PHARMACOLOGICAL SCREENING, PHYTOCHEMICAL EVALUATION AND PHARMACOGNOSTIC STUDIES OF INDIGOFERA GERARDIANA AND CRATAEGUS SONGRICA

Desertation

Submitted to the Department of Pharmacy, University of Peshawar in partial fulfillment of the requirements for the Degree of

DOCTOR OF PHILOSOPHY

IN

PHARMACEUTICAL SCEINCES

By SHAFIQ AHMAD TARIQ

DEPARTMENT OF PHARMACY UNIVERSITY OF PESHAWAR PAKISTAN 2009 Acknowledgements

ACKNOWLEDGEMENTS

All kinds of admiration is for Allah Almighty WHO created the universe and bequeath the mankind with knowledge and astuteness to search for his secret. The author is thankful to him who gave the impending, audacity and patience to complete this research work. I am indebted to my research supervisor Prof. Dr Mohammad Nisar Director Institute of chemistry university of Peshawar for his guidance, valuable, suggestions and stimulating discussions as well as enthusiastic support throughout the course of this investigation.

I appreciatively acknowledge Dr Raza Shah HEJ Research Institute of chemistry, International centre for chemical and Biological Sciences University of Karachi for his time and expertise that was always generously available during my research stay at HEJ Karachi.

The author’s thanks goes to faculty members, department of pharmacy, Prof. Dr Abdul Wadood and Prof Dr Riaz Naseem for their interest, moral support and encouragement.

With deep sense of gratitute and appreciation I would like to extend my thanks to Mr. Sirtaj and Mr Ishfaq Hameed.

I owe special thanks to Dr Manzoor Ahmad Malakand University to completing this assignment.

Special appreciation is due to my lab fellow Mr. Obaidollah, Mr. Saleem Ullah Khan, MrInamullah khan, Mr Ihsan Ali, Mr.Waquar Ahmad Kaleem and Mr.Mugal Qaym for their generous support and encouragement during my research period.

Thanks to my parents and my most profound apperception goes to my wife and kids Bilal Ahmad and Ajlal Ahmad who had been a springboard in my academic support.

i

DDeeddiiccaatteedd TToo MMyy GGrraanndd FFaatthheerr ((llaattee)) May their soul rest in piece (Amin) && PPaarreennttss

Contents

CONTENTS

Entry Title Page No.

ACKNOWLEDGMENT i LISTS OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS SUMMARY PART – A Pharmacognostic studies, Phytochemical Evaluation and Pharmacological Screening, of Indigofera gerardiana 1.0 Introduction 1 1.1 General Introductions 1 1.1.1 Plant as source of drug 2 1.1.2 Bioactive compounds from herbs 3 1.1.3 Role of natural products on economy 6 1.2 Genus Indigofera 7 1.2.1 Indigofera gerardiana 7 1.2.2 Natural habitat 8 1.2.3 Native distribution 8 1.2.4 Plant description 8 1.2.5 Folk medicinal use 8 1.3 Flavonoids 10 1.3.1 Introduction 10 1.3.2 Classification of Flavonoids 10 1.3.3 Structural behavior of Flavonoids 11 1.3.4 Biosynthesis of Flavonoids 15 1.3.5 General aspects of Flavonoids 15 1.3.6 Individual steps to Flavonoids classes 19 1.3.6.1. Flavanone Formation 19 1.3.6.2 Isofalovone Formation 20 1.3.6.3 Flavone Formation 21 1.3.6.4 Flavonol Formation 22 1.3.6.5 Glycosylation of Flavonoids 23 1.3.6.6 Methylation of Flavonoids 23

ii Contents

Entry Title Page No.

1.3.7 Biological Properties of Flavonoids 24 1.3.8 Important Sources of Flavonoids 27 1.4 Pharmacognositc Studies 28 1.4.1 Morphology and Anatomy 29 1.4.2 Nutrition 29 1.4.3 Microchemical Test 30 1.5 Pharmacological Studies 32 1.5.1 Antibacterial Assay 32 1.5.2 Antifungal Assay 33 1.5.3 Brine Shrimp Bioassay 34 1.5.4 Insecticidal Assay 35 1.5.5 Phytotoxic Assay 36 1.5.6 Anti leishmanial Assay 36 2.0 Pharmacognostic Investigations 37 2.1 Collection and preservation 37 2.1.1 Macroscopic features 37 2.1.2 Anatomical studies 38 2.1.3 Microscopic drug study 38 2.1.4 Nutrition analysis 38 2.1.4.1 Detection of ash 38 2.1.4.2 Determination of moisture 39 2.1.4.3 Determination of protein by macrojeldahl distillation method 40 2.1.4.4 Determination of fat or ether extract 41 2.1.4.5 Determination of crude fiber by fibertec 42 2.1.4.6 Carobhydrate contents 43 2.1.5 Microchemical tests 43 2.2 Phytochemical Studies 46 2.2.1 General notes 46 2.2.2 General experimental conditions 46 2.2.2.1 Physical constants 46 2.2.2.2 Spectroscopy 46 2.2.2.3 Plant material 47

iii Contents

Entry Title Page No.

2.2.2.4 Extraction, fractionation and isolation 47 2.3 Physical and spectroscopic data 51 2.3.1 S-1 51 2.3.2 S-2 52 2.3.3 S-3 53 2.3.4 -Sitosterol 54 2.4 Pharmacological investigation 55 2.4.1 Antibacterial assay 55 2.4.2 Antifungal assay 56 2.4.3 Brine Shrimp Bioassay 58 2.4.4 Insecticidal Activity 59 2.4.5 Phytotoxic Assay 60 2.4.6 Anti leishmanial Assay 63 2.4.7 Anti malarial Activity 64 3.1 Pharmacognostic Study 65 3.1.1 Macroscopic features of Indigefera gerardiana 65 3.1.2 Anatomical Studies of Indigefera gerardiana 67 3.1.3 Microscopic Study of Indigefera gerardiana 68 3.1.4 Nutritional Analysis of Indigefera gerardiana 68 3.1.5 Microchemical Tests of Indigefera gerardiana 69 3.2 Phytochemical Studies 70 3.2.1 Structural elucidation of new compounds 70 3.2.1.1 Compound (S-1) 70 3.2.1.2 Compound (S-2) 73 3.2.1.3 Compound (S-3) 75 3.2.2 Structural elucidation of known compound 77 3.2.2.1 -Sitosterol 77 3.3 Pharmacological Studies 80 3.3.1 Pharmacological studies on crude extract and fractions 80 3.3.1.1 Antibacterial Assay 80 3.3.1.2 Antifungal Assay 89 3.3.1.3 Brine Shrimp Bioassay 93

iv Contents

Entry Title Page No.

3.3.1.4 Insecticidal Assay 96 3.3.1.5 Phytotoxic Assay 99 3.3.1.6 Anti leishmanial Assay 102 3.3.1.7 Anti malarial assay 102 PART – B Pharmacognostic studies, Phytochemical Evaluation and Pharmacological Screening, of Crataegus songrica 4.0 Introduction 104 4.1 Genus Crataegus 104 4.1.1 Related Information 106 4.1.2 Natural habitat 106 4.1.3 Native distribution 106 4.1.4 Plant description 107 4.1.5 Medicinal uses 107 5.0 Phytochemical Studies 108 5.1 General Notes 108 5.1.1 General experimental conditions 108 5.1.1.1 Spectroscopy 108 5.1.1.2 Chromatography 109 5.1.1.3 Locating reagent 109 5.1.1.4 Collection and preservation 109 5.2 Isolation of chemical constituents 114 5.2.1 Physical and spectroscopic data of isolated compounds from 115 fruit of crataegus songrica 5.2.2 Stigmasterol 3-O--D-Glucoside (T-I) 115 5.2.3 Lupeol (T-II) 116 5.2.4 3,5, 7-Trihidroxy-6, 4-dimethoxy flavone (T-III) 117 5.2.5 5,4-Dhydroxy-3, 6, 7-trimethoxy flavone (T-IV) 117 6.1 Pahramcognostic Investigations 119 6.1.1 Macroscopic features of Crataegus songrica 119 6.1.2 Anatomical Studies of Crataegus songrica 119 6.1.3 Microscopic Study of Crataegus songrica 120 6.1.4 Nutritional Analysis of Crataegus songrica 120 6.1.5 Microchemical Tests of Crataegus songrica 121

v Contents

Entry Title Page No.

6.2 Phytochemical Studies 122 6.2.1 Structural elucidation of constituents isolated from barries of 122 Crataegus sangrica 6.2.1.1 Stigmasterol 3-O--D-Glucoside (T-I) 122 6.2.1.2 Lupeol (T-II) 123 6.2.1.3 3,5, 7-Trihidroxy-6, 4-dimethoxy flavone (T-III) 124 6.2.1.4 5,4-Dhydroxy-3, 6, 7-trimethoxy flavone (T-IV) 125 6.3 Pharmacological Studies 127 6.3.1 Antibacterial Assay 127 6.3.2 Antifungal Assay 133 6.3.3 Brine Shrimp Bioassay 137 6.3.4 Insecticidal Assay 140 6.3.5 Phytotoxic Assay 141 6.3.6 Anti leishmanial Assay 145 6.3.7 Anti malarial Activity 145 Plates of Anatomical and Microscopic Studies 147 (Part – A) Plates of Anatomical and Microscopic Studies 159 (Plates – B) References (Part – A) 164 References (Part – B) 176

vi List of Tables

LIST OF TABLES

Entry Table # Title Page # 1. 1.1 Active principles derived from ethno pharmacological source, 4 which are clinically used 2. 1.2 Pathogenesis caused by bacteria 33 3. 1.3 Pathogenesis caused by fungi 34 4. 2.1 Physical and spectroscopic data of S-1 51

5. 2.2 Physical and spectroscopic data of S-2 52 6. 2.3 Physical and spectroscopic data of S-3 53 7. 2.4 Physical and spectroscopic data of S-4 54 8. 2.5 Criteria for determining antibacterial assay 56 9. 2.6 Sabouraud dextrose agar (SDA) 57 10. 2.7 Criteria for determining antifungal assay 58 11. 2.8 Composition of medium for Lemna bioassay 62 12. 2.9 Criteria for determining Phytotoxicity 62 13. 3.1 Macroscopic features of the root of Indigofera geradiana 66 14. 3.2 Macroscopic features of the stem of Indigofera gerardiana 66 15. 3.3 Macroscopic features of the leaf of Indigofera gerardiana 66 16. 3.4 Nutritional analysis of the whole plant of Indigofera 69 gerardiana 17. 3.5 Microchemical tests of the Indigofera gerardiana 69 18. 3.6 1H (400 MHz) and 13C NMR (100MHz) Data of (S-1) in 72 MeOD 19. 3.7 1H (400 MHz) and 13C NMR (100 MHz) Data of (S-2) in 74 MeOD 20. 3.8 1H (400 MHz) and 13C NMR (100 MHz) Data of (S-2) in 77 MeOD 21. 3.9 1H (400 MHz) and 13C NMR (100 MHz) Data of in β- 79 sitosterol MeOD 22. 3.10 Result of antibacterial assay of crude extract and fractions 83 23. 3.11 Result of antifungal assay of crude extract and fractions 90 24. 3.12 Result of brine shrimp bioassay of crude extract and fractions 94 25. 3.13 Results of insecticidal assay of crude extract and fractions 97 26. 3.14 Phytotoxic assay of crude extract and fractions 100 27. 3.15 Results of anti leishmanial assay of crude extract and fractions 102

vii List of Tables

Entry Table # Title Page #

28. 3.16 Anti malarial assay of crude extract and fractions 103 29. 5.1 Physical and spectroscopic data of (T-I) 115 30. 5.2 Physical and spectroscopic data of(T-1I) 116 31. 5.3 Physical and spectroscopic data of (T-III) 117 32. 5.4 Physical and spectroscopic data of (T-1V) 118 33. 6.1 Macroscopical features of the fruit of Crataegus songarica 119 34. 6.2 Nutritional analysis of the fruit of Crataegus songrica. 120 35. 6.3 Microchemical tests of Crataegus sangrica 121 36. 6.4 Results of antibacterial assay of crude extract and fractions 128 37. 6.5 Result of antifungal assay of crude extract and fractions 134 38. 6.6 Results of brine shrimp bioassay of crude extract and fractions 138 39. 6.7 Results of insecticidal assay of crude extract and fractions 140 40. 6.8 Phytotoxicity bioassay of the crude extract and fractions of 142 Crataegus songrica 41. 6.9 Phytotoxic assay of crude extract and fractions 143 42. 6.10 Results of anti leishmanial assay of crude extract and fractions 145 43. 6.11 Anti malarial assay of crude extract and fractions 146

viii List of Figures

LIST OF FIGURES

Entry Figure # Title Page #

1. Picture of Indigofera geraediana 11 2. 1.1 Basic structure of flavonoid 12 3. 1. 2 Flavonoids having 1,2-diphenylpropane skeleton 12 4. 1.3 Flavonoids having 1,1-diphenylpropane skeleton 13 5. 1.4 Basic skeleton and numbering pattern in flavonoids 14 6. 1.5 Basic skeleton and numbering pattern in homoflavonoids 70 7. i Structure of S- (1) 72 8. ii Key HMBC interactions in S-(1) 73 9. iii Structure of S- (2) 74 10. iv Key HMBC interactions in S- (2) 75 11. v Structure of S- (3) 76 12. vi Key HMBC interactions in S- (3) 78 13. vii Structure of β-sigmasterol(4) 14. 3.1 Bacterial and fungal growth of some micro organisms on 81 solid medium 15. 3.2 Zone of inhibition by crude extract against different 84 bacteria 16. 3.3 Zone of inhibition by n-hexane fraction against different 84 bacteria 17. 3.4 Zone of inhibition by chloroform fraction against different 84 bacteria 18. 3.5 Zone of inhibition by ethyl acetate fraction against 84 different bacteria 19. 3.6 Zone of inhibition by n-butanol fraction against different 85 bacteria 20. 3.7 Zone of inhibition by aqueous fraction against different 85 bacteria 21. 3.8 Zone of inhibition of imipenum (standard drug) against 85 different bacteria 22. 3.9 Growth inhibition (%) by crude extract against different 86 bacteria 23. 3.10 Growth inhibition (%) by n-hexane fraction against 86 different bacteria 24. 3.11 Growth inhibition (%) by chloroform fraction against 86 different bacteria 25. 3.12 Growth inhibition (%) by ethyl acetate fraction against 86 different bacteria 26. 3.13 Growth inhibition (%) by n-butanol fraction against 86 different bacteria

ix List of Figures

Entry Figure # Title Page #

27. 3.14 Growth inhibition (%) by aqueous fraction against 86 different bacteria 28. 3.15` Zone of inhibition (mm) by crude extract, fractions 87 and imipenum (standard drug) against E.coli 29. 3.16 Zone of inhibition (mm) by crude extract, fractions 87 and imipenum (standard drug) against B.subtilus 30. 3.17 Zone of inhibition (mm) by crude extract, fractions 87 and imipenum (standard drug)against S.flexaniri 31. 3.18 Zone of inhibition (mm) by crude extract, fractions 87 and imipenum (standard drug)against S.aureus 32. 3.19 Zone of inhibition (mm) by crude extract, fractions 87 and imipenum (standard drug)against P.aeruginosa 33. 3.20 Zone of inhibition (mm) by crude extract, fractions 87 and imipenum (standard drug)against S.typhi 34. 3.21 Growth Inhibition (%) against E.coli 88 35. 3.22 Growth Inhibition (%) against B.subtilus 88 36. 3.23 Growth Inhibition (%) against S.flexenari 88 37. 3.24 Growth Inhibition (%) against S.aureus 88 38. 3.25 Growth Inhibition (%) against P.aeruginosa 88 39. 3.26 Growth Inhibition (%) against S.typhi 88 40. 3.27 Percent growth inhibition by crude extract 91 41. 3.28 Percent growth inhibition by n-hexane fraction 91 42. 3.29 Percent growth inhibition by chloroform fraction 91 43. 3.30 Percent growth inhibition by ethyl acetate fraction 91 44. 3.31 Percent growth inhibition by butanol 91 45. 3.32 Percent growth inhibition by aqueous 91 46. 3.33 Growth inhibition (%) against T.longifusisl 92 47. 3.34 Growth inhibition (%) against C.albicans 92 48. 3.35 Growth inhibition (%) against A.flavus 92 49. 3.36 Growth inhibition (%) against M.canis 92 50. 3.37 Growth inhibition (%) against F.solani 92 51. 3.38 Growth inhibition (%) against C.glabarata 92 52. 3.39 Comparison of larvae dead at 10g/ml 95 53. 3.40 Comparison of larvae dead at 100g/ml 95 54. 3.41 Comparison of larvae dead at 100g/ml 95 55. 3.42 Percent mortality of insect by crude extract 98 56. 3.43 Percent mortality of insect by n-hexane fraction 98 57. 3.44 Percent mortality of insect by chloroform fraction 98 58. 3.45 Percent mortality of insect by ethyl acetate fraction 98 59. 3.46 Percent mortality of insect by butanol fraction 98 60. 3.47 Percent mortality of insect by aqueous fraction 98

