TABLE OF CONTENTS

Acknowledgment .................................................................................................. iii

Abstract ..................................................................................................................vi

List of table ............................................................................................................ix

List of figure ........................................................................................................ xii

List of abbreviations ............................................................................................ xv

Chapter 1 General Introduction .......................................................................... 1

1.1 Introduction ....................................................................................................... 1

1.2 Diabetes Mellitus ............................................................................................ 2

1.21 Motivation and overview of diabetic research .......................................... 2

1.2.2 Diabetes epidemic in India ........................................................................ 3

1.2.3 Definition of diabetes ................................................................................ 3

1.2.4 Types of diabetic mellitus ......................................................................... 3

1.2.5 Diabetic complication ............................................................................... 4

1.2.6 Carbohydrate metabolism ......................................................................... 4

1.2.7 Lipid metabolism ...................................................................................... 5

1.2.8 Protein metabolism ................................................................................... 6

1.2.9 Type II Diabetes Mellitus ......................................................................... 6

1.2.9.1 Definition ........................................................................................... 6

1.2.9.2 Signs and symptoms ......................................................................... 6

1.2.9.3 Treatment and its disadvantage ......................................................... 7

1.3 Description of Costus igneus ......................................................................... 7

1.4 Review of literature ........................................................................................ 7

1.4.1 Pharmacognostical studies ....................................................................... 9

1.4.2 Phytochemical analysis ............................................................................ 9

1.4.2.1 Plant constituents-Saponin and sapogenin ..................................... 10

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 1.4.2.2 Flavonoids ....................................................................................... 11

1.4.3 Pharmacological study ........................................................................... 12

1.4.3.1 Antidiabetic potential of some important herbal plants. .................. 14

1.4.3.2 Biological activity of saponin ......................................................... 14

1.4.3.3 Biological activity of flavonoids ..................................................... 19

1.5. Objectives ............................................................................................ 20

1.6 Work plan .............................................................................................. 20

Chapter 2 Pharmacognosy: Morphological, anatomical and proximate

analysis of leaf, root and rhizome of Costus igneus ......................... 21

2.1 Introduction .................................................................................................. 21

2.2 Materials and methods ................................................................................. 22

2.2.1 Collection of plant material .................................................................... 22

2.2.2 Morphological and Anatomical studies of Costus igneus....................... 22

2.2.2.1 Specimens preparation .................................................................... 22

2.2.2.2 Sectioning ....................................................................................... 22

2.2.2.3 Photomicrography ........................................................................... 23

2.2.3 Proximate analysis ................................................................................. 23

2.2.4 Statistical analysis .................................................................................. 23

2.3 Results .......................................................................................................... 24

2.3.1 Morphological characters ....................................................................... 24

2.3.2 Anatomical studies of Costus igneus ..................................................... 24

2.3.2.1 Anatomy of Rhizome ...................................................................... 24

2.3.2.2 Anatomy of Leaf .............................................................................. 25

2.3.2.3 Anatomy of Root.............................................................................. 33

2.3.3 Proximate analysis .................................................................................. 33

2.4 Discussion ..................................................................................................... 37

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Chapter 3 Phytochemistry: Analytical studies of Costus igneus ..................... 39

3.1 Introduction .................................................................................................. 39

3.2 Materials and methods ................................................................................. 40

3.2.1 Cultivation of plant propagation of Costus igneus ................................ 40

3.2.2 Extraction preparation ........................................................................... 41

3.2.2.1 Solvent extractions .......................................................................... 41

3.2.2.2 Preparation of crude Sapogenin extract ........................................... 41

3.2.3 Qualitative analysis of phytochemicals in leaf, stem and rhizome

of Costus igneus ....................................................................................... 41

3.2.3.1 Test for tannins ............................................................................... 41

3.2.3.2 Test for phlobatannins ..................................................................... 42

3.2.3.3 Test for saponin................................................................................ 42

3.2.3.4 Test for flavonoids .......................................................................... 42

3.2.3.5 Test for steroids ............................................................................... 42

3.2.3.6 Test for terpenoids (Salkowski test)................................................. 43

3.2.3.7 Test for Cardiac Glyosides (Keller-Killani test) ............................. 43

3.2.4 Quantitative analysis of phytochemicals in leaf, stem and rhizome

of Costus igneus ...................................................................................... 43

3.2.4.1 Preparation of fat free sample ......................................................... 43

3.2.4.2 Determination of total phenols (by spectrophotometric method) .... 43

3.2.4.3 Alkaloid determination using Harborne (1973) method ................. 43

3.2.4.4 Tannin determination (by Van-Burden and Robinson

(1981) method) ............................................................................... 44

3.2.4.5 Saponin determination .................................................................. 44

3.2.4.6 Flavonoid determination ................................................................. 44

3.2.5 Isolation and characterization of diosgenin and flavonoid compound .. 44

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 3.2.5.1 Isolation of diosgenin compound .................................................... 45

3.2.5.2 Isolation of flavonoid compound .................................................... 45

3.2.5.3 Characterization of chemical compound ......................................... 46

3.2.6 Isolation and purification of diosgenin and flavonoid compounds

by High Performance Liquid Chromatography (HPLC) ........................ 46

3.2.6.1 Sample preparation for HPLC ......................................................... 46

3.2.6.2 HPLC separation condition .............................................................. 46

3.2.7 Quantification of flavonoids (quercetin, kaempferol) and

sapogenins (diosgenin, betasitosterol) by HPTLC................................ 46

