
maintained on normal, pelleted commercial feed. Test animals received extracts /
vehicle / standard lipid lowering drug, orally, (p.o), each day, between 10AM and -12
Noon, for three consecutive weeks. The grouping of animals was as follows.
26
 Group I: Normal control.

 Group II: Hyperlipidemic control / Pathologic Control
 Group III: Treatment Group: (D1 Group): Administered EEAM in the dose
of 150mg/kg
 Group IV: Treatment Group: (D2 Group): Administered EEAM in the dose
of 250mg/kg.
 Group V: Treatment Group: Administered Lovastatin in the dose of 10mg/kg.
4.5 Assessment of Parameters in hyperlipidemic test animals
4.5.1 Serum Lipid Profile
Serum lipid profile was determined on day 0 (pretreatment) and 24 hours after the last
dose i.e. on 22nd day (post treatment). Blood was withdrawn from the test animals by
retro orbital puncture under mild ether anesthesia, serum was separated and the total
cholesterol (TC), Triglycerides (TG) was estimated using semi auto analyzer [model:
MS500e, Maysen technology co. Ltd.] using commercial diagnostic kits, following
the method described in the product literature supplied with the kit.
4.5.2 HMG –CoA reductase
HMG-CoA reductase enzyme, which catalyses the rate limiting reaction of hepatic
cholesterol synthesis in animals is estimated by method followed by Rao &
Ramakrishnaan.40 The method estimates the HMG CoA /mevalonate ratio as an index
of activity of HMG CoA reductase enzyme.
This is the indirect method of estimating the HMG CoA reductase (NADPH)
activity in the liver homogenate. HMG CoA and mevalonate in the tissue samples is
taken as an index of activity of enzyme required to convert HMG CoA to mevalonate
ratio in the presence of NADPH.
27
Requirements are as follows and were prepared as follows.
 Saline Arsenate solution: 1Gram of sodium arsenate dissolved in a liter of

physiological saline.
 Dilute Hydrochloric Acid
 Hydroxylamine hydrochloride reagent: 2 mol/lt
 Ferric chloride reagent: Prepared by dissolving 5.2 Grams of Trichloro acetic
acid and 10g of Ferric chloride in 50ml of 0.65 mol/L hydrochloric acid and
diluted to 100ml.
Procedure:
 Liver tissues were collected from all the animals as quickly as possible and
10% homogenate was prepared in saline arsenate.
 Then homogenate was deproteinized using equal volume of diluted perchloric

acid.
 Then the mixture was allowed to stand for 5 min and centrifuged at 2000 rpm
for 10 minutes.
 1 ml of supernatant was treated with 0.5ml of freshly prepared hydroxylamine

hydrochloride reagent.
 Mixed well, and after 5 mins, 1.5 ml of ferric chloride reagent was added to
the same tube and was shaken well.
 Finally after 10 mins, readings were taken at 540nm against a similarly treated
saline/arsenate solution.
4.5.3 Fecal cholesterol excretion
Fecal cholesterol excretion in test animals, normal control and hyperlipidemic control
animals was estimated.41 Fecal matter was collected during the last 3 days of the
treatment period from all the six groups. Then dried pellets of fecal matter were
powdered and were then extracted analyzed for cholesterol content in a manner
28
similar to that of the serum. The cholesterol excreted in the fecal matter (mg/g) was
calculated.
4.5.4 Hematological parameters
RBC and WBC count in groups of animals in extract treated, standard lipid lowering
drug, hyperlipidemic control and in normal control group of animals was estimated at
the end of the study using Neubaur’s chamber following conventional procedure.42
24 hours after the last dose or last treatment day, blood sample was withdrawn
from each animal by retro orbital puncture under mild ether anesthesia, diluted with
respective diluting fluid and using a drop of diluted blood charged into the
chamber(after discarding first few drops of diluted blood sample).
RBC and WBC count was carried out in five representative chambers in case
of RBC and four representative chambers in case of WBC Count i.e., the blood count
was carried out in 1/400sq mm (for RBC) and 1/16 sq mm (for WBC) and Total count
was calculated using the formula (mentioned below) and expressed as
Numbers/ cu mm of undiluted blood sample.
No. of WBC in Undiluted blood is= N/4 X 10X20 = N x 50
N= Number in n1+n2+n3+n4
No. of RBC in undiluted blood is calculated by =N/80x4000x2000= Nx10000
4.5.5 Measurement of Lipid Peroxidation
Measurement of Lipid peroxidation in extract treated, standard lipid lowering drug,

Hyperlipidemic control and normal control animals, 24 hours after the last dose was
estimated to assess the degree of oxidative stress in hepatic tissue and consisted of
following steps
 Protein Extraction
 Determination of Hepatic LPO Level.
29
4.5.5.1 Protein Estimation in liver homogenate
Test animals from various groups were sacrificed under mild ether anesthesia; liver
was removed and immediately homogenized (in Perchloric acid solution, 2500 rpm
for 10min) and protein content was estimated by method suggested by lowry method
as modified by pomery.43
Principle
The principle involved was reactivity of peptide nitrogen with copper ions under the
alkaline conditions give deep blue colour. Further subsequent reduction of the Folin-
ciocalteu phosphomolybdic phosphotungustic acid to heteropolymolybdenum blue by
the copper-catalyzed oxidation of aromatic acids .Thus, producing intense blue-green
colour which was measured colorimetrically at 660 nm.
Reagents used
 0.1N NaOH: 2g of NaOH was dissolved in 500ml of water.
 Copper tatarate carbonate solution:
1. 1% copper sulphate solution.(CuSO4. 5H2O)
2. 2% potassium sodium tatarate solution (KNaC4H4O6 4H2O)
3. 1% sodium carbonate solution.(Na2CO3).
The above solutions were mixed in the following sequence.
1 ml of copper sulphate and 1 ml of potassium sodium tatarate was mixed in a
beaker. To that 100 ml sodium carbonate was added in a beaker (1:1:100) and
mixed thoroughly.
 1 N Folin-ciacteau reagent: 1 part of folin ciocalteu was mixed with 1 part of
distilled water gives 1N folin ciocalteu reagent.
 5% liver homogenate.
30
Procedure
 Liver homogenate was diluted upto 30ml of 0.1N NaOH then 0.5ml of
homogenate (liver) were pipette out in different test tubes labeled as standard,
test and blank.
 5ml of copper tatarate (1:1:2.5) solution; was added to each tube agitated for a
few seconds and allowed to stand for 10min.
 Folin - ciocalteu reagent was then added to all tubes and mixed vigorously
(allowed to stand for 2 hours).
 Optical density of intense, blue colour produced was measured