x List of Figures

Entry Figure # Title Page #

61. 3.48 Comparison of (%) mortality of T.castaneum 99 62. 3.49 Comparison of (%) mortality of S.oyrzae 99 63. 3.50 Comparison of (%) mortality of R.dominica 99 64. 3.51 Comparison of (%) mortality of C.analis 99 65. 3.52 Comparison of growth regulation (%) 10g/mL 101 66. 3.53 Comparison of growth regulation (%) 100g/mL 101 67. 3.54 Comparison of growth regulation (%) 100g/mL 101 68. 6.1 Zone of inhibition by crude extract against different 129 bacteria 69. 6.2 Zone of inhibition by n-hexane fraction against different 129 bacteria 70. 6.3 Zone of inhibition by chloroform fraction against different 129 bacteria 71. 6.4 Zone of inhibition by ethyl acetate fraction against 129 different bacteria 72. 6.5 Zone of inhibition by n-butanol fraction against different 129 bacteria 73. 6.6 Zone of inhibition by aqueous fraction against different 129 bacteria 74. 6.7 Growth inhibition (%) by crude extract against different 130 bacteria 75. 6.8 Growth inhibition (%) by n-hexane fraction against 130 different bacteria 76. 6.9 Growth inhibition (%) by chloroform fraction against 130 different bacteria 77. 6.10 Growth inhibition (%) by ethyl acetate fraction against 130 different bacteria 78. 6.11 Growth inhibition (%) by n-butanol fraction against 130 different bacteria 79. 6.12 Growth inhibition (%) by aqueous fraction against different bacteria 130 131 80. 6.13 Zone of inhibition (mm) by crude extract, fractions and imipenum (standard drug) against E.coli 81. 6.14 Zone of inhibition (mm) by crude extract, fractions 131 and imipenum (standard drug) against B.subtilus 131 82. 6.15 Zone of inhibition (mm) by crude extract, fractions and imipenum (standard drug)against S.flexaniri 131 83. 6.16 Zone of inhibition (mm) by crude extract, fractions and imipenum (standard drug)against S.aureus 84. 6.17 Zone of inhibition (mm) by crude extract, fractions 131 and imipenum (standard drug)against P.aeruginosa 131 85. 6.18 Zone of inhibition (mm) by crude extract, fractions and imipenum (standard drug)against S.typhi 131 86. 6.19 Growth Inhibition (%) against E.coli

xi List of Figures

Entry Figure # Title Page #

87. 6.20 Growth Inhibition (%) against B.subtilus 131 88. 6.20 Growth Inhibition (%) against B.subtilus 131 89. 6.21 Growth Inhibition (%) against S.flexenari 131 90. 6.22 Growth Inhibition (%) against S.aureus 131 91. 6.23 Growth Inhibition (%) against P.aeruginosa 131 92. 6.24 Growth Inhibition (%) against S.typhi 131 93. 6.25 Percent growth inhibition by crude extract 131 94. 6.26 Percent growth inhibition by crude extract. 135 95. 6.27 Percent growth inhibition by n-hexane fraction 135 96. 6.28 Percent growth inhibition by chloroform fraction 135 97. 6.29 Percent growth inhibition by ethyl acetate fraction 135 98. 6.30 Percent growth inhibition by butanol 135 99. 6.31 Percent growth inhibition by aqueous 135 100. 6.32 Growth inhibition (%) against T.longifusisl 136 101. 6.33 Growth inhibition (%) against C.albicans 136 102. 6.34 Growth inhibition (%) against A.flavus 136 103. 6.35 Growth inhibition (%) against M.canis 136 104. 6.36 Growth inhibition (%) against F.solani 136 105. 6.37 Growth inhibition (%) against C.glabarata 136 106. 6.38 Comparison of larvae dead at 10g/ml 139 107. 6.39 Comparison of larvae dead at 100g/ml 139 108. 6.40 Comparison of larvae dead at 100g/ml 139 109. 6.41 Comparison of growth regulation (%) 10g/mL 144 110. 6.42 Comparison of growth regulation (%) 100g/mL 144 111. 6.43 Comparison of growth regulation (%) 100g/mL 144

xii List of Abbreviations

LIST OF ABBREVIATIONS

Entry Abbreviation Full word

1. SI-1 Crude methanolic extract Indigofera gerardiana 2. SI-2 n-hexane fraction Indigofera gerardiana 3. SI-3 Chloroform fraction Indigofera gerardiana 4. SI-4 Ethylacetate fraction Indigofera gerardiana 5. SI-5 n-Butanol fraction Indigofera gerardiana 6. SI-6 Aqueous fraction Indigofera gerardiana 7. SC-1 Crude methanolic extract Crataegus songrica 8. SC-2 n-hexane fraction Bergenia cilliata 9. SC-3 Chloroform fraction Crataegus songrica 10. SC-4 Ethyl acetate fraction Crataegus songrica 11. SC-5 n-Butanol fraction Crataegus songrica 12. SC-6 Aqueous fraction Crataegus songrica 13. WHO World Health Organization 14. HTS High Throughput Screening 15. NASs New Active Substances 16. NCEs New Chemical Entities 17. FDA Food and Drugs Administration 18. NCI National Cancer Institute 19. HIV Human Immuno Deficiency Virus 20. UV Ultra violet 21. 2D NMR Two Dimensional Nuclear Magnetic Resonance 22. HMQC Heteronuclear Multiple Bond Connectivity 23. NOESY Nuclear Overhauser Effect Correlation Spectroscopy 24. HMBC Heteronuclear Multiple Bond Connectivity 25. MS Mass Spectroscopy 26. 1H NMR Proton Nuclear Magnetic Resonance 27. 13C NMR Carbon Nuclear Magnetic Resonance 28. FAB Fast Atom Bombardment 29. EI MS Electron Impact Mass Spectrum 30. IR Infrared 31. DEPT Distortionless Enhancement by Polarization Transfer 32. EI Electron Impact 33. CI Chemical Ionization

xiii List of Abbreviations

Entry Abbreviation Full word

34. FD Field Desorption 35. BB Broad Band 36. ppm Parts Per Million 37. Rf Relative flow 38. TLC Thin Layer Chromatography 39. GGPP Geranyl Geraniol Pyro Phosphate 40. E.coli Escherichia coli 41. B.subtulis Bacillus subtilus 42. S.flexenari Shigella flexenari 43. S.aureus Staphylococcus aureus 44. P. aeruginosa Pseudomonas aeruginosa 45. S. typhi Salmonella typhi 46. T. longifusus Trichophyton longifusus 47. C. albicans Candida albicans 48. A. flavus Aspergillus flavus 49. M. canis Mycosporum canis 50. F. solani Fusarium solani 51. C. glabarata Candida glabarata

52. LD50 Lethal Dose 50 53. SDA Sabouraud Dextrose Agar 54. SDB Sabouraud Dextrose Broth 55. ACE Angiotensin converting enzyme 56. AChE Acetylcholinesterase 57. BChE Butyrylcholinesterase 58. q.s Quantity Sufficient 59. DMSO Di Methyl Sulfoxide 60. FBS Foetal Bovine Serum 61. RBCs Red Blood Cells 62. STD Standard 63. EtOAc Ethyl acetate

64. CHCl3 Chloroform 65. BuOH n-Butanol

66. H2O Aqueous 67. n-Hx n-Hexane 68. TS Transverse section

xiv Summary

SUMMARY

The present study based on thorough literature survey and strongly supported by the local folkloric data pertains to detailed pharmacognostic, phytochemical and pharmacological investigations of two traditional medicinal plants. The thesis is alienated into two major parts namely Part-A and Part-B, each portion includes introduction, experimental besides results and discussion chapters.

The pharmacognostic studies involve morphological features, microscopic examination and histology of different parts of the Indigofera gerardiana and berries of Crategus songrica. Various physio-chemical parameters such as moisture, ash, fiber, proteins, fats, oils and carbohydrates were determined in the samples of Indigofera gerardiana (whole plant) and Crategus songrica (Beries). The percent levels of moisture (4.8), ash (4.79) and fats (3.03) were higher in Crateagus songrica compared to the levels of moisture (3.06), ash (4.23) and fats (2.37) in Indigofera gerardiana. The % levels of protein (3.7) and fibers (17.8) were same in both plants, while the level of carbohydrate (68.84) was higher in Indigofera gerardiana as compared to the levels of carbohydrate (65.88) in Crataegus songrica.

Phytochemical investigations included isolation, characterization and identification of potential biologically active constituents of two plants as indicated in the previous paragraph. These experimental procedures have lead to the isolation of three new and one known compounds from the ethyl acetate fraction of methanolic crude extract of Indigofera gerardiana while the ethyl acetate fraction of Crataegus songrica yielded four known compounds though isolated for the first time from this source. As regards identification of new compounds, they have been thoroughly characterized employing modern analytical techniques such as 1D- and 2D-NMR, HMBC and HMQC experiments and supported by EIHR Mass Spectrometry. Where applicable, purification of lead compounds was performed using various applied techniques of chromatography. While assigning appropriate framework to the newly isolated lead compounds, a reasonable attention was given to fully analyze the structural

xv Summary features through NMR based experiments. Two new compounds belonged to glycosides of acylated and alkylated simple phenols anchored to para-position of aromatic rings while one of them strongly corresponded to acylated Gallic acid.

The pharmacological activity of Crataegus songrica crude extract revealed potential antibacterial activity against E.coli, B.subtilisand S.flexener. Incase of anti-fungal assay crude extract and subsequent fractions exhibited marked anti- fungal activity against T.longifusus, A.flavous, M.canis and F.solani. on other hand both the Candida species were neither inhibited by crude extract nor by the fractions of the plant No ctotoxicity and nor insecticidal activity was observed in crude extract and other segments. Similarly crude extract and various fractions were unable to produce anti leishmanial and anti-malarial activity. While crude extract and chloroform fraction have good phytotoxic potential at higher dose.

The antibacterial, antifungal, acute cytotoxicity, phytotoxicity and insecticidal profile of the crude extract and various fractions of Indigofera gerardiana have been studied. In terms of susceptibility of organisms towards crude extract and different fractions thereof, n-hexane fraction remained the most active against Salmonella typhi, Pseudomonas aeruginosa and E.coli strains respectively. The most interesting anti-fungal activity was demonstrated by the methanolic crude extract, n-hexane and chloroform fractions. The crude extract and the fractions showed low insecticidal activity against Sitophilus oryzae, Rhyzopertha dominica and Callosbruchus analis but no activity against Tribolium castaneum. The brine shrimp lethality assay showed absence of any measurable cytotoxicity of the crude extract and fractions. Crude extract and other fractions had no significant ani-leismainal and anti-malarial activity.

The results of current investigation have added three new compounds to the global database of phyto-compounds and enhanced our knowledge on the phytochemistry of Indigofera gerardiana. As regards the plant Crateagus songrica, thorough phytochemical investigations on the same plant could not be performed for several unavoidable reasons such as poor access to the area known for this plant, non-availability of advanced instrumentation laboratories at the host institution and so on. However, the plant discussed in Part-A possesses a lot more room for further pharmacological and phytochemical studies.

xvi Chapter-1 Part- A Introduction

1.0 INTRODUCTION

1.1 General Introduction

Traditional medicine involves the use of plants for medicinal purposes. The term 'herb' includes leaves, stems, flowers, fruits, seeds, roots, rhizomes, and bark of any medicinal plant. Other naturally occurring substances including animal and mineral products are also used. In traditional system, there are three main methods through which herbal medicines are prescribed. The first is traditional Chinese medicine (TCM); herbal medicines are an essential part of TCM and are prescribed according to an individualized diagnosis, much as one would prescribe particular acupuncture points. Ayurvedic herbs are also prescribed according to the main underlying principles of Ayurvedic medicine on an individual basis. Western herbal prescriptions are individually formulated and usually involve a mixture of herbs. [Griggs and Barbara 1981].

The World Health Organization (WHO) estimates that about, 80 percent of the world population presently uses herbal medicine for some aspect of primary health care. Herbal medicine is a major component in all indigenous peoples’ traditional medicine and a common element in homeopathic, naturopathic and traditional oriental. WHO estimates that of 119 plant-derived pharmaceutical medicines, about 85 percent are used in modern medicine in a way that correlated directly with their traditional uses as plant medicines by native cultures [Akerele, 1988; Antonymous, 1975; 1978].

Pakistan, China and India relies on traditional medicine which makes this region different from the West [Rahman, 2000] and according to an estimate about 25% of all prescriptions in the United States are from natural products [Fransworth, 1994; Newman et al., 1997]. Ayurveda and Chinese medicinal system are the most popular traditional which contributed a lot in the field of Pharmacognosy, Chemistry, Pharmacology and Therapeutics [Parkashi, 2000 ; Gilani and Rahman, 2005].

1 Chapter-1 Part- A Introduction

1.1.1 Plants as source of drugs

Most of natural products derived drugs were originally discovered through the study of traditional and folk knowledge [Gilani et al., 1992, Pat Wardhan et al., 2004]. Nature has been the primary source to cure the disease and discomforts from the early era for existence. Plants which have been used for relieving symptoms of disease are called medicinal plants or herbal medicines. [Gilani et al, 1992; Pat wardhan et. al: 2004]

The treatment through herbal medicine is the oldest form of treatment known to mankind. Plants had been used by all cultures throughout history. It was an integral part of the development of modern civilization. [Weiss RFa and Fintelmann 2000]. The plants provided food, clothing, shelter, and medicine. Much of the medicinal use of plants seems to have been developed through observations of trial and error. Indeed, well into the 20th century much of the pharmacopoeia of scientific medicine was derived from the herbal lore of native peoples. Many drugs commonly used today are of herbal origin. Indeed, about 25 percent of the prescription drugs dispensed in the United States contain at least one active ingredient derived from plant material. Some are made from plant extracts and others are synthesized to mimic a natural plant compound.

Substances derived from the plants remain the basis for a large proportion of the commercial medications used today for the treatment of heart disease, hypertension, pain, asthma, and other problems. For example, ephedrine, the active ingredient in Ephedra, is used in the commercial pharmaceutical preparations for the relief of asthma symptoms and other respiratory problems. It helps the patient to breathe more easily.

Ayurveda and Chinese traditional or natural medicinal system have a lot of contribution in the research of Pharmacognosy, Chemistry, and Pharmacology. [Pakrashi, S. C 2000; Gilani A.H and Rahman A .2005] .The interest in the development and study of medicinal plants is due to ethno-medicine. It means to use the natural products as medicine [Farswerth. 1990]. But actually it is known as ethno-botanic medicine.

2 Chapter-1 Part- A Introduction

Natural products used in the traditional folk medicine provide an interesting and still largely unexplored source for the creation on development of potentially new drugs [Awadh. et al .2001].

1.1.2 Bioactive Compounds from Herbs

The idea of using the medicinal plants is not new. Many of the active ingredients is chemically manufactured drugs were originally derived from plant compounds. It is generally believed that purified natural products and synthetic compounds are more likely to cause side effect and have less healing power than the herbs themselves herbal medicines are successfully used to treat a full range of conditions that can no be treated with conventional drugs, since these medicines draw their strength from the long experiences of humankind.

Medicinal plants are used to isolate bioactive compounds and metabolites as a precursor for valuable drugs, as a pharmacological tool and as a source of herbal remedies. In herbal medicine the active principles will be found in their leaves, flowers, fruits, seeds, rhizomes, stems, roots, barks, flowers, buds, and young leaves. The part of the climate time of the day is also important to have the active ingredients with maximum therapeutic effect of the herbal medicine (Sammbamurty and Subrahmanyam, 2000). Natural products used in the traditional folk medicine provide an interesting and still largely unexplored source for the development of potentially new drugs. [Awadh Ali, 2001].