3.2.7.1 Sample preparation for HPTLC ...................................................... 46

3.2.7.2 Chromatography condition ............................................................. 47

3.2.7.3 HPTLC method for estimation of flavonoids (quercetin, kaempferol)

and sapogenins (diosgenin, betasitosterol) compounds ................... 47

3.2.7.3.1 Preparation of calibration curve of standard .......................... 47

3.2.7.3.2 Method Specifications ............................................................ 47

3.2.8 Isolation of fatty acid compounds from rhizome, stem and leaf

ethanolic extract of Costus igneus by GC-MS

(Gas Chromatography with Mass Spectroscopy) .................................... 48

3.2.8.1 Sample preparation for GCMS ........................................................ 48

3.2.8.2 GCMS separation condition ............................................................ 48

3.3 Results ........................................................................................................... 48

3.3.1Qualitative analysis of phytochemicals in leaf, stem and rhizome

of Costus igneus ...................................................................................... 48

3.3.2 Quantitative analysis of phytochemicals in leaf, stem and rhizome of

Costus igneus ....................................................................................... 51

3.3.3 Isolation and characterization of diosgenin compound........................... 51

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 3.3.4 Isolation and characterization of flavonoid compound ........................ 52

3.3.5 HPTLC method for estimation of flavonoid (quercetin, kaempferol)

and sapogenin (diosgenin, betasitosterol) compounds ............................ 52

3.3.5.1 Validation of HPTLC method ......................................................... 59

3.3.6 Isolation and purification of diosgenin and flavonoid compound by

High Performance Liquid Chromatography (HPLC).............................. 59

3.3.7 Isolation of fatty acid compounds from ethanolic extract of

rhizome, stem and leaf of Costus igneus by GC-MS

(Gas Chromatography with Mass Spectroscopy) .................................... 59

4.0 Discussion ..................................................................................................... 72

Chapter 4 Pharmacology: Antidiabetic study of Costus igneus rhizome in

streptozotocin induced diabetic rats ................................................................... 75

4.1 Introduction .................................................................................................. 75

4.2 Materials and methods ................................................................................. 77

4.2.1 Animals used .......................................................................................... 77

4.2.2 Plant materials ........................................................................................ 77

4.2.3 Preparation of extracts from Costus igneus rhizome ............................. 77

4.2.3.1 Preparation of CiREE ...................................................................... 77

4.2.3.2 Preparation of SECiR ....................................................................... 77

4.2.4 Animal study .......................................................................................... 78

4.2.4.1 Induction of diabetes mellitus in rats .............................................. 78

4.2.4.2 Acute toxicity evaluation in rat ........................................................ 78

4.2.4.3 Single dose study of CiREE, SECiR and isolated diosgenin............ 78

4.2.4.4 Effect of CiREE, SECiR and isolated diosgenin on oral glucose

tolerance in rats ............................................................................... 79

4.2.4.5 Experimental design for 30 days antidiabetic study of

CiREE, SECiR and isolated diosgenin ............................................. 80

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 4.2.4.5.1 Collection of liver, kidney, pancreas and blood ..................... 80

4.2.4.5.2 Estimation of biochemical constituents .................................. 80

4.2.4.5.3 Estimation of lipid profile ................................................... 81

4.2.4.5.3.1 Estimation of cholesterol ................................................ 81

4.2.4.5.3.2 Estimation of HDL ......................................................... 81

4.2.4.5.3.3 Estimation of LDL-Cholesterol ...................................... 81

4.2.4.5.3.4 Estimation of triglycerides .............................................. 81

4.2.4.5.4 Estimation of ALT and AST ................................................... 82

4.2.4.5.5 Assay for alkaline phosphatase ............................................... 82

4.2.4.5.6 Assay for SOD activity ............................................................ 82

4.2.4.5.7 Assay for catalase activity ....................................................... 83

4.2.4.5.8 Assay of glutathione peroxidase activity ................................. 83

4.2.4.5.9 Assay of reduced glutathione content (GSH) .......................... 84

4.2.4.5.10 Assay of sulfhydryl groups ................................................... 84

4.2.4.5.11 Assay of lipid peroxides ........................................................ 84

4.2.4.5.12 Determination of Vitamin A and Vitamin E ......................... 85

4.2.4.5.13 Histopathological Investigation ............................................ 85

4.2.4.5.14 Processing of tissue for electron microscopy (EM)...............85

4.2.4.6 Statistical analysis ............................................................................ 85

4.3 Results ........................................................................................................... 86

4.3.1 Acute toxicity studies ............................................................................. 86

4.3.2 Single dose and oral glucose tolerance study of CiREE, SECiR

and isolated diosgenin ............................................................................ 86

4.3.3 Antidiabetic study of CiREE, SECiR and isolated

diosgenin in STZ induced diabetic rats (30 days) ................................. 89

4.3.3.1 Biochemical analysis ....................................................................... 89

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 4.3.3.2 Histopathological investigation .................................................... 103

4.3.3.2.1 Histopathology of the pancreas ............................................. 103

4.3.3.2.2 Histopathology of the liver ................................................... 103

4.3.3.2.3 Histopathology of kidney ...................................................... 107

4.3.3.3 Electron microscopic change ......................................................... 107

4.4 Discussion .................................................................................................. 109

Chapter 5 Summary and Conclusion ............................................................... 117

5.1 Pharmcognosy ............................................................................................ 117

5.2 Phytochemistry .......................................................................................... 119

5.3 Pharmacology ............................................................................................ 120

References ........................................................................................................... 122

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