colorimetrically at 660nm with UV- visible spectrophotometer.
The below formulas were used to determine the protein content in the liver.
 Serum protein (g/dL) = (O.D)Test/(O.D)Std×10×100×1/1000.
 Liver homogenate protein (mg/dL) = (O.D)Test/(O.D)Std×10×30.
4.5.5.2 Determination of Hepatic LPO Level
Lipid peroxides formed in vivo usually causes severe cellular damage. It is firmly
established that homogenates of most animal tissues form thiobarbituric acid reactants
when incubated invitro under aerobic conditions. The determination of thiobarbituric
acid reactive substances was carried out.
Malondialdehyde (MDA) as an index of lipid peroxidation was carried out according

to the modified method of wills.44
Reagents required were as follows
 1.15M potassium chloride (Kcl).

 0.25 M Hydrochloric acid containing 15% trichloro acetic acid and 0.38%
thiobarbituric acid (TBA) and 0.055% butylated hydroxyl toluene and stored
at 2-8˚C.
31
Procedure mentioned below is followed.

 0.5ml liver homogenate was mixed with 2.5ml of trichloro acetic acid,
followed by boiling in water bath for 15min.
 After cooling to room temperature, centrifuged at 3000 rpm for 10min.
 2.0 ml supernatant of each sample was transferred to test tube containing 1.0
ml of TBA solution.
 All the tubes are placed in a boiling water bath for 15 mins, cooled to room
temperature.
 And the absorbance was measured at 532nm with respect to the blank
solution.
Calculation
MDA was calculated using following formula.
MDA (n mol/mg protein) = (O.D) Test× Total volume of reaction mixture

1.56×10-5 × sample volume× mg protein / ml
4.5.6. Weight of Visceral Organs

In test animals, after removing Liver, other visceral organs namely kidney, lungs,
heart carefully removed, freed from attached unwanted tissue and clot and were
weighed and recorded.11
4.6 Statistical Analysis

Data collected from above mentioned above were subjected to statistical analysis for
significance. All values are expressed as Mean±SEM, followed by One-Way
ANOVA, Post – hoc test – Student Newman Keul Multiple comparison test.
Values P< 0.05 or less were considered significant.
32
Results
5. RESULTS
5.1: Change in Serum Lipid Profile
Change in serum triglyceride and total cholesterol levels in test animals treated with
vehicle, EEAM of 150, 250mg/kg, Lovastatin(10mg/kg) as well untreated pathogenic
control/hyperlipidemic control at the end of the study are tabulated as shown in table
5.1 and graphically represented as shown in the figure 5.1.a and 5.1.b.
5.1.1 Change in triglyceride (TG)
 Vehicle treated normal control animals recorded an insignificant rise in the
TG 112.3±6.23 mg/dL to 114.34±6.17mg/dL and, on the contrary, in untreated
hyperlipidemic group II animals, a significant elevation (p<0.05) in TG level
was recorded, at the end of the study (124.00±5.61 to 142.16±3.61 mg/dL).
 Group III animals treated with 150mg/kg (dose 1) of EEAM recorded a
significant reduction in TG level (p<0.01, 137.66±4.06mg/dl to 110.33±3.92)
compared to its pretreatment level and was also found to be significant
(p<0.001) compared to pretreatment TG level of group II- hyperlipidemic
control group of animals.
 Group IV animals treated with 250mg/kg of EEAM, recorded a significant
reduction in TG levels (p<0.001) compared to post treatment level of untreated
hyperlipidemic group of animals (123.5±4.26 to 101.00±2.95mg/dl).
 Similarly group V animals treated with Lovastatin (10mg/kg), also recorded a
significant reduction in TG level (p<0.001) compared to post treatment level
of untreated hyperlipidemic group of animals (129.3±2.92 to
101.5±2.39mg/dL).
 Difference between TG level of normal control group of animals and extract
treated (dose 1 and 2) at the end of the study was found to be insignificant.
 Serum triglyceride level in Lovastatin treated animals, extract treated and
vehicle treated normal control animals do not significantly differ.
33
Results
TABLE 5.1(a) Change in serum triglyceride level of normal control, untreated

pathogenic control, extract and Lovastatin treated hyperlipidemic animals
Triglyceride ( mg/dl)
Group Treated with Pretreatment Post treatment
I Vehicle 112.30±6.23 114.33±6.17

Normal control 0.2 ml
II 124.00±5.61 142.16 ±3.60#
Hyperlipidemic control -
III EEAM 137.66±4.06 110.33±3.92##,***