Most of the research is focused on flowering plants used in traditional medicine but investigations into bioactive secondary metabolites. The structures of these compounds are determined by use of advanced spectroscopic techniques such as high resolution mass spectrometry and multinuclear magnetic resonance spectroscopy. Novel compounds are often isolated which could serve as lead molecules for pharmaceutical development. Compounds isolated include Dioxin from Digitalis purpora [Anonymous, 2003 and Bhattachargee .et al.2004]

There are many classes of secondary metabolites isolated from natural sources, which have been successfully used in different therapeutic preparations. Some of the common classes of natural compounds isolated from medicinal plants and other living sources along with their therapeutic categories are shown in table:

3 Chapter-1 Part- A Introduction

Table 1.1: Active principles derived from ethno pharmacological source, which are clinically used* Plant Family Drug Derived Pharmacological Class Rauwolfia serpentina Apocynaceae Ajmalicine Anti arrhythmic Areca catechu Palmae Arecoline Anthelmintic Artemisia annua Compositae Artemisinin Anti malarial Atropa belladonna Solanaceae Atropine Anti cholinergic Berberis vulgaris Berberidaceae Berberine Amebocidic Ardisia joponica Myrsinaceae Bergenin Anti asthmatic Coffea arabica Rubiaceae Caffeine CNS stimulant Cephaelis ipecacuanha Rubianceae Cephaeline Antibiotic Erythroxylum coca Erythroxylaceae Cocaine Local anesthetic Papaver somniferum Papveraceae Codeine Analgesic Colchicum autumnale Liliaceae Colchicine Anti inflammatory Curcuma longa Zingiberaceae Curcumine Choleretic Cynara scolaris Compositae Cynarin Choleretic Digitalis purpurea Scrophulariaceae Digoxin Cardiotonic Alangium lamavekii Alangiaceae Emetine Anti amoebic Ephedra sinica stapf Ephedraceae Ephedrine Anti asthmatic Vollota speciosa Amaryllidaceae Galanthamine Analgesic Simarouba glauca Simaroubaceae Glaucarubin Anti amoebic Corydolis fimbrillifera Fumariaceae Hydrastine Anti hypertensive Hyoscyamus niger Solanaceae Hyoscyamine Anti cholinergic Piper methysticum Piperaceae Kawain Tranquilizer Ammi visnaga Umbelliferae Khellin Anti asthmatic Vinca rosea Apocynaceae Leurocristine Anti cancer Lobelia inflate Companulaceae α-Lobeline Respiratory stimulant Papaver somniferum Papaveraceae Morphine Analgesic Corydalis cava Fumariaceae Narcotine Anti tussive Strophanthus gratus Apocynaceae Ouabain Cardiotonic Papver somniferum Papaveraceae Papaverine Vasodilator Carica papaya Caricaceae Papin Anthelmintic Hydrangea macrophylla Saxifragaceae Phyllodulcin Sweetener Lupinus pusillus Leguminosae Pachycarpine Cardiac

4 Chapter-1 Part- A Introduction

insufficiency Physostigma venenosum Leguminosae Physostigmine Cholinergic Anamirta cocculus Menispermaceae Picrotoxine CNS stimulant Pilocarpus microphyllus Rutaceae Pilocarpine Cholinergic Podophyllum peltatum Berberidaceae Podophyllotoxin Anti cancer Veratrum album Liliacceae Protoveratrine Anti hypertensive Ephedra sinica Stapf Ephedraceae Pseudoephaedrine Anti asthmatic Cinchona officinalis Rubiaceae Quinidine Anti arrhythemic Fagopyrum esculentum Polygonaceae Quercertin Capillary protective Rauwolfia vomitoria Apocynaceae Rescinnamine Anti hypertensive Rauwolfia serpentina Apocynaceae Reserpine Adrenolytic Rhododendron molle Ericaceae Rhomitoxine Anti hypertensive Fagophrum esculentum Polygonaceae Rutin Anti haemorrhagic Podocarpus saligna Taxaceae Saligenin Local anesthetic Sanguinaria canadensis Papaveraceae Sanguinarine Anti inflammatory Artemisia maritime Compositae Santonin Ascaricide Scilla maritime Lilliaceae Scillarens Cardiotonic Cassia acutifolia Leguminosae Sennosides A &B Laxative Silybum marianum Compositae Silymarine Anti hepatotoxic Strychnos nux-vomica Loganiaceae Strychnine CNS stimulant Taxus brerifolia Taxaceae Taxol Anti cancer Theobroma cacao Sterculiaceae Theobromine Vasodilator Camellia sinensis Theaceae Theophylline Vasodilator Trichosanthes kirilowii Cucurbitaceae Trichosanthin Abortifacient Chondodendron Menispermaceae Tubocurarine Muscle relaxant tomentosum Strychnos toxifera Loganiaceae Toxiferine Neuromuscular blocker Valeriana officinalis Valerianaceae Valeportiates Sedative Cantharanthus roseus Apocynaceae Vincristine Anti cancer Postina sativa Umbelliferae Xanthotoxine Radio protective Duphne genkwa Thymelaceae Yuanhuacine Abortifacient

* [Cordell et al., 1991; Duncan, 1987; Hoppe, 1975; Huxtable, 1992; Prasad, 1987; Watt, 1962; Buss, 2003; Grabley, 2000; Sneader. 1996; Mann, 2000].

5 Chapter-1 Part- A Introduction

1.1.3 Role of Natural Products on Economy

With the importance of natural products, their consumption has been increased world wide. Growing public interest in natural remedies has also carried enormous opportunities for scientist and industrialist. Herbal products demand has been increased through out the world for the last several years and got positive effect on the economy. The global sales of plants, exceeds to US $ 100 billion annually with a growth rate of over 10 %. The business of health and functional foods, dietary supplements, neutraceuticals, herbals teas and other herbal based personal care items worth over US $ 20 billion annually.

Pakistan is endowed with the wealth of medicinal plants. It has a valuable heritage of herbal remedies and like most developing countries, its rural population still relies mainly on the indigenous system of medicine for health related matters [Khattak et al., 1985]. Pakistan has about 6000 species of phanerogams and around 700 plant species are used as medicinal plants [Pei, 1992]. According to a survey conducted by Pakistan Forest Institute, 75 crude herbal drugs are extensively exported and more than 200 are locally traded in Pakistan. Indigenous people collect 85 % of these crude herbs from wild source. Crude medicinal plant materials worth more than Rs.150 million (US$ 2.3 million) per year are used in Pakistan. Similarly Pakistan exports large quantities of crude plants in international market of about US$ 6 million [Atta-ur-Rehman and Choudhary, 2003]. These health and trade statistics clearly indicate the growing global importance and economical value of herbal medicines.

6 Chapter-1 Part- A Introduction

1.2 Genus Indigofera

The Genus indigofera, comprises of 300 species. All of these are herbs or shrubs, distributed throughout the tropical regions of the globe. In Pakistan it is represented by 24 species [Awadh Ali, 2001].It belongs to family, Leguuminosae which is the third largest family of the flowering plants after orchidacneae and Compositae with approximately 650 genera and 18000 species. The family in general is characterized by the pod (legume) type of fruit developing from a single carpal with marginal placentaion. The family leguminosae is divided in to three sub-families. a) Caesalpiniodeae b) Mimosoideae c) Faboideae

1.2.1 Indigofera gerardiana Wall

The plant Indigofera gerardiana commonly known as Ghorega belongs to leguminoseae (Fabeaceae). It is widely distributed in northern parts of Pakistan and finds various medicinal uses in the indigenous system of medicine. In northern areas of Pakistan, this plant is traditionally used for relieving abdominal and spastic pains and the infectious diseases especially the skin infections involving microorganisms. The sister species of indigofera gerardiana were found to contain toxic nitro compounds of insecticidal activity.

Synonym Indigofera heterantha Wall [Khan, et al.2003]

Family Leguminosae

Genus Indigofera

Species Gerardiana

English name Indigo [Shinwari.et al.2006]

Local name Atees (Urdu), Ban-bal-nug (Kashmiri), Kainthi (Punjabi) Ghorejey (Pashto) S [Shinwari. et al.2006; Anonymous,2001]

Trade name Kainthi [Shinwari. et al .2006]

Habit Shrub[Shinwari. et al.2006]

Status Common

7 Chapter-1 Part- A Introduction

1.2.2 Natural Habitat

Dry sunny slopes, often forming dense scrub, and in forests 1500 - 3000 meters [Gamble, 1972]. Requires a light or medium well-drained soil and a sunny position [Huxley, 1992; Shinwari, et al.2006; Genders, 1994]. Succeeds on chalk [Shinwari. et al.2006]. Succeeds in full sun in most well-drained soils [Philips, 1989] including dry ones [Thoms, 1992; Genders, 1994].

1.2.3 Native Distribution

In Pakistan, the plant is found in mountainous areas of NWFP, AJK, Northern Areas of Dir, from 1500 to 3000 meters [Shinwari, et al.2006]. In India, it is available in Himalayas, Kasi Afghanistan and W. China [Anonymous, 2001]. Fast-growing when young but slowing with age [Huxley, 1992]. Tolerates1ight shade [Davis, 1990].

1.2.4 Plant Description

A tall shrub, 2.5m, covered bristly white hairs, copiously branched shrub with short imaripinnate, leaflets 9-33; flowers in axillary racemes, in erect often almost stalk less [Oleg and Rix, 1985] bright red or rosy or radish purple [Shinwari, e. al.2006; Anonymous, 2001] standard petal sessile, stamens diadelphous; pod cylindrical 10-12 seeded [Shinwari, et. al. 2006] have a vanillas cent [Eismon, 1999]; flowers are mostly 6-10mm; calyx bristal haired, with lobes as tubes; bracts minute. Leaves and leaflets very variable, leaflets elliptic to oblanceotate, mostly 4-12mm, with white hairs. Pod 1.3-2.5cm, straight, hairless [Oleg , 1985].

1.2.5 Folk Medicinal Use

In literature considerable work has been done on various species of the genus Indigofera. For instance, Indigofera daleoides Benth is used traditionally for the treatment of diarrhea and was found to be active against various pathogenic bacterial strains [Ethab, et al.2006] Similarly Indigofera oblongifolia has shown its antimicrobial [Dhot, 1999] hepatoprotective [Shahjahan, et al.2005] and lipoxygenase inihibitory activity [Sharif, et al.2005]. Abubakar et al. has reported the snake-venom neutralizing activity of Indigofera pulchra [(Abubakar et

8 Chapter-1 Part- A Introduction al.2006]. Antioxidant and free radical scavenging and anti-dyslipidemic activities of Indigofera tinctoria has been reported [Parkash, et al, 2007;Waako, et al, 2007]. Indigofera emarginella has shown in-vitro atimalarial activity against Plasmodium falciparum. Chakrabarti et al. have reported the antidiabetic activity of Indigofera mysorens [Chakarbarati, et al. 2006].

Whole plant is used in hepatitis, whooping cough [Shinwari, et.al. 2006] antispasmodic [Khan, et al 2003], tonic [Gamble, 1972], the extract prevents the development of hypoglycemia in the mouse [Nyarko et al., 1998]; the leaves, flowers and tender shoots are cooling and demulcent, they are used in the form of leprosry and cancerous infection. The leaves are applied to abscesses. The roots are chewed in toothache and apathy [Gamble, 1972]. The alcoholic extract of the dried shoots is reported anti – inflammatory activity [Amala Bhaskar et al., 1982]; the root bark is chewed in the mouth to relieve the abdominal pain [Esimon, 1999]; leaves, bark and roots have anti-bacterial activity [Umar, 1999; Khan, et. al. 2003]

9 Chapter-1 Part- A Introduction

1.3 FLAVONOIDS

1.3.1 Introduction

The term flavonoids refer to a class of plant secondary metabolites. ("Studies force new view on biology of flavonoids. They are widely distributed in plants are one of the most diverse and widespread group of natural products, occupy a prominent position among the natural phenols, fulfilling many functions including producing yellow or red/blue pigmentation in flowers and protection from attack by microbes and insects.

The name “falvonoid” is derived from Greek word flavus (yellow). These are important biologically active plant ingredients and about 2% of all the carbon photosynthesized is converted into flavonoids [Harbone et al., 1988]. Flavonoids are the coloring co-pigment of the plants [Zechmeister et al. 1957] which are also called anthoxanthins. The wide range of colours and shades in flowers are principally due to the presence of flavonoids. These usually occur as pigments in the cells of flower tissues [Manitto et al., 1981].

1.3.2 Classification of Flavonids

Over 5000 naturally occurring flavonids have been characterized from various plants. Flavonids have been classified according to their chemical structure, and are usually subdivided or classified into the following subgroups. Flavonoids, derived from 2-phenylchromen-4-one (2-phenyl-1, 4-benzopyrone) structure.

That is:

. FLAVONES use the 2-phenylchromen-4-one skeleton. Examples: Luteolin, Apigenin, Tangeritin

. FLAVONOLS or 3-hydroxyflavones use the 3-hydroxy-2-phenylchromen- 4-one skeleton. Examples: Quercetin, Kaempferol, Myricetin, Fisetin, Isorhamnetin, Pachypodol, Rhamnazin

. FLAVANONES use the 2,3-dihydro-2-phenylchromen-4-one skeleton..Examples: Hesperetin, Naringenin, Eriodictyol They are also

10 Chapter-1 Part- A Introduction

formed as 3-Hydroxyflavanones or 2,3-dihydroflavonols, 3- Hydroxyflavanones use the 3-hydroxy-2, 3-dihydro-2-phenylchromen-4- one skeleton. Examples: Dihydroquercetin, Dihydrokaempferol .

. ISOFLAVONES, derived from 3-phenylchromen-4-one (3-phenyl-1, 4-benzopyrone) structure. Examples: Genistein, Daidzein, Glycitein.

. NEOFLAVONOIDS, derived from 4-phenylcoumarine (4-phenyl-1, 2-benzopyrone) structure. Anthocyanidins use the flavylium (2-phenylchromenylium) ion skeleton: Examples: Cyanidin, Delphinidin, Malvidin, Pelargonidin, Peonidin, Petunidin .

1.3.3 Structural Behavior of Flavonoids

They occur in a different structural form, may be in the form of aglycon, glycosides and methylated derivatives. All contain fifteen carbon atoms in their parent nucleus and share the common structural feature of two phenyl rings linked by a three carbon chain (diphenyl propane derivatives). The compounds possessing a 1, 3 –diphenylpropance, skeletons are regarded as chalconoids. The three carbon chain may be formed into a third (five or six membered) ring thought oxygen on one of these phenyl rings generating a tricyclic system. The tricyclic compound possessing a five membered heterovylic ring are referred to auronoiods wherease those possessing a six memebered heterocyclic ring are designated as flavonoids.

Fig. 1.1: Basic structure of flavonoid.

The three-carbon chain may be cyclized into a third (five or six membered) ring through an oxygen atom with one of the preexisting phenyl rings, generating a tricyclic system. The tricyclic compounds possessing a five membered

11 Chapter-1 Part- A Introduction heterocyclic ring are referred to auronoids, whereas those possessing a six membered heterocyclic ring are designated as flavonoids (Fig. 1.1).

O O O O A B

C

Isoflavonoid Isoflavonoid Isoflavonoid 3-Phenylcoumarin3-Phenylcoumarin

Figure 4.2 Flavonoids having 1, 2-diphenylpropane skeleton

Fig.1.2: Flavonoids having 1, 2-diphenypropane skeleton

The tricyclic compounds derived from 1, 2-diphenylpropane system are known as isoflavonoids and 3-phenylcoumarins (Fig.1.2), whereas those derived from 1,1-diphenylpropane are called neoflavonoids (Fig.1.3).

O O

Neoflavonoids

Figure 4.3 Flavonoids having 1, 1-diphenylpropane skeleton

Fig.1.3: Flavonoids having1, 1-diphenylpropane skeleton

12 Chapter-1 Part- A Introduction

5 6 4 5' O 1 4' 6' 3  2 1' 3'  2' ' Chalcone '-Chalcanol '-Chalcanone OH O HO O

O -Chalcanol Chalcan-1,3-dione -Chalcanone O O 8 9 H 7 O 2 1 6' OH O 4 3 O 6 5' 10 1' 5 O 2' H O Aurone 4' Aurononol Auronol 3' O O 5' O 6' 4'

O 8 1' 9 O 3' O 7 1 2 2'

6 4 3 10 OH H 5 Flavanone Isoaurone O O Flavanonol

O O O Flavonol OCH3 OH Flavone O 3-Methoxyflavone O O

O O O

Flavan OH Flavan-3-ol Anthocynanidin O O O

O O Isoflavan Isoflavanone Isoflavone

Figure 4.4 Basic skeleton and numbering patterns in flavonoids

Fig.1.4: Basic skeleton and numbering patterns in flavonoids

13 Chapter-1 Part- A Introduction

In tricyclic compounds of the flavonoid, auronoid, and isoflavonoid types, the rings are labeled as A, B, and C and the individual carbon atoms are referred by a numbering system which utilizes ordinary numerals for the ring A and ring C, and primed numerals for the ring B (Fig. 1.4).

8 11 2' O O 9 O 1' 1 3' 7 2

6 4 3 4' 10 6' 5 5' O O O Homoflavone Homoisoflavone Homoisoflavanone

8 9 O 2 11 O O 7 1 O O 2' 6 4 1' 10 3 3' 5 O 6' 4' O Homoisoflavan Rotenoid 5' Dehydrorotenoid

Figure 4.5 Basic skeleton and numbering patterns in homoflavonoids

Fig. 1.5: Basic skeleton and numbering patterns in homoflavonoids

Natural flavonoids and isoflavonoids are usually oxygenated and bear hydroxy and/or methoxy substituents. The structures and numbering of some common naturally occurring classes of monomeric flavonoids are illustrated in Fig. 1.5.

Homoflavonoids (Fig.1.5) form a class of flavonoids, which contain an additional carbon in their skeleton. This additional carbon is designated as C 11. A large number of flavonoids occur as O-glycosides in which one or more of the hydroxyl groups of the flavonoid are bound to a sugar or sugars via an acid labile hemiacetal bond. In flavonoid with C-glycosides [Harbone et al., 1975; Harborne and Mabry, 1982], the sugar is C-linked and this linkage is acid resistant. The effect of glycosylation makes the flavonoid less reactive and more water soluble.

14 Chapter-1 Part- A Introduction

1.3.4 Biosynthesis of Flavonoids

1.3.5 General Aspects

The biosynthesis of all flavonoids involves the formation of central C15 intermediate known as chalcone. The central role of chalcones in flavonoid biosynthesis has been confirmed in a number of investigations [Grisebach, 1965; Wong, 1965]. The formation of chalcone is catalyzed by chalcone synthase, which is the key enzyme in flavonoid biosynthesis.

The precursors for chalcone formation are malonyl-CoA and 4-coumaroyl- CoA (hydroxycinnamic acid CoA ester). Both flavonoid precursors are derived from carbohydrates. Malonyl-CoA is synthesized from the glycolysis intermediate acetyl-CoA and carbon dioxide, the reaction being catalyzed by acetyl-CoA carboxylase (Scheme 1.1). The synthesis of 4-coumaroyl-CoA is more complex and involves the shikimate pathway (Scheme 1.2), which is the main route to aromatic amino acid, phenylalanine and tyrosine in higher plants. The biosynthesis of shikimic acid (Scheme 1.3) begins with the condensation of D- erythrose-4-phosphate and phosphoenolpyruvic acid [Monitto, 1981].

15 Chapter-1 Part- A Introduction

O SCoA 3 HOOC CH3 C SCoA Carbohydrates O Acetyl-CoA Malonyl-CoA

OH OH

OSCoA CoASH O CoAS HO O 4-hydroxy -H2O O O coumaric acid O O

CO2

OH OH

O SCoA HO OH Chalcone Synthase O Chalcone

O O OH O Scheme 4.1 Biosynthesis of chalcones through malonyl-CoA

Scheme 1.1: Biosyenthesis of chalcones through malonyl-CoA

16 Chapter-1 Part- A Introduction

D-Glucose + CO2 Pentose phosphate Glycoslysis cycle

O P O HO CH H H H2O H O C H POH C OH 2 2 Phosphoenol pyruvic D-Erythrose-4-phosphate acid (PEP)

Pi O H O HO CO2 HOOC OOC OP

O OH OH HO OH OH HO OH OH H2O Syn elimination

CO2 NADP NADPH CO2

HO OH O OH H OH OH

Shikimate

Continued

17 Chapter-1 Part- A Introduction

CO2 CO2

PEP ATP PO CO2

HO OH PO OH.. OH OH Shikimate Pi

CO2 CO2 PO H

O CO2 PO O CO2 OH OH Chorismate O CH2COCO2 O O 2C CH2COCO2 NAD NADH

H

CO OH 2 O CO2 CH2COCO2 H 2C NH3 NH3

OH OH CO H CO H 2 2 HC H C 2 NH H 2

NH2

OH OH p-hydroxy coumaric acid Scheme 4.2 Biosynthesis of p-hydroxy coumaric acid through Shikimate pathway

Scheme 1.2: Biosynthesis of p-hydroxy coumaric acid through Shikimate pathway

18 Chapter-1 Part- A Introduction

1.3.6 Individual Steps to Flavonoid Classes

1.3.6.1 Flavanone Formation

Flavanones are formed from chalcones by isomerization. There is a good evidence for the in vitro and in vivo existence of equilibrium between flavanones and the corresponding chalcones [Monitto, 1981]. The interconversion between chalcones and flavanones is catalyzed in vivo by enzyme known as chalcone isomerase. The stereospecificity of this enzymatic reaction is apparent in the (S) chirality of C 2 in flavanone derivative. Therefore, it is not accidental that all the flavanones found in nature have the (S) configuration at C 2 and are levorotatory.

.. OH OH B H HO O HO O BH

HOH* OH O HA OH O Chalcone OH H .. A HO O OH

* H OH O Flavanone Scheme 4.3 Biosynthesis of flavanones

Scheme 1.3: Biosynthesis of flavanones

With chalcones having at least two free hydroxyl groups at C 2 and C 6, the equilibrium in an aqueous solution is shifted completely and rapidly to the flavanone (Scheme1.3). The stabilization energy of the strong hydrogen bond between the carbonyl group and the ortho-phenolic hydroxyl group greatly influence the position of equilibrium and the interconversion rate. When only one hydroxy group is available, either for the cyclization or for hydrogen bonding, the system tends to remain in the open form (chalcone form) [Barnes.et al., 2002].

19 Chapter-1 Part- A Introduction

1.3.6.2 Isoflavone Formation

The key step in isoflavone formation is the 2,3-migration of the aryl side chain of a flavanone intermediate, chalcone (Scheme1.3). An enzyme activity catalyzing this transformation was recently found in microsomal preparations from elicitor-challenged soybean cell suspension cultures. It transforms (2S)- naringenin (flavanone) into genistein (isoflavone) (Scheme 1.4).

It was found that two enzymatic steps are involved in this transformation. The first step comprises of oxidation and rearrangement of naringenin to 2- hydroxy-2, 3-dihydrogenistein. This step is strictly dependent on NADPH and molecular oxygen. Another enzyme which catalyses the elimination of water from the 2-hydroxyisoflavanone was isolated but has not yet been characterized [Kochs and Grisebach, 1986]. The sequence of events may be initiated by an epoxidation step. Protonation and subsequent cleavage of the epoxide would render a positive charge to the ring B. Keto-enol tautomerism, as indicated by proton exchange at C 3 in flavanones [Grisebach and Zilg, 1968], allows homoallylic interaction between C 3 and C 1′, and rearrangement of the structure then takes place. Addition of hydroxyl ion to C 2 leads to the 2-hydroxyisoflavanone intermediate, which is transformed to the isoflavone by elimination of water molecule.

OH OH

HO O HO O

. OH O OH OH . BEnz Naringenin NADPH H O2 O H2O.. HO O HO O

O -H2O

O HO O HB Enz HO HO

H HO O OH HO O

H2O

OH O OH O OH Genistein OH 2-Hydroxydihydrogenistein (Isoflavone)

Scheme 4.4 Biosynthesis of isoflavones Scheme 1.4: Biosynthesis of isoflavones

20 Chapter-1 Part- A Introduction

1.3.6.3 Flavone Formation

The in vitro conversion of flavanones to flavones was first observed in parsley plants. The reaction has been studied in more detail in parsley cell suspension cultures [Britsch et al., 1981] and in Antirrhinum flowers [Stotz and Forkmann, 1981]. The parsley enzyme requires 2-oxoglutarate, Fe2+ and possibly ascorbate as co-factors. This co-factor requirement would classify it as a 2- oxoglutarate dependent dioxogenase. Ascorbate stimulates this and other 2- oxoglutarate dependent dioxygenases involved in the flavonoid pathway and, in addition, exhibits a remarkable stabilizing effect on the enzyme activity [Britsch and Grisebach, 1986].

OH OH OH HO O HO O [O]

HO O OH HO O Naringenin HO O H O H2 2

HO O Apigenin (Flavone)

Scheme 4.5 Biosynthesis of flavones

Scheme 1.5: Biosynthesis of flavones

Both the parsley and the flower enzyme catalyzed the conversion of (2S)- naringenin (flavanone) to apigenin (flavone) (Scheme 1.5). The mechanism of double bond formation is still unclear. It has been suggested that 2-hydroxyflavanone is formed in the first step, and water is then eliminated via a dehydratase [Britsch et al., 1981; Stotz and Forkmann, 1981]. However, no such 2-hydroxy intermediate has yet been isolated even with a nearly homogenous enzyme protein [Britsch and Grisebach, 1986]. On the other hand, 2-hydroxyflavones certainly exist as plant metabolites and they are indeed, the substrates in C-glycosylflavones formation [Kerscher and Franz, 1987].

21 Chapter-1 Part- A Introduction

1. 3.6.4 Flavonols Formation

Enzymatic conversion of dihydroflavonols to flavonols was first observed with enzyme preparations from parsley cell suspension cultures [Britsch et al., 1981]. The synthesis of flavonols was found to be catalyzed by a soluble 2- oxoglutarate dependent oxygenase. Flavonol synthesis, most probably, proceeds via a 2-hydroxy intermediate such as; 2-hydroxydihydrokaempferol with subsequent dehydration, giving rise to the respective flavonols [Hauteville et al., 1979] (Scheme 1.6). Flavonol synthase has also been identified in flower extract from Matthiola [Spribille and Forkmann, 1984] and Petunia [Forkmann and Sratz, 1984]. As in parsley, flavonol formation in these flowers is catalyzed by a soluble 2-oxoglutarate dependent dioxygenase.

OH OH

HO O HO O [O]

OH HO O HO O Naringenin Dihydrokaempferol [O] OH OH OH HO O HO O -H O 2

OH OH HO O HO O Kaempferol (Flavonol) 2-Hydroxydihydrokaempferol Scheme 4.6 Biosynthesis of flavonols

Scheme 1.6: Biosynthesis of flavonols

22 Chapter-1 Part- A Introduction

1.3.6.5 Glycosylation of Flavonoids

A vast number of flavonoid glycosides are found in nature indicating the occurance of a range of glycosyltransferases with varying substrate specificities [Harbone et al., 1975].

Novel flavonol O-glycosyltransferases were demonstrated in enzyme preparations from tulip anthers [Kleinehollenhorst et al., 1982], Pisum flowers, Chrysosplenium shoots [Bajaj et al., 1983; Khouri et al., 1986], and Anethum cell cultures [Mohle et al, 1985]. These enzymes exhibit a pronounced specificity with regard to the substrate, the position and the sugar transferred. The isoflavone 7-O- glucosyltransferase, isolated from Cicer, shows a similar high specificity [Koster and Barz, 1981].

Two enzymes isolated from Chrysosplenium extracts glycosylate the B- ring of highly methoxylated flavonols in the 2′ and 5′ positions [Ibrahim, 1986]. A particular interesting situation has been found during extensive genetic and biochemical studies of the glycosylation of isovitexin (6-C-glucosylapigenin) in Silene paratensis and Silene dioica [Steyns et al., 1984]. Eleven functional alleles, spread over six loci, have now been identified. These codes for different glycosyltransferases which catalyzed glucosylation, galactosylation, and xylosylation of the 7-hydroxyl group or glucosylation, rhamnosylation, xylosylation, and arabinosylation of the 2-hydroxyl group of the carbon bound glucose of isovitexin.

1.3.6.6 Methylation of Flavonoids

Many enzymes catalyzing a methyl transfer from S-adenosylmethionine to the various hydroxyl groups of flavonoid substrates [Harborne and Mabry, 1982]. Some of them require Mg+2 as an obligatory co-factor. S-Adenosylhomocysteine, formed in the reaction, is an inhibitor of these enzymes. An 8-hydroxyflavonol-8- O-methyltransferase was reported from Lotus corniculatus flowers [Jay et al., 1985; 1983]. Lotus flowers also contain 3- and 3′-O-methyltransferases, which have been shown to catalyse the methylation of the flavones (luteolin) and flavonols (quercetin, myricetin and isorhamnetin) in the relevant positions [Jay et al., 1983].

23 Chapter-1 Part- A Introduction

1.3.7 Biological Properties of Flavonoids

Flavonoids have been referred to as "nature's biological response modifiers" because of strong experimental evidence of their inherent ability to modify the body's reaction to allergens, viruses, and carcinogens. They show anti- allergic, anti-inflammatory [Yamamoto and Gaynor 2006], anti-microbial and anti-cancer activity. The widespread distribution of flavonoids, their variety and their relatively low toxicity compared to other active plant compounds (for instance alkaloids) mean that many animals, including humans, ingest significant quantities in their diet. Consumers and food manufacturers are interested in flavonoids for their medicinal properties, especially their potential role in the prevention of cancers and cardiovascular disease.

The biologic activities of flavonoids include action against allergies, inflammation, free radicals, hepatotoxins, platelet aggregation, microbes, ulcers, viruses and tumors [Kinsella, J.E., et al. 1993.] Flavonoids also inhibit specific enzymes. For example, flavonoids block the angiotensin-converting enzyme (ACE) that raises blood pressure: By blocking the "suicide" enzyme cyclooxygenase that breaks down prostaglandins, they prevent platelet stickiness and hence platelet aggregation. Flavonoids also protect the vascular system and strengthen the tiny capillaries that carry oxygen and essential nutrients to all cells. [Flavonoids (isoflavonoids and neoflavonoids). IUPAC Compendium of Chemical Terminology]. Flavonoids block the enzymes that produce estrogen, thus reducing the risk of estrogen-induced cancers. One way they do this is by blocking estrogen synthsase, an enzyme that works overtime in binding estrogen to receptors in several organs. [ Northrup, C. Women's Bodies, Women's Wisdom: 305. New York; Bantam Books, 1994].

Phytonutrients of this phenol subclass come from beans and other legumes and are distant cousins of flavonoids. Isoflavones function much like flavonoids in that they effectively block enzymes that promote tumor growth. Best known isoflavones are genistein and daidzein found in soy products and the herb Pueraria lobata (Kudzu). People who consume traditional diets rich in soy foods rarely experience breast, uterine and prostate cancers. Pueraria has gained

24 Chapter-1 Part- A Introduction popularity as an aid for those who consume alcohol because it appears to alter the activity of alcohol detoxification enzymes, namely the speed at which alcohol dehydrogenase converts alcohol into aldehydes. The result is a lowered tolerance for alcohol and reduction of the pleasure response to drinking it. [Xie, C.I., et al.1994].

Different classes of flavonoids have been analyzed for different physiological activities. No universal function for the flavones and/or flavonols in plants has yet been established, in spite of the fact that these are the most common and widely distributed flavonoids. [Harborne and Mabry, 1982]. Willaman et al has reviewed, the biological effects of the flavonoids and listed thirty-three different manifestations of activity under the heading “Bioflavonids” [Willaman et al., 981]. Rutin and hesperdin, also called vitamin P or permeability factors, are used in the treatment of various diseases, like capillary bleeding, increased capollary fragility, diabetes, allergic manifestation and hypertension. [Tyler et al., 1988]. Phenolic substances are well known to have anti-inflammatory. Some flavonoids like myrcetin and kaempferol-3-glucoside have an anti HIV-I Potency at non toxic concentration [Hostettman at al., 1995].

The position of the substitution also affects the properties. The flavonols containing two ortho or para hudroxyl in the 2-phenyl ring have anti-oxidant properties, while free hydroxyl at the 5, 7-positions have a pro-oxidant effect [Zechmeister et al., 1957]. Apigenin and genkwanin, which are present in the Chinese, drug “Yue hua are believed to have diuretic and anthelmintic properties. These actions become more pronounced with an increasing number of hydroxyl groups. Fluka has reported the cardiac stimulation and vasoconstriction of the flavones and their glycosides [Zechmeister et al., 1957].

Some flavones have the properties of prevention of anaphylactic shock, protection against X-rays and cure from frostbite [Willaman 1981]. The simple isoflavone and coumestans have oesterogenic activities, rotenoids have insecticide properties while isoflavonoid phytoalexins have anti-fungal and anti-bacterial activities [Ingham et al., 1983].

Some of the minor flavonoids have very interesting activities. They have anti- microbial, anti-fungal and cytotoxic properties [Harborne et al., 1988]. Tricin

25 Chapter-1 Part- A Introduction has smooth muscle movements activity [ferguson et al.1950]. Anthocyanin peonin and isorhamnetin of the algae Chlamydomonas are highly potent sex determining hormone [Kuhn et al., 1994]

The flavonoid glycosides have also a vital pharmacological role. De Rodrigues et. al., reported an anti-giral activity for the 5-0 methylgecistein-7-0- - D- glucopyranoside. This isoflavonide glycoside was isolates from the Ulex europaeus L. [De Rodriguez et al., 1990]. Similarly, prenylflavonol glycoside, epimesokoreanoside isolates from the Epimedium Koreamum (Berberidaceae) showed a hypotensive activity [Pachaly et al.1990].

Anthocyanins have wide application in the food industry. It helps in determining wine quality. It is used as natural colourant to replace synthetic red dyes [Harbone et al.1988]. Anthocyanins are also used for the inhibigion of larval growth in insects [Paerpoint et al. 1986]. Anthocyanidin (aglycones of anthocyanins) have been investigited for biological activity. It was found that delphindin chloride has a microvascular protective activity [Gabetta et al. 1990].

Chalcones have been thoroughly investigated by G. Michiro and it was found that these have anti-microbial activity, which is enhanced by the bromination. Dihydroxychalcones, which is a usual class of flavonoids are generally considered physiologically inactive but 2, 6-dihydroxy-4-methy-3-5- dimethylnydrochalcone has lipoxygenase inhibiting activity –3, 5 [Michihiro et al., 1990]. C-methylated dihydochalcones are very rare as natural products. The exudeates of myrica gale fruits are a rich source of c-methylated dihydrochalcones [Yoshiaka al, 1973]. One of the functions of flavonoids in plants is to protect them from the diseases caused by microorganism. Uvaritin and isouvaritin have anti- microbial and cytotoxic activities [Malterud, et al. 1990, Alcoraz et al., 1990 Ruzicka 1953]. Some dihydrochalcones have unconupling and inhibitory activities on isolated mitochondria [Harbone et al., 1982 and Nicholas et al., 1967].

Some flavonoids and dihydroxychalcones have taste properties. Their different taste normally depends upon the point of attachment of the substitutent. Isoflavonoids act as venom antidote. Some flavonoids are used as inhibitors of

CCI4 induced cytotoxicity in isolated hepatocytes. The isoflavones genistein has oesterogenic activities, which is important for sheep breeding in the dairy areas of

26 Chapter-1 Part- A Introduction

Western Australia. The biosynthesis of coumosterol in leaves is induced by UV light, so its level is used for the indication of damage of UV to the plant system about Bioflavonoids functions as UV filters. These functions guard on the leaf from insect and microbial attack. These inhibit adhesion of blood platelets and blocking of inflammatory effects. These also act as heart stimulant [Ruzicka 1953].

1.3.8 Important sources of flavonoids

Good sources of flavonids include all citrus fruits, berries, onions, parsley, legumes, green tea, red wine, sea buckthorn, and dark chocolate.