Treatment group 150 mg/kg BW
IV EEAM 123.50±4.26 101.00±2.95#,***

Treatment group 250mg/ kg BW
V Lovastatin 129.30±2.92 101.50±2.39##,***

Treatment group 10 mg/Kg BW
All the values are Mean ± SEM of n=6, one way ANOVA followed by Newman keul
multiple comparison test.
 # p<0.05 and ## p<0.01Vs pretreatment in respective group.
 *** p<0.001 Vs the post treatment value of hyperlipidemic control
200
Serum Triglyceride concentartion (mg/dL)
TG pre treatment
TG post treatment
150
***
100 *** ***
50
0
NC PC 150mg/kg 250mg/kg LST 10 mg/kg
Group and treatment
Figure 5.1(a) Change in serum triglyceride levels of NC,PC,extract and Lovastatin treated animals at the end of study.
*** Vs pathogenic control group(post treatment).
NC= normal control; PC=pathogenic control
150mg/kg and 250mg/kg are the doses of EEAM,LST= lovastatin
34
Results
5.1.2 Change in total cholesterol level (TC)
 Vehicle treated normal control group I animals recorded an insignificant rise

in TC levels compared to its initial value (49.00±2.30mg/dL to
54.66±2.77mg/dL) at the end of study.
 On the contrary, untreated hyperlipidemic animals recorded a significant
elevation (p<0.001, 101.00±5.06 to 128.5±2.63mg/dl) compared to post
treatment value of vehicle treated group I animals.
 Similarly insignificant rise was found in the TC levels of group II –
hyperlipidemic control animals (101.00±5.06 to 128.5±2.63mg/dL).
 Group III animals treated with 150mg/kg bw of EEAM recorded an
insignificant (p>0.05) reduction in TC level, however it was found to be
significantly higher (p<0.001) compared to group I animals (vehicle treated) in
during post treatment phase.
 Group IV animals treated with 250mg/kg bw of EEAM recorded a significant
(p<0.05) reduction compared to TC level of untreated hyperlipidemic control.
However, in TC level, the change was insignificant (p>0.05) compared to its
pretreatment level. TC levels was remaining significantly elevated (p<0.001)
compared to group I animals treated with vehicle.
 In Lovastatin treated group V animals, reduction in TC was compared to that
of the hyperlipidemic control animals i.e., p< 0.001 and reduction in levels
ranged from 128.5±2.63 to 79.5±3. Reduction was insignificant compared to
that of pretreatment value.
35
Results
TABLE 5.1(b): Change in total cholesterol in normal control, untreated pathogenic

control, extract and Lovastatin treated hyperlipidemic animals.
Total cholesterol(mg/dL)
Group Treated with
Pretreatment post treatment
I Vehicle 49.00±2.30 54.66 ±2.77

II 101.10±5.06 128.50± 2.63†††
III EEAM 109.66±19.86 101.83±2.41†††
IV EEAM 117.16±4.21 95.00±2.00*,†††
V Lovastatin 99.50±2.59 79.50±3.28***
All the values are Mean ± SEM of n=6.

One way ANOVA followed by Newman keul multiple comparison test.
 ††† p<0.001 Vs post treatment value of vehicle control.
 * p<0.05 and ***p<0.001Vs the post treatment value of hyperlipidemic
control.
TC pre treatment
TC post treatment
150
Serum total cholesterol
100 *
***
50
0
NC PC 150mg/kg 250mg/kg LST 10 mg/kg
Group and treatment

Figure 5.1(b) Change in total cholesterol of NC,PC,extract and Lovastatin treated animals at the end of study.
*, *** Vs pathogenic control group. NC= normal control; PC=pathogenic control,
36
Results
To summarize,
 EEAM treatment in the selected doses (150 and 250 mg/kg bw) produced
significant (p<0.01 and p<0.05) reduction in the TG level compared to
pretreatment (corresponding values) and reduction was significant
(p<0.001) compared to post treatment TG concentration of hyperlipidemic
control group (group II) of animals. In Lovastatin treated group, the
reduction was also significant (p<0.001).
 EEAM treatment in the selected dose (150mg and 250mg/kg bw) did not
significantly altered the TC level, compared to respective values in the
pretreatment phase. Level of TC was significantly higher (p<0.001)
compared to TC level of group I animals treated with vehicle, at the
similar interval of time. However, post treatment TC level of 250 mg/kg
bw treated animals was found to significantly (p<0.01) less compared to
TC level of group II animals at similar interval of time. Similarly in case
of total cholesterol values, the significance (p<0.001) was found when
compared to that of the hyperlipidemic control and insignificant with the
pretreatment value.
5.2 HMG CoA reductase enzyme levels estimation
HMG CoA reductase is the enzyme that controls the synthesis of cholesterol. The
levels of HMG CoA reductase were found to be significant in measuring the synthesis
of cholesterol. When compared with the different drug treated groups, the following
outcomes have been noticed. The results are represented as table in table 5.2 and as
graph in fig.5.2
 The group I was considered as normal control and the value recorded was
0.320±0.02.
37
Results
 In untreated hyperlipidemic group II animals, relatively significant rise in the

HMG CoA enzyme levels was seen (i.e., p<0.05) compared with that of group
I animals. The reduction ranged from 0.320±0.02 to 0.379±0.07.
 In group III, EEAM 150mg/kg dose treated animals, more significant

reduction in the enzyme levels was observed when compared to that of
hyperlipidemic control (p<0.01) reduction from 0.379±0.07 to 0.129±0.08 as
well when compared to normal group reduction was (p<0.05).
 In group IV, the EEAM 250mg/kg dose treated animals, a significant

reduction (p<0.001) was observed when compared to that of untreated
hyperlipidemic control (0.379±0.07 to 0.05±0.007).
 Dose dependent influence of extract on HMG CoA reductase inhibition was

observed.
 Group V, Lovastatin 10mg/kg treated animals, the reduction was of

significance (p<0.001), when compared with that of hyperlipidemic control
animals. (0.379±0.07 to 0.05±0.007). When compared with the normal
treated group, significance of p<0.01 and reduction was from 0.320±0.02 to
0.05±0.002. Difference in the levels of HMG CoA reductase enzyme level of
Lovastatin and extracts treated animals was found to be insignificant.
38
Results
TABLE 5.2 Effect on HMG CoA reductase levels in normal control, untreated
pathogenic control, extract and Lovastatin treated hyperlipidemic animals.
Group Treated with Levels of HMG CoA reductase