Quercetin; flavonoids and more specifically a flavonol consist of aglycone of the glycoside rutin. Which are most active of the flavonoids, possessing the anti-inflammatory activity by the production and release of histamine and other allergic/inflammatory mediators. It has been found in the herbal products such as Cateagus, which are used for acute symptoms of Congestive heart failure. It also exerts potent antioxidant activity. [Lee et al. 2003].

27 Chapter-1 Part- A Introduction

1. 4 PHARMACOGNOSTIC STUDIES

Pharmacognosy is the study of the physical, chemical, biochemical and biological properties of drugs, drug substances or potential drugs or drug substances of natural origin as well as the search for new drugs from natural sources plant and animal kingdom [Tyler, 1981] [The American Society of Pharmacognosy].

It is closely related to both botany and plant chemistry. It also plays an important role in curing disease but has been generally pursued for utilization ends and may thus be called an applied science [Trease and Evans, 1992]. In herbal medicine, the product is important apart from its other plant parts. For example the active biological constituents will be found in their leaves, flowers, fruits, seeds, rhizomes, stems, roots, bark, flower buds, and young leaves. Not only this, the timing of the active principle found in which part of the season and also the time of the day are important sometimes to get maximum effect of the herbal medicine [Sammbamurty and Subrahmanyam, 2000].

While studying the compound mixtures (crude extract or partially purified by fractionation), isolated pure compounds from nature possessing biologically activity, i.e. species-specific chemical agents that can be grouped into various categories. A typical protocol to isolate a pure chemical agent from natural origin is bioassay-guided fractionation, meaning step-by-step separation of extracted components based on differences in their physicochemical properties, and assessing the biological activity. The most common means for separation is chromatographic methods, such as thin-layer chromatography (TLC) is used for these preparative purposes. After isolation of a pure substance, the structure can be determined by subjecting them to combination of modern instrumental techniques such as nuclear magnetic resonance spectroscopy (NMR) and mass spectroscopy (MS).

The study includes collection, preservation, macroscopical, anatomical, microscopic characters of powdered drug, nutritional and elemental analyses, microchemical tests, biological activities and phyto-chemical study.

28 Chapter-1 Part- A Introduction

1.4.1 Morphology and Anatomy

Morphology and histology makes the first step to get knowledge about the diagnostic features, which are ascertained through the study of the tissue and their arrangement, cell wall and cell content [Youngken, 1950]. Microscopy of the powder drug has the advantage of material. This study provides useful references not only for forensic scientists and for others engaged in the evaluation of powder drug but also to analysts who are concerned with standardization and authentication of prepared drug material [Jackson, 1992].

1.4.2 Nutrition

Feed of high nutritive value promote a high level of production. [Eagan et al. 1986]. That is determined by the ability to provide the nutrients required by the animals for maintenance, growth and reproduction. The nutritive values depend upon the voluntary intake of feeds consumed and extent to which the quantity of dry matter consumed by animals supplements, dietary energy, proteins, minerals and vitamins. Much will depend on actual quantity of feed eaten by animals on daily basis. [Dougell et al. 1964]. The nutritive values are estimated by the measurements of digestibility either directly or by using standard equations derived from a large number of feeding trials. [NRC, 1985].

The essential life nutrients are proteins, fat and carbohydrates. They contribute to caloric content of the dietary, minerals including trace elements, vitamins and water. Numerous studies in man have demonstrated clearly that life may be sustained by nutrient mixtures in which every component is definable chemically and soluble in water [Underwood, 1994]. The quality and quantity of protein in the seed are basic factors important in the selection of plants for nutritive value, systematic classification and plant improvement programs [Siddique, 1998].

Most of the countries of the world are facing malnutrition problems. The deficiency of protein in human food and animal feed is well recognized. The need of the good quality of proteins is increasing due to rapid growth of population. It has been reported that in Pakistan the protein gap would continue to increase

29 Chapter-1 Part- A Introduction unless well-planned measures are adopted to tackle the situation. It is therefore imperative to increase protein production by utilizing all the available ways and means. In addition to increase in conventional production, a great of work has been done in recent years in developing new chemical and biological methods for the production of protein foods and feeds [Shah and Khalil, 1988]. The quality and quantity of protein in the seed are basic factors important in the selection of plants for nutritive value systematic classification and plant improvement programs [Siddique, 1998].

Indigofera contains the pigment indigo, which is used as grazing forage as well as in and high quality food supplement for ruminants [Haude, 1997]. Indigofera spicata is a useful tropical legume but contain hepatotoxic amino acids (Inospicine) which interfere with the metabolism [Hutton, 1970]

1.4.3 Microchemical Tests

These tests may provide considerable knowledge about the chemical constituents, which are responsible for the therapeutic effects. Preliminary screening of plant samples for alkaloids, saponins, tri-terpines and steroids is the usual initial step undertaken by a phytochemist in the quest of novel or bioactive compound [Ahmed, 1995].

Microchemical tests, nutritive and elemental analysis may provide considerable knowledge about the chemical contents and active biological constituents, which could be responsible for the medicinal properties. The use of the medicinal herbs along with other metallic preparations can be traced from prehistoric times. The medicinal plants have great importance both from professional as well as economic point of view. Literature survey reveals that a very few plants have been analyzed for their chemical constituents and large no of them remain unanalyzed [Sondhi and Agarwal, 1995].

Feed of high nutritive value promote a high level of production. [Eagan et al. 1986]. That is determined by the ability to provide the nutrients required by the animals for maintenance, growth and reproduction. The nutritive values depends upon the voluntary intake of feeds consumed and extent to which the quantity of dry matter consumed by animals supplements, dietary energy, proteins, minerals

30 Chapter-1 Part- A Introduction and vitamins. Much will depend on actual quantity of feed eaten by animals on daily basis.

[Dougell et al. 1964]. The nutritive values are estimated by the measurements of digestibility either directly or by using standard equations derived from a large number of feeding trials. [NRC, 1985].

Most of the countries of the world have been facing malnutrition problems. The deficiency of protein in human food and animal feed is well recognized. The need of the good quality of proteins has been increasing due to rapid growth of population. It has been reported that in Pakistan the protein gap would continue to increase unless well-planned measures are adopted to tackle the situation. It is therefore imperative to increase protein production by utilizing all the available ways and means. In addition to increase in conventional production, a great of work has been done in recent years in developing new chemical and biological methods for the production of protein foods and feeds [Shah and Khalil, 1988]. The quality and quantity of protein in the seed are basic factors important in the selection of plants for nutritive value systematic classification and plant improvement programs [Siddique, 1998].

31 Chapter-1 Part- A Introduction

1.5 PAHRMACOLOGICAL STUDIES

1.5.1 Antibacterial Assay

According to WHO, 43 % of total deaths in developing countries occurred due to the infectious diseases. The search for new effective antimicrobial agents is necessary due to the microbial resistance and occurrence of opportunistic infections. The drug resistant bacteria have further complicated treatment of infectious diseases in immuno-compromised and cancer patients [Kawamori et al., 1999; Limitrakul et al., 1997].

Ethno botanical data have proved to be useful in search for new antimicrobial agents and many antibiotics were isolated from natural sources (microbes or medicinal plants). The substances that can either inhibit growth of pathogen or kill them and have no or least toxicity to host cells are considered candidates for developing new anti-bacterial drugs [Kivack et al., 2001].

Bacteria are classified as Gram positive and Gram negative on basis of their cell wall structures. During this study, antibacterial activity was carried out against Escherichia coli, Bacillus subtilus, Shigella flexenari, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhi. Pathogenesis caused by these bacteria is discussed in Table 1.2

32 Chapter-1 Part- A Introduction

Table 1.2 Pathogenesis caused by bacteria*

Entry Bacteria Pathogenesis

1 Escherichia coli Gastroenteritis, Urinary tract Infection, Wound infections (necrotizing fasciitis).

2 Bacillus species Urinary tract Infection, wound ulceration, septicemia.

3 Shigella species Dysentery and shigellosis.

4 Staphylococcus Pneumonia, meningitis, sinusitis, pharyngitis, tonsillitis aureus and food poisoning.

5 Pseudomonas Respiratory tract infections, ear and wound infection, aeruginosa urinary tract infections, dermatitis, soft tissue infections. Systemic infections are common particularly in patients with severe burns, cancer and immunosuppressed patients (AIDS)

6 Salmonella typhi Enteric fever, septicemia, food poisoning, bone infection. * [Bisno and Stevens, 1996; Walker et al., 2005; Shanson D.C., 1989; Brooks, 1996].

1.5.2 Antifungal Assay

There is considerable need to discover new antifungal compounds, in view of many plant and human fungal diseases. There are various fungal pathogens that infect human, animal and plants. In study we have selected various fungal strains. Pathogenesis caused due to these fungi is explained in Table 1.3

33 Chapter-1 Part- A Introduction

Table 1.3 Pathogenesis caused by Fungi*

Entry Fungi Nature Pathogenesis

1 Trichophyton Human Causes cutaneous mycosis. Results in longifusus pathogen severe inflammatory infection of hair and follicle called Favus. It results in permanent hair loss. It also infects nails and skin. 2 Candida Human Causes candidasis (opportunistic mycosis), albicans pathogen which is an acute, sub-acute or chronic infection involving any part of body like lungs, ear and skin. 3 Aspergillus Human Opportunistic mycosis. It is a contaminant flavus pathogen but can cause disease in debilitated patients, particularly infection of lung, ear, skin, joints and central nervous system. 4 Mycosporum Animal It is one of the most common species of canis pathogen dermatophytes and is cause of Tinea capitis and Tinea corporis.Human infections are caused by contact with animals, soil or indirectly from other human colonized with the fungus. In human, it causes superficial fungal infections of skin [Korstanje and Stata, 1994; Zaror et al., 1986]. 5 Fusarium Plant pathogen It is seed born pathogen and is food solani contaminant. It causes disease after ingestion of food prepared from grain/seed contaminated by fungi that produce toxins. It is known to effect cornea. * [Bisno and Stevens, 1996; Walker et al., 2005; Shanson, 1989; Brooks, 1996].

1.5.3 Brine Shrimp Assay

The brine shrimp bioassay was first developed as a convenient method for detecting insecticide residues. Subsequently, the assay was applied for the screening of mycotoxins and bioassay guided fractionation of toxic fungal cultures. Later, this bioassay was adopted to provide a quick and inexpensive means for screening plant extract for general toxicity and bioassay guided fractionation of cytotoxic and antiplasmodial extracts. Significant correlations

34 Chapter-1 Part- A Introduction with brine shrimp toxicity have been shown for cytotoxicity and antitumor activity. Conversely, the use of brine shrimp toxicity assay in plant screening as an adjuvant to several biocidal assays, including cytotoxicity, in order to deselect generally toxic extracts has been suggested [Horgen et al., 2001]. In nut shell, today, the brine shrimp lethality bioassay is an efficient, rapid and inexpensive test that requires only a relatively small amount of sample and above all has a good correlation with cytotoxic activity in some human solid tumors and with pesticidal activity, and has led to the discovery of the annonaceous acetogenins as a new class of natural pesticides and active antitumoral agents [McLaughlin et al., 1998].

1.5.4 Insecticidal Assay

Freedom from insect infestation and contamination has become an important consideration in storage of grain and to maintain a high quality product. Almost any stored material, whether of plant or animal origin, is subject to attack by insects.

The interest in the discovery of botanical insecticides as an alternative to the synthetic ones, which possess well known adverse effects on agroecological systems, has been increased in the last few decades in integrated pest management program. Development of resistance against conventional pesticides is another problem associated with the use of insecticides in grain protection. This interest has led to increasing number of investigations on plant insect chemical interactions, which have revealed the potential of secondary plant metabolites as pest control agents [Pavela, 2004]. Numerous plants have demonstrated to produce pesticidal compounds, either as a chemical defense mechanism against predation or infection [Ciccia et al., 2000]. Botanical insecticides are relatively safe and biodegradable and are readily available sources of bioinsecticides [Shafeek et al., 2004]. Consequently, the use of plant allelochemicals has been accepted as a promising approach for pest management. Therefore, in recent years, agrochemical companies have focused on the study of natural products for the development of new insecticides [Addor, 1995].

35 Chapter-1 Part- A Introduction

1.5.5 Phytotoxic Assay

Weeds are one of the major factors of poor agricultural productivity in developing world. There is a need to discover new herbicides since the number of herbicide resistant weeds is increasing and conventional synthetic herbicides are often expensive, non-specific and less effective against the resistant weed biotypes [Bhowmik, 2003], in addition to environmental and health related concerns. Therefore, new herbicides from natural sources are currently receiving more attention, which could be appropriate and non-hazardous alternatives to the currently used synthetic agrochemicals. The natural products are effective, biodegradable and thus posing less threat to the environment. A number of plants, their extracts or purified active constituents can act as allelochemicals to other plants and thus could be candidates for application of agricultural purposes [Khan et al., 2002].

1.5.6 Anti leishmanial Assay

Leishmaniasis is one of the major health problems of tropical, subtropical and Mediterranean regions. It is a parasitic disease caused by trypanosomated protozoan leishmania, which is transmitted by the female phlebotomus sand fly. It has an elaborate life cycle, in which it undergoes morphological changes in two successive hosts. Upon introduction into blood stream of a mammalian host through an infected sandfly bite, it transformed into a non-motile form that invades and multiplies within lysosomal compartment of macrophages. Heavily parasitized cell rupture, releasing the amastigote, which are dispersed to different organ depending on strain tissues specificity, resulting in the different clinical forms of leishmaniasis. As no vaccine is yet available for any type of leishmaniasis, chemotherapy is the only means of controlling the disease. Many novel compounds isolated from various medicinal plants have been reported for their anti-leishmanial activity.

36 Chapter-2 Part- A Experimental

2.0 PHARMACOGNOSTIC INVESRIGATION

The procedures adopted for the Pharmacognostic studies are grouped under the fowling headings:

. Collection and preservation

. Macroscopic study

. Anatomy

. Powder drug study

. Nutritional value

. Elemental analysis

. Microchemical analysis

2.1 Collection and Preservation

The fresh specimens of both plants Indigofera gerardiana Wall and Crateagus songrica K. Koch were collected from upper Dir and Chitral (Bonni), respectively. Each plant sample was washed, separated and dried in air for about two weeks at room temperature and was used for following tests and analyses. These plants were ground to 60 mesh size and were preserved in airtight bottles. Some fresh specimens were used to study morphological characters and some of it was utilized for section cutting.

2.1.1 Macroscopical Features

The morphological observations of roots, stems and leaves included colour, odour, taste, size, shape, fracture, fracture surface, texture, duration and rootlets. The study of petiole included colour, odour, taste, size, shape, fracture, fracture surface and texture., leaf study included colour, taste, odour, size, shape, fracture, texture, margin, venation, surface, apex, duration, type, phyllotaxis, presence and absence of petiole and stipule [Trease and Evans, 1992].

37 Chapter-2 Part- A Experimental

2.1.2 Anatomical Studies

The hand-section was made of fresh specimens. The material was mounted on the center of potato pith by making a whole, cylindrical or longitudinal according to the type of material. Then a large number of transverse cuts were made across the material with the help of sharp razor and was kept moist with the help of water. The thin sections were selected for staining, which was carried out on a microscopic glass slide [Puruis et al. 1966].

The staining was achieved by putting the section in safranin for 2-3 minutes. Then it was dehydrated in different grades of alcohol i.e. 10 %, 20 %, 30 %, 50 %, 75 %, and 90 %. After this, the dehydrated section was dipped in to a drop of light green stain and then again dehydrated in absolute alcohol for 2-3 minutes. The sections were finally mounted on Canada–balsam to make them permanent [Johanson, 1940]

2.1.3 Microscopic Study

The powder drug studies included two aspects; macroscopic and microscopic studies. Macroscopic studies included colour, odour, size and taste of all specimens [Trease and Evans, 1992]. While microscopic study of different tissues of powder drug was performed by using electronic microscope at power 40X. The powder material was macerated with chloral hydrate solution. A drop of this solution was taken on a slide and then it was heated on spirit lamp fallowed by examination under microscope. Different tissues were observed under the microscope and were sketched [Wallis, 1985].

2.1.4 Nutritional Analysis

The nutritional analysis included the determination of moisture, crude proteins, carbohydrates and ash content [Lenahen and Smith, 1986, Jeffery, 1989]

2.1.4.1 Determination of Ash

The ash is an analytical term used for inorganic residue that remains after the choring of organic matter present in the food. The ash may not be the same as

38 Chapter-2 Part- A Experimental the organic matter present in the original food. There may be loss of organic matters due to the chemical reactions among the different constituents.

Equipment and glassware

Muffle furnace, silica dish, electric balance, desiccators, Bunsen burner

Ash contents were determined by heating sample at 550 oC in muffle furnace. The procedure is given below.

Procedure

A clean flat bottom silica dish was taken and was held in a hot burner flame for 1 minute. After that it was transferred to a desiccator then cooled weighed (W). A suitable quantity of food sample was weighed in china dish (W1) and heated gently on the Bunsen burner. Then the charred mass was transferred to a muffle furnace at 550oC.

The sample was heated until all the carbons were burned. The dish

(containing sample) was placed in desiccater, cooled weighed (W2).