(µl)
I Vehicle
Normal control 0.2 ml 0.320±0.020
II
Hyperlipidemic - 0.379±0.070
control
III EEAM
Treatment group 150 mg/kg 0.129±0.080**,†
BW
IV EEAM
Treatment group 250mg/ kg 0.050±0.007***
BW
V Lovastatin
Treatment group 10 mg/Kg BW 0.050±0.002***,††
 **p<0.01 and ***p<0.001 Vs the value of hyperlipidemic control group.
 † p<0.05 and †† p< 0.01 Vs the untreated normal group.
0.5
0.4
HMG CoA Reductase
0.3
**
0.2
0.1
*** ***
0.0
NC PC 150mg/kg 250mg/kg LST 10mg/kg
Group and treatment
Figure 5.2: Levels of HMG CoA reductase NC,PC,extract and Lovastatin treated animals at the end of study.
** and *** Vs pathogenic control group. NC= normal control; PC=pathogenic control,
39
Results
5.3: Fecal Cholesterol Excretion
On the last 3 to 4 days of the treatment period, the fecal matter was collected from the
animals and the cholesterol content in it was estimated and tabulated as shown in table
5.3 and figure 5.3.
 In group I, normal vehicle treated animals, the total fecal excretion was found
to be 138mg/dl.
 In group II, the untreated hyperlipidemic control animals, total fecal

cholesterol excretion was found to be 134mg/dL. Though the reduction was
not much significant, numerically it confirms that the cholesterol excretion
was reduced.
 In group III, the EEAM 150mg/kg treated group, the total cholesterol
excretion was found to be 151mg/dL which was significantly increased when
compared to that of hyperlipidemic control i.e.,134mg/dL.
 In group IV, the EEAM 250mg/kg treated group, the total cholesterol
excretion was found to be 158mg/dL which was significantly increased when
compared to that of hyperlipidemic control i.e.,134mg/dL.
 In group V, Lovastatin treated animals, the total cholesterol excretion was

found to be significant i.e.,171mg/dL, when compared with that of
hyperlipidemic group and even those of EEAM treated groups.
40
Results
TABLE 5.3 Effect on the levels of fecal cholesterol excretion in normal control,
untreated pathogenic control, extract and Lovastatin treated hyperlipidemic animals.
Group Treated with Fecal cholesterol(mg/g)

I Vehicle
Normal control 0.2 ml 138
II
Hyperlipidemic control - 134
III EEAM
Treatment group 150 mg/kg BW 151
IV EEAM
Treatment group 250mg/ kg BW 154
V Lovastatin
Treatment group 10 mg/Kg BW 171
All the values are Mean ± SEM of n=6, one way ANOVA followed by
Newman keul multiple comparison test.
200
fecal cholesterol (mg/g)
150
100
50
0
Group and treatment
Figure 5.3: Level of fecal cholesterol exrcretion in NC,PC,extract and Lovastatin treated animals at the end of study.
NC= normal control; PC=pathogenic control,
41
Results
5.4 Change in Lipid Peroxidation Levels (MDA levels)
Lipid peroxidation status was measured as MDA levels. Though the readings of all
the groups were found to be statistically insignificant, there is numerical variation
compared between each groups.
 In vehicle treated group, the level was found to be 1.118±0.01.
 In untreated hyperlipidemic control, the MDA levels were raised to

1.137±0.01.
 In EEAM treated groups, the reduction in MDA levels was seen i.e.,
1.130±0.006 in 150mg/kg bw dose treated animals and 1.125±0.009 in
250mg/kg bw treated animals.
 Lovastatin treated group has shown the value of 1.122±0.007 which was
relatively lesser than that of EEAM treated group of hyperlipidemic animals.
42
Results
TABLE 5.4 Change in MDA levels in normal, pathogenic control, extract and
standard lipid lowering drug treated group of animals.
Group Treated with MDA(nmol/mg of protein)
I Vehicle 1.118±0.01
II 1.137±0.01
III EEAM 1.130±0.006

IV EEAM 1.125±0.009
V Lovastatin 1.122±0.007
1.5
MDA levels (nmol/mg of protein)
1.0
0.5
0.0
Group and treatment
Figure 5.4: Levels of MDA of NC,PC,extract and Lovastatin treated animals at the end of study.
43
Results
5.5: Change in Body Weight
Change in body weight in animals treated with vehicle control, extracts (two doses)
and lipid lowering drugs as well as pathogenic control/hyperlipidemic control are
tabulated in table 5.5 and graphically represented in the figure 5.5.
 Only group I animals recorded a significant elevation in (p<0.001) body

weight at the end of the study and EEAM treatment in the dose 150mg/kg and
250mg/kg bw did not significantly changed (elevated) body weight of animals
despite, no change in feed consumed. Although group II, group III and group
IV animals recorded a marginal rise, it was found to be insignificant compared
to its pretreatment values.
 The weight changes of animals of group V was found to be insignificant when
compared with that of all other groups used in the experiment.
44
Results
TABLE 5.5 Change in body weights in normal control, untreated pathogenic control,
extract and Lovastatin treated hyperlipidemic animals.
Body weight (in gms)

Group Treated with Pretreatment Post treatment
I Vehicle 164.16±2.38 194.16±7.23###