Weight of the empty dish = W

Weight of the empty dish + sample = W1

Weight of the empty dish + ash = W2

Calculations

W1 - W2 % Ash  x 100 (A. O. A. C, 2000) Wt of the sample

2.1.4.2 Determination of moisture

Equipment and glassware

Electric oven, Petri dish, desiccators, electric balance

Procedure

2 g of sample was taken in a clean weighed Petri-dish (W1), place the Petri-dish partially covered with a lid. Then a Petri dish was heated in oven at 105 oC, for 5 hours, until constant weight was obtained.

39 Chapter-2 Part- A Experimental

Then cover of Petri dish was removed and was placed in desiccator for 30 minutes in order to cool it. After cooling we got the weigh the dish (W2). Percent moisture was calculated as follow

Calculations

W1 - W2 % Moisture  x 100 (A.O.A.C, 2000) Wt of the sample

2.1.4.3 Determination of proteins by “Macrojeldahl distillation method

The proteins determination depends upon the decomposition of organic nitrogen compounds by boiling with sulphuric acid that causes the oxidation of organic matters to CO2 and H2O, the sulphuric acid is also reduced to O2 which in turn reduces the nitrogen to ammonia. Ammonia combines with H2SO4 and remains as (NH4)2SO4. The ammonia liberated by the addition of NaOH, is distilled into boric acid which is titraed against a standard acid.

Reagents

Conc H2SO4, 4% boric acid, 32% NaOH, K2SO4, CuSO4 and 0.1 N standard HCl solution.

Mixed indicator

Methyl red (0.016 g) and bromocresol green was dissolved in alcohol (100 ml) at room temperature and stored.

Apparatus

Kjeldahln flask, digestion and distillation apparatus, burette etc. Protein (% Nx6.25) was determined by Macro Kjeldahl method. The procedure is given below. a. Digestion

0.5 gram of dry ground sample was taken in a Kjeldahl flask. 18 gram of

H2SO4, 1gm of CuSO4 and 20-25 ml of conc. H2SO4 was added. It digested in

40 Chapter-2 Part- A Experimental

Kjeldahl digestion unit (Kjeldatherm) for 6 hours so that the complete evaluation of SO2 occurred and the solution became clear. The mixture was cooled. b. Distillation

50 ml of 4% boric acid solution was taken in a receiving flask. 4 drops of mixed indicator was added, and then the flask was placed under the condenser of Kjeldahl.

Condenser tube was placed beneath the surface of the acid in the flask. 50 mls of water and 60 ml of 32% NaOH solution was added. After distillation 200 mls of the sample was collected in flask and titrated. c. Titration

The content of flak was titrated against the 0.1N Hcl. The reading was noted and the % was determined by the following formula.

Calculations

% Protein = % Nitrogen x 6.25. (A.O.A.C, 2000)

2.1.4.4 Determination of fat or ether extract

Equipment and glassware

Petroleum ether B.P (40-60 oC), extraction thimbles, sox let extraction apparatus.

Determination of fat was carried out by Soxhlet extraction using petroleum either (B.P 40-60 oC) in Soxhlet system H.T (Tecator). The procedure is given below.

Procedure

Soxhelt’s apparatus was used in this method. 2-4 gram of extraction thimble was taken, plugged with absorbent cotton wool and was placed in extraction chamber. Pre weighed clean and dried round bottom flask (250 mls) was taken, 1/3 part was filled with solvent and connected to the extraction tube. After the setting of whole apparatus, flow of tap water and burner was put on. The sample was extracted for 5h, then Siphoning occurred after 6 minutes on condensation and the rate was 4 drops / second.

41 Chapter-2 Part- A Experimental

Subsequent to completion of the whole process the thimble was winkle out from the extractor. The flask was heated in order to collect the solvent. Finally the flak was dried at 105 oC and weighed again after cooling.

Calculation

W1 - W2 % Crude fibers  x 100 (A.O. A.C, 2000) W0

2.1.4.5 Determination of crude fiber by Fibertec

The crude fibers are the washed, dried organic residue that remains after boiling the defatted material with sulphuric acid and sodium hydroxide.

Equipment and Glassware

Crude fiber extraction apparatus (Fiber Tec System M. Tecator), muffle furnace, sustion pump, oven and analytical balance.

Reagents

n-Octanol, KOH solution, water

Procedure

1g of sample (W0) was taken in crucible and was placed in a crucible stand. A few drops of octonal were added in order to prevent foaming and then the sample was heated for 30 minutes. After that the samples was filtered and then rinsed with hot water, with the help of spray device and dried.

Preheated 150mls solution of KOH was added in the second reagent system. Few drops of octanol were supplemented and the assortment was boiled as above for 30 minutes.

Calculations

W1 - W2 % Crude fibers  x 100 (A.O. A.C, 2000) W0

42 Chapter-2 Part- A Experimental

2.1.4.6 Carbohydrate contents

Carbohydrate content was determined by subtracting the weights of protein fats, crude fibers, ash, and moisture contents from 100.

2.1.5 Microchemical Tests a. Detection of Alkaloid

The detection of alkaloids was carried out by the method adopted by Trease and Evans (1989). By precipitation with Dragnodorffs reagent (Solution of potassium bismuth iodide), the reddish brown or pinkish purple indicated the presence of alkaloids (Trease and Evans, 1992). b. Detection of Mucilage

A small quantity of powder drug was mixed with a few drops of water and allowed to stand; if the powder was not dissolved completely and became gummy, mucilage were detected [Trease and Evans, 1992]. c. Detection of Anthraquinone

A small quantity of powder was macerated with ether, the presence of pink, red, or violet colour, in aqueous layer after shaking indicated the presence of anthraquinone derivations [Trease and Evans, 1992]. d. Detection of Calcium Oxalate

The crystals may be identified as calcium oxalate, if these are soluble in

HCl, and H2SO4 without effervescence [Trease and Evans, 1992]. e. Detection of Saponin

Powdered drug was shaken in a test tube half filled with water any marked frothing suspected saponin [Trease and Evans, 1992].

43 Chapter-2 Part- A Experimental

f. Detection of Tannin

The presence of bluish black or greenish colour after treating the drug with diluted solution of ferric chloride indicated the presence of tannin [Trease and Evans, 1992]. g. Detection of Starch

The drug was treated with iodine solution and the appearance of deep blue colour marked the presence of starch [Trease and Evans, 1992]. h. Detection of Fat and Oil

A small quantity of powdered drug was pressed between filter papers; the appearance of an oily stain indicated the presence of fats and oil [Trease and Evans, 1992]. i. Detection of Protein

The drug was treated with aqueous solution of picric acid the appearance of intense yellow precipitate indicated the appearance of proteins [Johnson, 1940]. j. Detection of Lignin

The appearance of pink or red colour after treating the drug with phloroglucinol and hydrochloric acid indicated the presence of lignin [Trease and Evans, 1992]. k. Detection of Cutin

The appearance of yellow brown colour after treating the drug with chloro- zinc iodide solution indicated the presence of cutin [Trease and Evans, 1992]. l. Detection of Cellulose

The appearance of blue colour on treating the drug with chloro-zinc iodide solution indicated the presence of cellulose [Trease and Evans, 1992]. m. Detection of Catechin

44 Chapter-2 Part- A Experimental

A matchstick was dipped in plant extract. Dried and moistened with HCl (Conc) and warned near a flame the appearance of pink or red colour indicated the presence. [Trease and Evans, 1992].

45 Chapter-2 Part- A Experimental

2.2 PHYTOCHEMICAL STUDY

2.2.1 General Notes

The experimental work started with extraction of fresh material of the entire plant of Indigofera gerardiana to furnish a dark brown pasty liquid.

Most of the research work was carried out at the Department of Pharmacy, University of Peshawar, like extraction, fractionation of crude extract, isolation and purification of the natural constituents. Where as analytical studies and screening for pharmacological/biological activities was performed at Hussain Ebrahim Jamal Research Institute of International Center for Chemical and Biological Sciences, University of Karachi under co-supervision of Dr. Raza Shah.

2.2.2 General experimental conditions 2.2.2.1 Physical Constants Melting points were noted in glass tube using Buchi 535 melting point equipment and are uncorrected.

2. 2.2.2 Spectroscopy The compound so isolated in pure form were subjected to extensive spectroscopy such as Infrared (IR) band were traced in CH3OH on JASCO A- 320 Infrared spectrophotometer. The proton magnetic resonance (1H NMR) were recorded in MeOD at 400 MHz on Bruker, AM- 300, AM-400 and AMX-500 nuclear magnetic resonance spectrometer with feature 3000 data systems at a digital resolution of 32 K, while the 13C NMR bands were traced in MeOD on 100 MHz. The Distortionless Enhancement by Polarization Transfer (DEPT) o o o experiments were carried out at 45 , 90 , and 135 for determination of CH, CH2, and CH3 groups, while the quaternary carbons were determined by subtracting the signals of these spectra from broad band (BB) 13C NMR spectrum.

Low resolution electron impact mass (EIMS) bands were traced on a Finnegan MAT 311 with MASPEC Data system. Peak matching, field

46 Chapter-2 Part- A Experimental dsorptions (FD) and field ionization (FI) was performed on the Finnegan MAT 312 mass spectrometer. High-resolution mass measurement and fast atom bombardment (FAB) mass measurements were recorded on Jeol JMS HX 110 mass spectrometer. FAB source, using glycerol or thioglycerol as the matrix and cesium iodide (CsI) as internal standard were used for accurate mass measurement.

2.2.2.3 Plant Material

Indegofera gerardiana was collected as an whole plant from upper Dir, NWFP (Pakistan) during the month of spring season of 2005. The plant was recognized by Prof Dr Jahandar Shah, plant taxonomist University of Malakand, Chakdara. The plant specimen number HA-014 was placed in the herbarium of botany department, Jahanzeb Post Graduate College, Swat. The air-dried plant material was crushed in a grinder. The powdered plant material was then used for extraction and isolation.

2.2.2.4 Extraction, Fractionation and Isolation

The plant sample with an appropriate quantity was obtained after the preliminary necessary preparations such as drying under shadow for three weeks then comminuted into fine particles and pulverized into a fine powder. The grinded plant (20 kg) was dipped in MeOH (80% v/v) with rare stirring at room temperature. After two weeks, the materials dissolved in MeOH were separated through process of filtration. The procedure was replicated 3 times and the clear material obtained was converted in to syrupy liquid in vaco at 40 OC to bestow dark brown paste. The crude MeOH extort (463.5g) was suspended in distilled water and consecutively extracted with n-hexane (20.71 % w/w), chloroform (15.96 % w/w), ethyl acetate (12.94 %), n-butanol (19.41 % w/w), and finally the aqueous (30.96 % w/w) fraction was obtained. Each organic extract was then evaporated to dryness. Stock extracts solutions were prepared at 200 mg/ml in distilled water and stored at ambient temperature for further investigation.

47 Chapter-2 Part- A Experimental

Crushed whole plant Indigofrera gerardiana 30kg +80% MeOH (70litrs)

Process was repeated Percolate for 15 days at room temperature, three times filter through Whatman filter paper

Percolate

MeOH Evaporated in vacuo at 40 ºC

Crude extract (463.5g)

23.5g separate for activities 410g for phytochemical studies

Scheme 2.1: Extraction of Indigofera gerardiana

48 Chapter-2 Part- A Experimental

Crude Methanolic extract (410g)

Extracted with n-hexane

Hexane fraction Aqueous fraction (70g)

Extracted with CHCL3

CHCL3 fraction Aqueous fraction (65g)

Extracted with EtOAc

EtOAc fraction Aqueous fraction (62g)

Extract with n-BuOH

BuOH fraction Aqueous fraction (73g) (97g)

Scheme 2.2: Fractionation of Indigofera gerardiana

49 Chapter-2 Part- A Experimental

EtOAc fraction S-4 (60g)

ElutedEluted with with ColuColumn mn ch chromatography ro matog raphy n-hexane, CHCl3, MeOH n-hexane:CHCl3: MeOH

Fraction K Fraction L Fraction M Fraction N

One major spot Two major spots One major spot

C CCC elutedwith eluted with CC eluted with CC eluted with C C eluted with MeOH: CHCl3 (3::97) C C eluted with MeOH: CHCl3 (9::91) Me OH: CHCl3 MeOH: CHCl (7::93) MeOH: CHCl3 MeOH: CHCl3 3 (9:91) (9:91) (10:90)

Compund Compund S-1 S-4

Compund Compund S-2 S-3

Scheme...Isolation of Chemical Constituents Scheme 2.3: Isolation of Chemical Constituents

50 Chapter-2 Part- A Experimental

2.3 Physical and Spectroscopic Data

2.3.1 (S- 1)

The sub-fraction K (2 g) was loaded on column using silica gel, 40g and eluted with methanol: chloroform (3:97) to afford a gummy black solid (Scheme.2.3). It was visualized under UV light on the precoated silica gel TLC plates to determine the purity.

Table 2.1 Physical and spectroscopic data of (S-1)

Entry Parameter Observation

01 Physical state Black gummy solid

02 Rf 0.28 [methanol: chloroform; 3: 97]

03 Yield 3.5 x 10-3 %

-1 04 IRmax cm (CH3OH) 35.21(OH groups), 1687 (keto-carbonyl),

05 UV max (MeOH) 575 nm

06 1H-NMR (400 MHz, MeOD) (see Table.3.1, Page.72)

13 07 C-NMR (MeOD, 100 MHz) (see Table.3.1, Page.72)

08 HREI-MS (m/z) 382.4487 (observed), C20H30O7,

382.4481 (calcd.)

51 Chapter-2 Part- A Experimental

2.3.2 (S-2)

The sub-fraction L (2 g) was inclined to flash column chromatography (silica gel, 20g) and the elution was performed with methanol: chloroform (7: 93) to give a gummy black solid (Scheme2.3). It was envisaged under UV light on the pre coated silica gel TLC plates.

Table 2.2. Physical and spectroscopic data of (S-2)

Entry Parameter Observation

01 Physical state Yellow amorphous powder

02 Melting point 140-142 OC

03 Rf 0.36 [methanol : chloroform 7 : 93 ]

04 Yield 1.94 x 10-3 %

-1 05 IRmax cm (CH3OH) 6545-3402 (OH groups),

1690 (keto-carbonyl )

06 UV max (MeOH) 556 nm

07 1H-NMR (400 MHz, MeOD) (see Table 3.2, Page 74)

13 08 C-NMR (CDCl3, 100 MHz) (see Table 3.2, Page.74)

09 HREI-MS (m/z) 268.3066 (observed), C14H20O5,

268.3061 (calcd.)

52 Chapter-2 Part- A Experimental

2.3.3 (S-3)

The L (7g) as a sub-fraction was adsorbed on column chromatography (silica gel, 70g) and eluted by methanol: chloroform (7:93) to have a brown powder (Scheme.2.3). It was visualized below UV light on the pre coated TLC plates.

Table 2.3 Physical and spectroscopic data of (S-3)

Entry Parameter Observation

01 Physical state brown powder

02 Melting point 145-147 OC

03 Rf 0.40 [methanol: chloroform; 7:93]

04 Yield 3.0 x 10-3 %

-1 05 IRmax cm (CH3 OH) 2922 (C-H), 1623-1517 (aromatic C = C), 1230 (C=O).

06 UV max (MeOH) 577 nm

07 1H-NMR (400 MHz, MeOD) (see Table.3.2, Page.77)

13 08 C-NMR (MeOD, 100MHz) (see Table.3.2, Page.77)

09 HREI-MS (m/z) 354.4384 (observed), C19H30O6,

354.4380 (calcd.)

53 Chapter-2 Part- A Experimental

2.3.4 β-Sitosterol

The segment M (3g) obtained was loaded to column chromatography (silica gel, 30g) and eluted with methanol: chloroform (9:91) to afford colourless needle like crystals (Scheme.2.3). It was recognized as β-sitosterol by comparison with an authentic sample (CO-TLC) and spectroscopic data.

Table 2.4 Physical and spectroscopic data of β-Sitosterol

Entry Parameter Observation

01 Physical state Colourless needle like crystals

02 Melting point 141-143 0C

03 Rf 0.6 [methanol: chloroform; 9:91]

04 Yield 4.9×10-3 %

-1 05 IRmax cm (CH3OH) 3492 (OH groups), 2905 (C-H), 1642 (C=C)

06 1H-NMR (400 MHz, MeOD) (see Table.3.4, Page.79)

13 07 C-NMR (MeOD, 100 MHz) (see Table.3.4, Page.79)

08 HREI-MS (m/z) 414.3855 (observed), C29H50O,

414.3861 (calcd.)

54 Chapter-2 Part- A Experimental

2.4 PHARMACOLOGICAL INVESTIGATION

2.4.1 Antibacterial Assay

Antibacterial assay was performed by agar well diffusion method [Atta-ur- Rehman et al., 1999]. Methanolic extract and different portions dissolved in various organic solvent were used in amount of three mg/mL of DMSO and pure compounds were used in dose of 1 mg/mL. Antibacterial activity was carried out against various human pathogens including Escherichia coli (ATCC 25922), Bacillus subtilus (ATCC 6633), Shigella flexenari (clinical isolate), Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa(ATCC 27853) and Salmonella typhi (ATCC 19430).