II 173.33±6.41 190.00±4.66
III EEAM 185.83±2.00 187.50±5.73
IV EEAM 183.33±2.10 201.66±2.78
V Lovastatin 190.00±10.65 202.00±8.34
 ### p<0.001 Vs the pretreatment value in respective group.
250
body weight-pretreatment
###
200 body weight-post treatment
body weight(g)
150
100
50
0
Group and treatment
Figure 5.5: Change in body weights of NC,PC,extract and Lovastatin treated animals at the end of study.
### Vs pretreatment value of NC; NC= normal control; PC=pathogenic control,
45
Results
5.6: Change in Weights of Different Visceral Organs
In this investigation, the weight of several visceral organs like liver, kidney, heart and
lungs were observed and their relative weights were compared to know the effect of
treatment. Weight of organs per se, as well as relative weight (%) are as tabulated as
shown in the table 5.6 and graphically represented in the figures 5.6.a (liver), 5.6.b
(lungs), 5.6.c (heart), 5.6.d (kidney).
Weight of liver
 Weight of liver per se in group II animals (hyperlipidemic control) did not

significantly differed from group I animals treated with vehicle. However,
group II animals treated with 150mg/kg bw resulted in higher and significantly
(p<0.05) heavier liver compared to group IV animals treated with 250mg/kg
bw.
 Weight of liver in 250mg/kg bw (group IV) treated animals were significantly
lower (p<0.05) compared to 150mg/kg bw (group III) treated animals.
 Although the weight of liver of group I and group II animals differed, however
the difference was found to be statistically insignificant.
 Liver weight of group I animals and that of group IV animals were found to be
more or less similar and did not significant differed.
 Lovastatin treated hyperlipidemic animals recorded significant reduction in
weight of liver compared to 150mg/kg bw extract treated animals and did not
significantly differed from vehicle treated animals.
Weight of lungs
 Although the weight of lungs per se in various groups of animals was found to
be slightly different, the difference between them was found to be
insignificant.
46
Results
Weight of heart
 EEAM treatment in the dose of 150 mg/kg bw resulted in significant reduction
(p<0.01) in the mean weight of heart, compared to untreated hyperlipidemic
control animals (group II). Further, it was found to be significantly (p<0.05)
less than vehicle control group of animals.
 In the EEAM 250mg/kg bw dose treated animals, the change was found to be
significant (p<0.05) compared to that of hyperlipidemic control.
 In Lovastatin 10mg/kg treated animals, the weight of liver per se statistically
differed from untreated hyperlipidemic group of animals (p<0.05).
Weight of kidney
 Hyperlipidemic control (group II) animals, as well EEAM treated (150mg/kg
bw) animals had significantly heavier kidney compared to vehicle treated
group I animals (p<0.05).
 The weight of kidneys between group I and group IV did not differ
significantly.
 Weight of kidney of EEAM treated animals was found to be slightly heavier
than that of group II animals.
 Lovastatin treated group showed insignificant change in weight of kidneys
when compared with that of EEAM treated groups and pathogenic control
group.
47
Results
TABLE NO 5.6: Change in visceral organ weight per se and relative weights of
different visceral organs in normal control, untreated pathogenic control, extract and
Lovastatin
treated hyperlipidemic animals.
Group Treated with Liver per se(gm) Lungs per se(gm) Heart per se(gm) Kidney per
se(gm)
I Vehicle 6.69±0.50 1.50±0.16 0.738±0.046 1.45±0.07
Normal 0.2 ml (3.48±0.33) (0.78±0.10) (0.37±0.01) (0.74±0.05)
control
II 8.02±0.16 1.52±0.10 0.796±0.01 1.69±0.04†
Hyperlipidem - (4.13±0.14) (0.78±0.06) (0.40±0.05) (0.86±0.06)
ic control
III EEAM 8.75±0.62†† 1.26±0.05 0.656±0.02** 1.72±0.08†
Treatment 150 mg/kg (4.75±0.39) (0.68±0.03) (0.35±0.01) (0.92±0.05)
group BW
IV EEAM 7.22±0.15¥ 1.24±0.04 0.669±0.02* 1.54±0.06
Treatment 250mg/ kg (3.75±0.10) (0.64±0.01) (0.34±0.01) (0.79±0.03)
group BW
V Lovastatin 7.117±0.29¥ 1.608±0.07 0.70±0.02* 1.66±0.04
Treatment 10 mg/Kg (3.53±0.18) (0.80±0.05) (0.34±0.02) (0.82±0.04)
group BW
Note: Values in the parenthesis refer to % of body weight i.e, relative weight.
All the values are Mean ± SEM of n=6, one way ANOVA followed by Newman
Keul multiple comparison test.
 * p<0.05 Vs the hyperlipidemic control

 ** p<0.01 Vs hyperlipidemic control
 # p<0.05 between the group III and group IV
 † p<0.05 and ††p<0.01 Vs vehicle control
 ¥ p<0.05 Vs that of EEAM 150mg/kg treated group
48
Results
10
8
* *
weight of liver(gm)
6
0
Group and treatment
Figure 5.6(a) Weight of livers of NC,PC,extract and Lovastatin treated animals at the end of study.
* Vs PC; NC= normal control; PC=pathogenic control,
2.0
weight of lungs(gms)
1.5
1.0
0.5
0.0
Group and treatment
Figure 5.6(b) Weight of lungs of NC,PC,extract and Lovastatin treated animals at the end of study.
49
Results
1.0
0.8
weight of heart(gms) *** * *
0.6
0.4
0.2
0.0
Group and treatment
Figure 5.6(c) Change in weights of heart in NC,PC,extract and Lovastatin treated animals at the end of study.
*,*** Vs pathogenic control group. NC= normal control; PC=pathogenic control,
2.0
weight of kidneys(gms)
1.5
1.0
0.5
0.0
NC PC 150mg/kg 250mg/kg LST10mg/kg
Group and treatment
Figure 5.6.d: Change in weights of kidneys of NC,PC,extract and Lovastatin treated animals
at the end of study.
50
Results
5.7: Change in Hematological Profile:
5.7.1 Changes in RBC level
 RBC count did not significantly differ between groups.
5.7.2 Changes in WBC level
 Group II hyperlipidemic control animals had significantly (p<0.05) elevated

WBC count compared to group I vehicle treated animals.
 Group III and group IV animals treated with 150mg/kg and 250mg/kg bw had
significantly lesser WBC, compared to group II (p<0.001) and group I
(p<0.001) animals.
 However, difference between WBC count between group III and group IV did
not significantly differed.
 The significant reduction in WBC count was seen to maximum extent in the
Lovastatin treated group when compared with that of untreated hyperlipidemic
control group. The significance was (p<0.001) and the reduction was seen
from 16474.83±825.03 to 5431±205.8.
51
Results
TABLE 5.7 Effect on hematological parameters in normal control, untreated

pathogenic control, extract and Lovastatin treated hyperlipidemic animals.
Group Treated with RBC (×106/mm3) WBC (cells/mm3)
I Vehicle 5.63±0.61 13,779.5±1239.8

II 4.67±0.62 16474.83±825.03†
Hyperlipidemic -
control
III EEAM 6.08±1.09 8629.33±698.64***,†††
IV EEAM 5.05±0.69 9190.83±497.10***,†††
V Lovastatin 6.272±0.435 5431±205.8***,†††,¥¥
 *** p<0.001 Vs hyperlipidemic control.

 † p<0.05, †† p<0.01 and ††† p<0.001 Vs vehicle control.
 ¥ ¥ p<0.001 Vs that of EEAM treated groups.
52
Results
RBC levels(106/mm3)
6
0
Group and treatment
Figure 5.7.a: Levels of RBC in NC,PC,extract and Lovastatin treated animals at the end of study.
20000
15000
WBC level(/mm3)
10000 *** ***

***
5000
0
Group and treatment
Figure 5.7.b: Levels of WBC in NC,PC,extract and Lovastatin treated animals at the end of study.
*** Vs pathogenic control group. NC= normal control; PC=pathogenic control,
53
Discussion
6. DISCUSSION
Review of literature for herbs possessing anti hyperlipidemic and anti oxidant
property revealed that several herbs from traditional system of medicine, folklore
possess significant lipid lowering properties and in addition antioxidant property –
i.e., elevated endogenous antioxidant enzyme levels and levels of lipid peroxidation,
thus extending beneficial effects.
Recently, Vijaya et al reported that the extract of leaves of Aegle marmelos

possess significant anti hyperlipidemic effect in high fat diet fed laboratory animals.15
In brief the male wistar rats of weight range 150-180g were rendered hyperlipidemic
by feeding them with high fat diet for 4 weeks and in triton induced hyperlipidemia
with 200mg/kg Triton WR 1339 was given to animals. The Aegle marmelos leaves
were extracted in 50% ethanol by cold maceration and were given in dose 125mg/kg
and 250 mg/kg once a day for a week and compared with Gemfibrozil (50mg/kg bw)
treated group of animals for one week. All four groups were kept on same high fat
diet throughout the study. Serum total cholesterol and triglycerides were estimated.
Atherogenic index was calculated. But several associated physiological and
biochemical changes during the measurement of anti hyperlipidemic effect have not
been investigated.
The current study was envisaged with the intention of arriving at a broader
picture of its antihyperlipidemic activity where other parameters like fecal cholesterol,
effect of drug and cholesterol on weights of different visceral organs, lipid
peroxidation and effect on hematological parameters like RBC and WBC levels were
assessed.
The effect of extract treatment on products of lipid peroxidation was

essentially an important part since lipid peroxidation levels are likely to be elevated in
hyperlipidemic state.45 In case, if extract treatment were to reduce the same, the
extract is likely to be a potential antihyperlipidemic drug.
Investigation of antihyperlipidemic property was carried out in cholesterol fed

hyperlipidemic animals, such animals were treated for a month and lipid profile of
extract treated hyperlipidemic animals was assessed at the end of the study. With an
54
Discussion
already significantly reduced triglyceride level, in a dose dependent manner. The

reduction being statistically differing from pretreatment or basal value, as well
compared to untreated hyperlipidemic control animals.
However, low dose i.e., 150mg/kg produced a significant fall in triglyceride

level compared to post treatment level of normal control group and not to its
pretreatment value. Higher dose i.e.,250mg/kg treatment produced a significant fall in
total cholesterol compared to untreated hyperlipidemic group of animals. This further
confirms that EEAM possess anti hyperlipidemic activity and can effectively reduce
the elevated lipid profile in hyperlipidemic animals, thus held potential for further
investigation.
HMG CoA is an enzyme that catalyses the sole rate limiting step of hepatic
cholesterol synthesis in animals.40 The levels of these enzymes were estimated in all
the groups. In hyperlipidemic control animals, the significant rise in enzyme levels
confirm the enhanced activity of HMG CoA reductase and increase the synthesis of
cholesterol. In the EEAM 150mg/kg and 250mg/kg bw treated groups, the levels were
significantly reduced when compared to those of hyperlipidemic control indicating a
significant influence on important enzyme associated with cholesterol synthesis. This
is usually a target for statins. It is likely that, extract is eliciting this observed
beneficial effect in hyperlipidemic state by exerting an influence like a statin.
A reduction in intestinal absorption prevents the accumulation of cholesterol

in the liver which leads to increase in the clearance LDL-C from the circulation, thus
lowering the blood cholesterol.46 In the investigation, fecal cholesterol excretion was
estimated in groups of animals being treated with extracts. In pathogenic control the
cholesterol excretion was found to be 138mg/dL. Where as in the EEAM treated
groups, raise in cholesterol excretion was seen significantly that extract treatment
leads to anti hyperlipidemic activity via fecal excretion.
Peroxidation is one of the consequences of oxidative stress which finally leads