In this bio assay, three types of media are required viz. solid medium (nutrient agar), semi-solid medium (soft agar) and liquid medium (nutrient broth).  Solid medium (Nutrient agar) Nutrient agar 28 g Distilled water 1 L (q.s) Nutrient agar was dissolved in distilled water and volume was made up to 1 liter. It was then placed in auto clave at 121 0C for 15 minutes. Media was then chilled to 40 0C and poured in sterile petri dishes and media was then left to solidify at room temperature.  Semi solid medium (Soft agar) Soft agar 0.8 g Distilled water 100 mL (q.s) Soft agar was dissolved in distilled water and volume was made up to 100 mL. It was then dispensed in 7 mL quantity to screw capped test tubes. Then placed in auto clave at 121 0C for 15 minutes and then cooled.  Liquid medium (Nutrient broth) Nutrient broth 0.8 g Distilled water 100 mL (q.s)

55 Chapter-2 Part- A Experimental

Nutrient broth was dissolved in distilled water and volume was made up to 100 mL. Prepared broth was dispensed in 3 ml quantity to screw capped test tubes, which was placed in auto clave at 121 0C for 15 minutes and then refrigerated.

The culture of bacterial organisms was maintained on stock culture agar. A colony of bacterial culture was implanted in nutrient bisque and incubate at 371 0C for 24 hours. Next day soft agar was melted and then cooled to 40 0C and then add 100 μL of bacterial culture, shake it well and then pour it on nutrient agar containing plate. Rotate the plate to make even distribution of culture and allow solidifying the lawn. Hole (6 mm diameter) was made in medium in each plate using a sterile sharp tool with centers at least 24 mm apart. Samples (3 mg/ml of DMSO) were then added in respective well using sterilized dropping pipettes. DMSO was added other holes and standard anti bacterial drug provided as -ve and +ve control, correspondingly. Plates were then incubated at 371 0C for 24 hours. Activity was calculated by measuring diameter of zones depicting resistance (mm). Antibacterial potential of sample was then determined as per criteria mentioned in Table 2.5. Percent enlargement of resistance was calculated with standard to the +ve control.

Table: 2.5 Criteria for Determining Antibacterial Assay

Entry Diameter Activity

1 Below 9 mm No activity

2 9-12 mm Non-Significant

3 13-15 mm Low

4 16-18 mm Good

5 Above 18 mm Significant

2.4.2 Antifungal Assay

Antifungal activity was carried out following agar tube dilution protocol [Paxton, 1991]. Methanolic extract and various fractions were used in dose of 24 mg/mL and pure compounds 12 mg/mL of sterile DMSO, provided as stock

56 Chapter-2 Part- A Experimental solution. The agar dilution method is the most convenient method for routine testing of samples such as plant extracts. The method is suitable for testing non- sterile plant extracts, because aerobic organisms do not develop well under the solidified agar. However, the occasional culture that develops on the surface of the agar can be easily recognized. Non-polar extracts, essential oils, suspensions of solids or emulsions and antimicrobial substances, which do not diffuse through agar media, can be tested directly by incorporating them with the agar media as if they were aqueous solutions. This method has an advantage that unlike the diffusion method, no concentration gradient occurs during the testing procedure. More over, several different test microorganisms may be tested simultaneously on the same dilution, which makes the agar dilution method very efficient.

Antifungal activity was carried out against clinical specimen of human pathogens namely Candida albicans (ATCC 2091), Candida glabarata (ATCC 90030), Aspergillus flavus (ATCC 32611), Trichophyton longifusis (clinical isolate), Mycosporum canis (ATCC 11622) and Fusarium solani (ATCC 11712).

Table: 2.6 Sabouraud Dextrose Agar (SDA)

Formula G/Litre

Mycological peptone 10.0

Glucose 40.0

Agar 15.0

PH 5.60.2

(15 g) was dissolved in distilled water and volume was made to 1 liter, the mixture was heated for at least 10 minutes and then sterilized in autoclave (15 lbs/in2 pressure and at 121C temperature for 15 minutes).

The culture of organisms was maintained on Sabouraud Dextrose Agar (SDA). Sabouraud Dextrose Agar (SDA), 4 ml was distributed into attached cap tubes, that were then placed in autoclave at 121 0C for fifteen minutes and then

57 Chapter-2 Part- A Experimental temperature was brought to 50 0C. The stock solution (66.6 μL) was then added to non-solidified SDA media, giving final concentration of 400 μg of extract per ml of SDA. Tubes were then left to solidifying in angled pose at room temperature. Every tube was implanted with a piece of inoculums (4 mm diameter), detached from seven days old culture of respective fungi. For non mycelial growth, an agar surface band was used. Resistance of fungal growth was recorded after seven days of incubation at 28+1 0C. Media supplemented with DMSO and standard anti fungal drugs are used as +ve and -ive control correspondingly. Miconazole was used as reference antifungal drug for all pathogens except C.albicans for which Amphotericin-B was used as a reference drug. Growth in medium containing extracts was calculated by determining linear growth in mm and then percent growth inhibition was measured with standard to –ve control using formula:

Growth in sample tube (mm) % inhibition  x 100 Growth in control tube (mm)

Following criteria was followed for activity of sample.

Table: 2.7 Criteria for Determining Antifungal Assay

Entry Percent Inhibition Activity

1 30-40 Low

2 50-60 Moderate

3 61-70 Good

4 Above 70 Significant

2.4.3 Brine Shrimp Bioassay

Natural products are often toxic to Artemia salina (Leach) shrimp larvae. The eggs of the brine shrimp Artemia salina are readily available as fish food in pet shops [Kivack et al., 2001; Carballo et al., 2002]. When placed in artificial seawater, the eggs hatch within 48 hours, providing large numbers of larvae. Shrimp can be used 48-72 hours after the initiation of hatching. After 72 hours they should be discarded.

58 Chapter-2 Part- A Experimental

This method is a rapid, inexpensive, general bioassay, which has been developed for screening, fractionation and monitoring of physiologically active natural products. The crude extract and fractions of plant extracts were prepared for the estimation of LD50 activity in brine shrimps. The procedure described by Mayer et al. and followed by McLaughlin et al. and Arnason et al. was adopted for this work.

Three different concentrations 10, 100 and 1000 g/ml of test samples were prepared. Sea water was prepared by mixing sea salt available in the market (38 g) in distilled water (up to 1 L) to have pH 7.4. Brine shrimp eggs (Artemia salina) were hatched in sea water medium at 25- 27 0C for 48 hours in a perforating hatching tray under illumination. After 2-days of hatching and maturation as nauplii, 10 larvae were placed in each sample vial, using a Pasteur pipette, and the volume was made up to 5 ml with seawater. Dry yeast suspension was added as food to each vial. Different concentrations of test samples were then added accordingly. Two vials were filled with solvent and the standard cytotoxic drug gave as +ve and -ve control correspondingly. Etoposide (LD 50= 7.465 μg/mL) was used as reference cytotoxic drug. All vials were incubated in growth chamber (Fisons Fi-Totron 600H) at 26+1 0C for 24 hours. After completion of incubation period, the survived brine shrimps were counted in each vial. The data was analysed with Finney computer program to determine LD 50 values with 95 % confidence interval.

2.4.4 Insecticidal Activity

Insecticidal activity of plant extract and fractions was determined by direct contact application using filter paper, also called Impregnated filter paper method [Ahn et al., 1995].

Test insects are Tribolium castaneum, Sitophilus oryzea, Rhyzopertha dominica, Trogoderma granarium and Callosobruchus analis. Standard insecticide used is Permethrin. Crude extract and fractions 200 mg test sample was dissolved in 3 ml volatile solvent. The stored grain pests are reared in the laboratory under controlled conditions (temperature and humidity) in plastic bottles containing

59 Chapter-2 Part- A Experimental sterile breeding media. Insects of uniform age and size are used for the experiment. The filter paper was cut as per size of petri plate (9 cm or 90 mm) and then placed in respective plate. Prepared sample was applied to filter paper with micropipette and allowed to evaporate solvent completely in 24 hours. After evaporation of solvent, 10 insects of each species were placed in each plate (test and control) with the help of a clean brush. These insects were healthy, active and of same size and age. Cover each plate with a lid. A check batch delighted with solvent was prepared for calculation of solvent action. Similarly a blank lot was placed for calculation of environmental effects; other lot was added with standard insecticide (Permetherin 235.71 μg/cm2). These plates were incubated at 27 °C for 24 hours with 50% relative humidity in growth chamber, without food. On third day, results were noted in terms of survival of the insects by counting the number of survivals in each plate. Percentage Mortality was calculated with the help of the following formula.

No. of insects survived in test Percent Mortality  100 - x 100 No of insects survived in control 2.4.5 Phytotoxic Assay

The Lemna bioassay is a quick measurement of phytotoxicity of the materials. It is primary screening for pesticides research. In this bioassay the growth stimulating effects of test samples are also determined. It has been observed that natural antitumor compounds can inhibit the lemna growth. In addition it was also discovered that some substances stimulate frond proliferation, and the assay might be useful to detect new plant growth stimulants. For cultivation of lamneaceae generally includes, cleaning of the new clone in water and cultivation under optical conditions for one or two days. In this way a good number of healthy fronds will be obtained. After washing in water, these are transferred to nutrient solution.

Medium was prepared by mixing various constituents (Table 2.10) in 1L distilled water (stock solution), and pH is adjusted 5.5 to 7.0 by the addition of potassium hydroxide beds and finally placed in autoclave at 121°C for 15 minutes. Working medium is prepared by mixing 100 ml of stock solution and 900 ml of

60 Chapter-2 Part- A Experimental distilled water. 30 mg of sample was mixed in 1.5 ml of solvent (methanol/ethanol) used as stored solution.

Three sterilized flasks were inoculated with 10, 100, and 1000 l of stock solution to have 10, 100 and 1000 g/ml of concentration respectively. The solvent evaporated for the whole night under sterile conditions. Every flask, twenty milliliters of working medium and then ten plants of Lemna minor, each containing a rosette of 2-3 fronds, to every flask, was added. Flasks containing medium and plants was used as negative control, while flask containing solvents, standard (paraquat) plant growth promotes and inhibitors serving as positive control. All flasks were caped with cotton and incubated in growth cabinet (Fisons Fi Totron 600H) at 30 °C, 56 + 10 % relative humidity and 9000 lux light intensity for 12 hours daily for seven days. The number of fronds for each flask were calculated and documented on the seventh day. The results were analyzed as growth regulation in percentage, calculated with srandard to the -ve control by following formula. Activity was determined as per criteria mentioned in Table 2.11 [Atta-ur-Rehman, 1991; Lewis, 1995; Finny, 1971; Hideji et al., 1982].

No. of fronds in test flask % Growth inhbition  100 - x 100 No. of fronds in negative control

61 Chapter-2 Part- A Experimental

No of fronds in control 40

Table : 2.8 Composition of medium for Lemna bioassay

Entry Constituents Formula mg/ml

1 Potassium dihydrogen phosphate KH2PO4 680

2 Potassium nitrate KNO3 1515

3 Calcium nitrate Ca(NO3)2.4H2O 1180

4 Magnesium sulphate MgSo4.7H2O 492

5 Boric acid H3BO3 2.86

6 Manganous chloride MnCl2.4H2O 3.62

7 Ferric chloride FeCl3.4H2O 5.40

8 Zinc sulphate ZnSO4.5H2O 0.22

9 Copper sulphate CuSO4.5H2O 0.22

10 Sodium molybdate Na2MoO4.2H2O 0.12

11 Ethylene diamine tetraacetic acid EDTA 11.20

Table: 2.9 Criteria for Determining Phytotoxicity

Entry Percent inhibition Activity

1 40 Low

2 50 Moderate

3 60-70 Good

4 Above 70 Significant

62 Chapter-2 Part- A Experimental

2.4.6 Anti leishmanial Assay It was performed by 96-Well Micro Titer Plate Bioassay (Ash and Orithel, 1987; Solomon, 2003). Leishmania Promastigotes are grown in bulk early in modified NNN biphasic medium using normal physiological saline. Leishmania parasite Promastigotes were cultured with RPMI 1640 medium added with ten percent heat inactivated foetal bovine serum (FBS). Parasites at log phase are centrifuged at 2000 rpm for 10 minutes, and washed three times with saline at same speed and time. Parasites are diluted with fresh culture medium to a final density of 106 cells /ml.

In a 96-hole micro titer plate, 180l of medium was supplemented in various holes. 20l of the experimental compound was supplemented in medium and diluted in sequence. 100l of parasite culture was added in all wells. Two rows should be left for negative and positive control. Negative controls receives medium while the positive control contains varying concentrations of standard antileishmanial compound e.g. Amphotericin B, Pantamidine. The plate was incubated to 21-22 C for 72 hrs. The culture was observed under microscope on an enhanced neubauer counting chamber and IC50 values of compounds having antileishmanial activity were determined by Software Ezfit 5.03 Perella Scientific. All assays were run in duplicate.

63 Chapter-2 Part- A Experimental

2.4.7 Anti malarial Activity

It was done by method described by Makler and Hinrichs (1993), Makler and Ries (1993) and Trager and Jensen (1976).

To sterile 96 well plate 100 µl of CCM was added by multichannel pipette in each well (from 1-12 and from A to H). 100 µl of sample (1mg/ml) was added in triplicate in well A1-A3, Sample 2 from A4-A6 and standard drug Chloroquine from A7 to A9 and A10-A12 total 14 dilutions (stock will be of 1 µg/ml). Serial two fold dilution from A1 to F1, A2 to F2 and so on (total seven dilutions) was then made. Infected RBCs solution was added with 2 % parasitemia and 1 % hematocrit. The total volume became 200 µl in each well and Parasitemia will be 1% and 0.5 % hematocrit. In last row, well H1 to H6 contained infected RBCs and

H7-H12 have non-infected RBCs. Plate was incubated in candle jar with 5 % CO2 at 37º C for 72 hours. After 72 hours, plate was placed in freezer for complete lyses of cell for 24 hours and then after 24 hour Malstat reaction was done.

After lysis of cell, the plate was taken out from freezer and placed in water bath at 37º C for 1 hour. 100 µl of Malstat solution was then added in each well of 96 well plates. Take the pre-read in ELISA plate reader at 650 nm. 20 µl from each well of plate 1 was added to respective well of Malstat plate and then placed in shaking water bath at 37º C for 30 minutes. Solution of NBT (2mg/ml) and PES (0.1mg/ml) in ratio 1:1 and 25 uL of this solution were supplemented to every hole and the plate was placed in the dark for complete reaction. The plate was read at 650 nm and OD was noted. Percent inhibition was calculated by following formula. LD50 was calculated by EZfit computer program.

64 Chapter-3 Part- A Results and Discussion

3.1 PHARMACOGNOSTIC STUDY

3.1.1 Macroscopic features of Indigofera gerardiana. Wall

Study of the fresh and dry forms of the roots, stems and leaves were carried out.

The morphological features are tabulated as fallows:

Entry Feature Morphology

1 Fresh root Indigofera gerardiana whitish in colour; odour is indistinct and taste is irritating. Average length is 2.5m and average width is 4cm; shape is cylindrical and features smooth. Fracture surface is fibrous; surface, hairs are absent and texture is smooth.

2 Dry root All the morphological features are the same except the

average length i.e. 5.8cm and average width is 2mm on dryness.

3 Fresh stem The macroscopic features of are, colour is brownies; odour is indistinct and taste is irritating

It has all the same features as the fresh one but the 4 Dry stem difference is only average length i.e. 1.8m while the

average width is 5mm

The morphological characteristics of the shows that, the 5 Fresh leaf colour of both surfaces are of dark green colour; taste is

chloropphyllous and odour is indistinct. Average length is 1.2cm, while the average width is 8mm.Shape is chorodate; fracture is short and texture is hairy. Margin is entire venation is reticulate and apex is acute. Duration is perennial, phyllotaxis is imeripinn and stipules are present; Gray green, grayish green, feathery in nature.

65 Chapter-3 Part- A Results and Discussion

65 Chapter-3 Part- A Results and Discussion

Table: 3.1. Macroscopic Features of the root of Indigofera geradiana (Wall. ex. Bakcer) Status Colour Odour Taste Size Shape Fracture Fracture Surface Texture Ave. Length Ave. Width Surface Fresh Whitish Indistinct Irritating 20cm 4cm Cylindrical Smooth Fibrous Hair are absent Smooth Dried Whitish Indistinct Irritating 17cm 2 cm Cylindrical Smooth Fibrous Hair are absent Smooth

Table: 3.2. Macroscopic Features of the stem of Indigofera gerardiana (Wall. ex Baker) Status Colour Odour Taste Size Shape Fracture Fracture Surface Texture Surface Ave. Length Ave. Width Fresh Brownish Indistinct Irritating 1.9 m 6 cm Cylindrical Short Fibrous Hair are absent Smooth Dry Brownish Indistinct Irritating 1.8 m 5 cm Cylindrical Short Fibrous Hair are absent Smooth

Table: 3.3 Macroscopic Features of the leaf of Indigofera gerardiana (Wall. ex Baker) Status Stipules Colour Taste Odour Size Shape Fracture Texture Margin Veination Apex Duration Phyllotaxis Fresh Both surfaces Chlorophyllous Indistinct Ave. Length: 1.2 cm Chordate Short Hairy Entire Reticulate Acute Perennial Imperipinnate Present are dark green Av. Width = 8 mm Dry Both Surfaces Indistinct Indistinct Ave. Length = 1.1 cm Chorodate Short Hairy Entire Reticulate Acute Perennial Imperipinnate Present are Light Green Ave. Width = 7 mm

66 Chapter-3 Part- A Results and Discussion

3.1.2 Anatomical Studies of Indigofera gerardiana Wall.ex.Brand’s Root: The transaction of the root is wavy in out line (Plate 1). The outermost layer is the periderm. The periderm comprises brown tubercular cells having some what irregular arrangement. The cortex (Plate 2) is parenchymatus in nature with collenchymas. The secondary phloem consists of sieve elements, phloem parenchyma, phloem fibers, rays and phloem, fallowed by distinct cambium (Plate 3 and 4).