to cell damage. Both isolated poly unsaturated fatty acids and those incorporated into
lipids are readily attacked by free radicals becoming oxidized into lipid
peroxidation.47
55
Discussion
There has not been yet a definitive explanation of mechanism of

hypocholesteremic effect of polyunsaturated fat in diet. Theoretically this action could
result from any or combination of the following alterations in cholesterol
metabolism.48
a) A decrease in endogenous cholesterol synthesis,

b) Reduction in absorption of cholesterol or bile acids,
c) An increase in fecal excretion of cholesterol and
d) A redistribution of blood cholesterol between plasma and other body tissues
either directly or by way of an alteration in lipoprotein metabolism.
Dietary fats, after being digested and absorbed, transported through the body
via lipoproteins. More cholesterol rich lipoprotein, LDL contains approximately 2700
fatty acids/molecules half of which are PUFAs that are sensitive to oxidation. The
fluidity of cell membranes decrease with lipid peroxidation.
Increase in concentration of superoxide, hydrogen peroxide and presence of

metal ions make conditions favorable for the production of hydroxyl radical (OH-)
which results from addition of one or more electron to hydrogen peroxide. This OH- is
capable of interacting with almost every type of molecule found in living cell. It
reacts by abstraction, addition and electron transfer from non-radical molecules thus
initiating oxidation of macromolecules such as lipids resulting in a lipid radical (L)
that in presence of oxygen generate lipid peroxyl radical (LOO). This LOO continues
chain reaction by attacking another lipid and oxidation process continues.
Reaction begins when methylene group (-CH2-) of PUFA (poly unsaturated fatty
acids) is attacked by a free radical to abstract a hydrogen atom and an electron.
Adjacent double bond weakens the attachment of hydrogen atoms present on the next
carbon, especially if there is double bond on either side of methylene group. With
subsequent rearrangement and reaction with more oxygen, lipid peroxyl radicals
(LOO) are formed. The lipid peroxyl radicals oxidize adjacent lipids in cell membrane
or LDL molecule. Now oxidatively modified, the LDL-C particle is identified by
receptor of macrophage and engulfed leading to foam cell formation. Through this
chain reaction, a single initiating radical can result in conversion of hundreds of fatty
56
Discussion
acid side chains into lipid peroxides that alter the integrity of biochemical function of
cell membrane.
Another fate of oxidized fatty acid is the formation of cyclic peroxide that continues
to be oxidized to Malondialdehyde (MDA), F2- isoprostanes or other oxidation
products.49
At the end of study, products of lipid peroxidation measured as MDA was

measured in hepatic tissue and compared between different groups of animals.
Untreated hyperlipidemic animals did record a marginal elevation than vehicle treated
(normal control) groups, but was statistically insignificant. It is likely that duration of
treatment may be of shorter period to produce a significant change or increase in dose
may be required to produce a significant change in lipid peroxidation level.
In post treatment phase, the body weights of animals were not significantly
varied. The only alteration was seen in case of normal untreated animals and no
significance was seen in other EEAM treated and standard treated groups though the
food intake was increased in all groups during the treatment period. This might be due
to excess excretion of cholesterol through fecal matter. Thus the reported result
confirms the significant effect of EEAM doses and standard (Lovastatin) drugs. This
indicates that extract treatment has no deleterious effect on metabolism and liver and
also confirms the safety of extract on long term use.
The effect of drugs on the weights of visceral organs was also recorded at the
end of the study- of liver, lungs, heart and kidney.19 Untreated hyperlipidemic animals
had relatively heavier liver, heart and kidney per se as well relative weight compared
to normal animals except for weight of lungs. Vehicle treated did influence the weight
of visceral organs, especially that of heart and kidney. EEAM in the dose of
250mg/kg produced a significant change. In other words, extract treatment reversed to
some extent, influence of hyperlipidemic effect on visceral organs.
A high total of WBC count is commonly found with major predictions of

atherosclerosis. It is possible that increase in WBC like lymphocyte and neutrophils
and also circulating monocytes.50, 42
57
Discussion
These significant reductions in WBC levels confirm their reduced activation

which is responsible for formation of atheromatous plaques. Thus, the anti
hyperlipidemic activity was found to be satisfactory. Untreated hyperlipidemic
animals recorded a significantly higher WBC count compared to normal control.
Marginal and significant difference in RBC among test groups (pathogenic control,
lower RBC than normal) indicates insignificant influence on these parameters on
RBC.
At the end of the study, untreated hyperlipidemic animals recorded a relatively

higher WBC number, than vehicle control. Extract treatment recorded relatively lower
levels of WBC than untreated pathogenic control group.
Such a significant influence on associated profile, thus, makes EEAM of

leaves fit further detailed study.
In view of significant alterations in parameters reflecting antihyperlipidemic

property of the herb, current investigations revealed hereto unreported properties of
Aegle marmelos.
58
Conclusion
7. CONCLUSION
Current study further strengthened reported antihyperlipidemic effect of ethanolic
extract of Aegle marmelos in a dose dependent manner in animal model of
hyperlipidemia and significantly altering associated physiological and biochemical
parameters.
59
Summary
8. SUMMARY
Hyperlipidemia is considered as one of the major predisposing factors for

atherosclerosis. Many synthetic drugs are used for the therapy of is risk factor. Plant
products are frequently considered as less toxic and are also free from unwanted
effects than those of synthetic drugs in treating hyperlipidemia.
 Aegle marmelos is a well known medicinal herb with varied therapeutic

activities. In this study the ethanolic extract of Aegle marmelos was used to
investigate its antihyperlipidemic activity on various parameters.
 Fresh leaves of Aegle marmelos were collecting in Bangalore, later air dried
and were extracted in 50% ethanol following cold maceration technique. For
this experiment 150mg/kg and 250mg/kg were used.
 Hyperlipidemic animals were assigned into various groups –untreated
hyperlipidemic control (Group II), two groups treated with lower and higher
doses of ethanolic extract of Aegle marmelos (Group III and Group IV) and
standard group (Group V) treated with Lovastatin and age and sex matched
normal control (vehicle)(Group I). In hyperlipidemic treated and untreated
animals , various parameters, as envisaged were assessed after 30days
 Serum triglyceride (TG) levels and total cholesterol (TC) levels were
estimated in all the groups after the completion of their treatment periods by
withdrawing the blood retroorbitally. The effect of two doses of extract was
found to be significant in reducing the elevated TG and TC levels.
 The liver enzyme HMG CoA reductase is an enzyme that controls the
synthesis of cholesterol. Increase in the levels of this enzyme leads to more
synthesis of cholesterol. The EEAM treated animals and Lovastatin treated
animals’ recorded significant reduction in HMG CoA reductase enzyme.
 The fecal cholesterol excretion was measured by collecting the fecal matter
from all the groups during the last 3-4 days of treatment period. Further they
were dried, made in powder and were extracted in chloroform and methanol in
ratio 2:1. The obtained filterate was estimated for measuring the cholesterol
content and a significant increase in fecal cholesterol excretion was seen in
EEAM treated and Lovastatin treated groups.
60
Summary
 The increase in lipid peroxidation end products lead to atherosclerosis. They

were measured as MDA (Malondialdehyde) levels or TBARS (Thiobarbituric
acid reactive substances). There was significant reduction in MDA levels in
EEAM treated and Lovastatin treated groups compared to that of
hyperlipidemic control.
 Hematological parameters like RBC and WBC levels were estimated and
change in RBC was insignificant, whereas change in WBC levels was
significant in EEAM treated groups.
 The weights of different visceral organs like liver, kidneys, heart and lungs
were estimated at the end of the study in order to observe the change in their
weights, the EEAM treated and Lovastatin treated groups recorded lighter
livers, where as the hyperlipidemic control animals had slightly heavier livers.
 From these obtained results, it can be concluded that ethanolic extract of
leaves of Aegle marmelos has antihyperlipidemic effect and potential to reduce
the damages due to lipid peroxidation - thus extending its role in the
prevention of atherosclerosis accelerated by hyperlipidemia.
61
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FURTHER INVESTIGATION ON

MADURI SHASHANKA RAO

KLE Academy of Higher Education & Research – Deemed University, Belgaum,

in partial fulfilment of the requirements for the degree of

DECLARATION BY THE CANDIDATE

I hereby declare that this dissertation entitled

CERTIFICATE BY THE GUIDE

This is to certify that the dissertation entitled

Mr. PRASANNA GS,

ENDORSEMENT BY THE HEAD OF THE DEPARTMENT AND

This is to certify that the dissertation entitled

Under the guidance of

Dr. Purnima Ashok, Dr. B.G. Desai

DECLARATION BY THE CANDIDATE

I hereby declare that the KLE Academy of Higher Education &

Place: Bangalore MADURI SHASHANKA RAO

© KLE Academy of Higher education and Research-Deemed University

I am indebted to my parents Shri Maduri Srinivasa Rao and Maduri

I wish to express my special thanks to my brother Sunil and my granny for

I would like to convey my sincere gratitude to my guide Prasanna GS for his

I am especially grateful to Dr. Purnima Ashok, Head of the Department,

I would like to convey my token of gratitude to Prof. Dr. B.G. Desai,

I am thankful to my ever caring lecturers Mrs G P Rajani, Mrs Vanitha S

My friends of chemistry Viswa, Garvita, Gayathri, Paramesh, Ramjith and

My friends of pharmaceutical technology, Dhaval, Chirag and all other

My seniors Radhakrishna, Rajesh, Vivek, Mrs Komala, kayur, Sowjanya,

My beloved juniors Deepthi, Nishi, Ramakrishna, Sagar, Rajsekhar,

ANOVA Analysis of variance

EDTA Ethylene diamine tetra acetic acid

EEAM Ethanolic extract of Aegle marmelos type of cholesterol

g/dl Gram per deciliter

HDL-C High density lipoprotein type of cholesterol

HMG CoA Hydroxyl methyl glutaruyl coenzyme A

Kcl Potassium chloride

LDL-C Low density lipoprotein type of cholesterol

LPO Lipid peroxidation

mg/dL Milligram per decilitre

NADPH Reduced nicotinamide adenine dinucleotide phosphate

NaOH Sodium hydroxide

nmol Nano mole

P.O Per oral

PPARα Peroxyzome proliferator receptor activator α

PUFA Poly unsaturated fatty acids

RBC Red blood corpuscles

SEM Standard error mean

TBA Thiobarbituric acid

TCA Trichloro acetic acid

TNF Tumour necrosis factor

VCAM Vascular cell adhesion molecule

VLDL Very low density lipoprotein

WBC White blood corpuscles

Materials and methods:

Test animals were rendered hyperlipidemic by oral administration of 500mg/kg

In hyperlipidemic animals, ethanolic extract of leaf of Aegle marmelos produced

Changes are suggestive of antihyperlipidemic effect of ethanolic extract of Aegle

Aegle marmelos, hyperlipidemia, fecal cholesterol excretion, lipid peroxidation,

Sl. No CONTENTS PAGES

2 Aim and objectives 5

3 Review of literature 6-23

4 Materials and methods 24-32

Sl. Title of the table Page

1 Change in the levels of serum triglycerides in 34

2 Change in the levels of total cholesterol in NC, 36

3 Effect of extract treatment on the levels HMG 39