Grains of starch and crystals of calcium oxalate can be seen in parenchymatus cells (Plate 5). Xylem vessels, tracheds, fibers and xylem parenchyma are present towards the inner side having uniseriate and multiseriate rays. Pith is composed of scelerified parenchyma (Plate 6 and 6a)

Stem:

The outer most layer of the stem is crushed epidermis and illustrates lenticels. Epidermis is followed by parenchymatus cortex (Plate 7 and 7 a). Parenchymatus cells contain tannins, crystals of ca-oxalate and starch granules (Plate 8). Scattered collenchymas can be seen in cortical region. Vascular bundle is bounded by endodermal cells fallowed by pericycle. Phloem is composed of sieve elements, fibers and parenchyma. Xylem contains vessel elements parenchyma, fibers and sclereds. (Plate 9 and 9 a). Vessels are filed with tyloses (10).

Leaf:

The upper epidermis is composed of compactly packed cells. Which are cutinized and the cells are rectangular in shape. Compactly packed palisades and parenchyma are present below the epidermis (Plate 11). Vascular bundle is bounded by bundle sheath cells. Bundle sheath extension cells can also be seen. (Plate 12). Endodermis and pericycle are single layered. Pith is composed of few cells only. Lower epidermis contains sunken anomocytic and paracytic stomata. (Plate 13) Unicellular hocked and T-shaped hairs are present on the epidermis. (Plate 14). Vascular supply of the leaf was also observed (Plate 15).

67 Chapter-3 Part- A Results and Discussion

Rhizome:

The outer most layers are periderm. Corky layer is fallowed by Phellowderm, below which phllogen (phellem) is present. The periderm is fallowed cortex. Cortical cells are irregular in shape and contain crystals of calcium–oxalate, tannins, and starch granules. Sclereds of different shapes can also be seen. Distinct cambial layers separate, phloem from the xylem (annual rings). Phloem consists of sieve elements and fibers. Xylem, parenchyma, vessels, tracheds and fibers can also be seen. Pith is composed of sclerenchymatus parenchyma (16).

3.1.3 Microscopic Study of Indigofera gerardiana

The powdered drug of Indigofera gerardiana contained multicelliate, atrichome (Plate 17), parenchyma, phloem, endodermis, rays and fibers (Plate). The sample also contained hooked hair (Plate 18), erenchymatous cells, pith, pericycles, epidermal cells and paracytic stoma (Plate 19.). Crystals of ca-oxalate and vessel elements were also found (Plate 20 and 21). [Geeta et al. 2006] reported epidermis, colenchymatous cells and stomata in Acyranthes rubrofusa Linn.

3.1.4 Nutritional Analysis of Indigofera gerardiana

The fats and oils present in the Indigofera gerardiana are to be 2.37%. Gupta (1962) reported water (9.3%), albumenoids (34.3%), carbohydrate (43.4%), oil (3.0%), fiber (6.5%) and ash (3.5%) in Indigofera linifolia. Crude fiber is 17.8% while carbohydrate is 66.00%. Protein present in the fruit is 3.7%. Moisture content is 3.06% and ash value found to be 4.23%; Irvine (1992) reported carbohydrates, fats and ash in Polygonum hydropiper. According to Naseem et al., (2006) the percent levels of various ingredients in Crotalaria burhia were carbohydrate 7.16 (2.55 reducing and 4.61 non-reducing), starch contents 5.45, crude fibers 27.2, crude fats 6.36, moisture 63.10 and ash 5.67.

68 Chapter-3 Part- A Results and Discussion

Table: 3.4. Nutritional analysis of the whole plant of Indigofera gerardiana.

Plant Part Moisture Ash Fat and Oils Proteins Fiber Carbohydrate Whole plant 3.06 4.23 2.37 3.7 17.8 66.0 (68.84) Proteins, fats, oils, crude fiber, moisture content and ash value were determined by following AOAC methods [Anon, 2000]

3.1.5 Microchemical Tests of Indigofera gerardiana

Different types of chemical substances were present in powdered drug. Anthraquinone was present in the plant sample on shaking gave violet colour indicating the presence of anthraquinone [Trease and Evans, 1992].

Saponins was not found in Indigofera gerardiana,by producing violet colour. Alkaloid was found in sample plant sample that gave reddish brown colour on test. Naseem et al., [2006] reported that alkaloids, flavonoids and unbounded anthraquinones were absent from Crotalaria burhia. Calcium oxalate and starch were also found. Taninns was present and gave bluish colour. Protein was present plant and gave yellowish precipitate. Udayakumar et al., [2005] reported protein from Tridax procumbens. Lignin was present in Indigofera gerardiana and gave pink colour. Celloluse and mucilage were absenting from Indigofera gerardiana and gave yellowish brown colour. Gurbuz et al., [2005] reported mucilage from the inflorescence of some Verbascum species.

Fats and oils were found in this specie. Geetha et al. [2006] reported lignin, cellulose, protein, starch and Ca-oxalate from Achyranthes rubrofusca. Catechin was found in Indigofera gerardiana.

Table:3.5 Microchemical tests of the Indigofera gerardiana Whole Anthr Sap Alk Cao Tan Sta Pro Lig Cell Fat Cat Muc plant Indi + + + + + + + + - + + - Anthr: Anthraquinone derivatives, Sap: Saponins, Alk: Alkaloids, Cao: Ca-oxalate, Tan: Tannins, Sta: Starch, Pro: Protein, Lig: Lignins, Cell: Cellulose, Cat: Catechin, Muc: Mucilage Key: Indi: Indigofera gerardiana

69 Chapter-3 Part- A Results and Discussion

3.2 PHYTOCHEMICAL STUDIES

The present Phytochemical study in Part-A describes isolation and characterization of three new phloroglucinol type compounds 1-3, along with one known compounds were secluded from the EtOAc soluble part of the MeOH extort of Indigofera gerardiana.

The structures of the compounds 1-4 were elucidated through extensive spectroscopic studies including EI-MS, HREI-MS, 1H-NMR, 13C-NMR, DEPT, and 2D-NMR techniques including 1H-1H COSY, HMQC and HMBC.

3.2.1 Structure elucidation of new compounds

3.2.1.1 Compound (S-1)

Compound (S-1) was obtained from the EtOAc segment soluble part of the methanolic extorts of Indigofera gerardiana (see page 49) as black gummy solid.

The EI-MS spectrum of compound (S-1) illustrated the molecular ion peak + [M ] at m/z 382. The molecular formula C20H30O7 was establish by HREI-MS, awarding the molecular ion peak at m/z 382.447 (calcd for C20H30O7, 382.4481), having six degrees of unsaturation.

Fig.i. Structure of compound (S-1) 6-methyl-1-(4-((2S, 3S, 4S, 5S, 6R)-3, 4, 5-trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yloxy) phenyl) heptan-1-one

The IR spectrum of chemical constituent (S-1) illustrated the fascination band at 3521 and 1687 for hydroxyl and keto carbonyl groups.

The study of 1H-NMR spectrum of compound S-1 proved signal for side chain including two methyl, four methylene and one methine protons. In the upper

70 Chapter-3 Part- A Results and Discussion region of the band two doublets each of three protons integration resonating at δ 0.87 and 0.89 and having coupling constants 6.6 Hz (each ) were assigned to the terminal methyl protons. Similarly three multiplets each of two protons integration ` ` ` at δ 1.21, 1.38 and 1.56 were allocated to the methylen H of C-5 , C-4 , and C-3 . While a multiplet of one H integration, resonating at δ 1.46 was allocated to the methine proton of C-6`. Two d d each of single H integration at δ 3 and 2.97 having coupling constants J = 15.4, 6.6 Hz and 15.4, 7.1 Hz were allotted to the α- methylene H. In the down field region, signals for aromatic protons at δ 7.03-7.83 were also observed. Signals for the sugar moiety observed at δ 5.50 (1H, d, J = 7.4 Hz) was given to the anomeric H-1``. Three multiplets each of one H integration at δ 3.54, 3.51 and 3.49 were assigned to the methine protons. Likewise 2 broad d, each of one H integration at δ 4.40 and 4.52 having coupling constant J 11.7 Hz (each) were allotted to the methylene protons at C- 6`` of sugar moiety. The glucose was recognized as β-D- glucose by means of its 1H- and 13C-NMR data (Table3.6). The acid hydrolysis of compound (S-1) provided glycone, which was separated and identified as D-glucose.

The 13C-NMR spectrum (BB, DEPT) (Table.3.6) of compound S-1 established twenty signal, excluding 2 methyl, five methylene, ten methine and three quaternary carbons. The 1H-13C- association was resoluted by HMQC spectrum. Similarly the extended range 1H-13C connectivites were established via HMBC technique (Fig 3.1). In the 13C-NMR band the downfield resonance at δ 203.5 may be attributed to the carbonylic carbons, while the signals appeared at δ 128.6 and 131.5 may be assigned to the aromatic carbons. Similarly, resonance at δ 101.1 was due to the C-1`` of glucose clan.

` HMBC spectrum (Fig.3.2), H-6 proton (δ 146) illustrated cumulation with C-5` (δ 39.5), C-7` (δ 21.9), C-8` (δ 21.0) and C-6` (δ 38.0). While H-2` (δ 3.00) exhibited correlations with C-1` (δ 203.5), C-4 (δ 131.6), C-2` δ 39.0) and C-3` (δ 25.2). Likewise, H-1`` proton (δ 5.50) showed its correlation with C-2`` (δ 72.5) and C-1 (δ 158.4).

71 Chapter-3 Part- A Results and Discussion

Fig.ii. Key HMBC interactions in (S-1) Due to spectral evidences the structure of chemical constituent (S-1) was established. (Indigofyrin). Table 3.6 1H (400 MHz) and 13C NMR (100MHz) Data of (S-1) in MeOD

Entry C # 1H δ (J Hz) 13C (δ) Multiplicity HMBC correlations

1 1 - 158.4 C - 2 2 7.03 131.5 CH 1, 2, 3 3 3 7.83 128.6 CH 2 , 3 ,4 4 4 - 131.6 C - 5 5 7.83 128.6 CH - 6 6 7.03 131.5 CH 1 ,6 ,5 7 1` - 203.5 C -

8 2` 3, dd,15.4,6.6 39.0 CH2 4, 1`, 2`,3` 2.97, dd,15.4,7.1

9 3` 1.56, m 25.2 CH2 -

10 4` 1.38 , m 28.2 CH2 -

11 5` 1.21 , m 39.5 CH2 - 12 6` 1.46 , m 38.0 CH 5` ,6` ,7` ,8`

13 7` 0.89, d , 6.6 21.9 CH3 -

14 8` 0.87 , d , 6.6 21.0 CH3 - 15 1`` 5.50 , d , 7.4 101.1 CH 1 , 7`` , 2`` 16 2`` 3.54 , m 72.5 CH - 17 3`` 3.51 , m 77.5 CH - 18 4`` 3.49 , m 71.4 CH - 19 5`` 3.46 , m 79.1 CH -

20 6`` 4.4, brd,12.3 67.4 CH2 - 4.52 , d d , 11.9, 4.9

72 Chapter-3 Part- A Results and Discussion

3.2.1.2 Compound (S-2)

Compound (S-2) was secluded from the EtOAc soluble part of the of Indigofera gerardiana Wall (see page49) as yellow amorphous powder.

In EI-MS spectrum of compound (S-2) the molecular ion peak was observed at m/z 268. The high resolution electron impact mass spectrum (HR- + EIMS) customized the molecular formula to be C14H20O5, viewing M peak at m/z

268.3066 (calcd.for C14H20O5, 268.3061), having five degree of unsaturation. The IR spectrum compound (S-2) showed the assimilation bands at 3545-3402 and 1690 cm-1 for hydroxyl and keto carboxylic faction respectively.

Fig.iii. Structure of (S-2) 6-hydroxy-1-(2, 4, 6-trihydroxyphenyl) heptan-1-one

The study of H1-NMR spectrum of compound (S-2) illustrated singlet for methyl, methylene and methine protons. In the downward ground region of the band, two doublets each of one H integration resonating at δ 6.15 and 5.80 having the coupling constant J = 2.2 Hz, were assigned to the aromatic protons. The up field shifts and the smaller coupling constant of these protons indicated that these meta coupled protons are between oxygenated quartenary carbons [Kosasi., 1989]. In the upper ground area of the spectrum a doublet of the three H integration resonating at δ 1.09 with a coupling constant 6.6 Hz was assigned to the methyl protons, while, three multiplets each of two protons integration resonating at δ 3.09 and 2.86 having coupling constant J = 15.7, 6.8 Hz and J = 15.7, 7.4 Hz were assigned to the methylene protons. The protons α- to carbonyl group resonated at low field, but such a phenomenon is commonly observed in case of acylphloroglucinols when the 1H-NMR spectra are recorded in polar deuterated solvents [Kosasi., 1999 and Fung., 1994].

73 Chapter-3 Part- A Results and Discussion

The 13C-NMR spectrum (Broad-Bond BB, DEPT) (Table.3.2) of compound

(S-2) indicated fourteen signals, including single CH3, five methylenes, three methine and five quaternary carbons. The 1H-13C relationships are determined by hetero multiple quantum coherence (HMQC) were established through hetero multiple bond connectivites (HMBC) technique (Fig3.4). In the 13C-NMR band, up field resonance at δ 164.5, 165.6 and 164.4 were allocated to the C-1, C-3 and C-5 aromatic carbons having hydroxyl groups. ` 1 2 In the HMBC spectrum (Fig-3.4) H-2 (δ =3.09) showed J and J relationship with C-1` (δ 208.1), C-2` (δ 42.8), C-3` (δ 24.9) and C-2 (δ 106.5), H- 6` (δ 1.89) illustrated the interaction with C-5` (δ 34.5), C-6` (δ 38.0), C-7` (δ 68.5) and C- 8` (δ 17.2). The C-4 methine protons (aromatic) (δ 5.80) exhibited relationship with C-3 (δ 164.4), C-4 (δ 95.4), C-5 (δ 165.6) and C-2 (δ 106.5).

Fig.iv. Key HMBC interactions in (S-2) Spectroscopic evidences helps to establish the structure of compound (S-2) as Indigoferin. Table 3.7 1H (400 MHz) and 13C NMR (100 MHz) Data of (S-2) in MeOD Entry C # 1H δ (J Hz) 13C (δ) Multiplicity HMBC Correlations 1 1 - - C - 2 2 - 106.5 C - 3 3 - 164.4 C - 4 4 6.15, d , 2.2 95.4 CH 3 , 2 ,4 , 5 5 5 - 165.6 C - 6 6 5.80, d ,2.2 95.9 CH 5 , 6, 1 7 1` - 208.1 C -

8 2` 3.09, dd, 42.8 CH2 2 , 1` , 2`, 3` 15.7,6.81 2.86, dd, 15.7,7.4 9 3` 1.53, m 24.9 CH2 -

10 4` 1.48 , m 27.7 CH2 - 11 5` 1.52, m 34.5 CH2 - 12 6` 1.89, m 38.0 CH 5` ,6` , 7`, 8`

13 7` 3.78,br,m 68.5 CH2 - 14 8` 1.09 , d, 6.6 17.2 CH3 -

74 Chapter-3 Part- A Results and Discussion

3.2.1.3 Compound (S-3)

Compound (S-3) obtained from the EtOAc soluble part of the methanolic extorts of Indigofera gerardiana as brown particles.

The EI-MS spectrum of compound (S-3) exhibited molecular ion peak + [M ] at m/z 354. The molecular formula C19H30O6 was customized by HREI-MS, exhibiting the molecular ion peak at m/z 354.4384 (calcd for C19H30O6, 354.4380), having five degree of unsaturation.

The IR band of chemical constituent (S-3) explained absorption bands at 2922 (C-H), 1623, 1517 (aromatic C=C) and 1230 (C-O) cm1.

CH3

OH OH CH3

H O H O H H OH HO H Fig.v. Structure of (S-3) (2R,3R,4R,5R,6S)-2-(hydroxymethyl)-6-(4-(5-methylhexyl)phenoxy)tetrahydro- 2H-pyran-3,4,5-triol

The study of the 1H-NMR band of compound (S-3) showed close resemblance to that of compound 2, in the up field region of the spectrum two doublets each of three protons integration at δ 0.87 and 0.91 having coupling constant 6.8 Hz (each) were assigned to methyl protons. Similarly, four multiplets each of two protons integration resonating at δ 2.61,1.21, 1.19 and 1.63 were allocated to the methylene H of C1`, C-2` , C-3` , and C-4`, so a multiplet of one H integration resonating at δ 1.46 was assigned to C-5` methine proton . The signals for sugar moiety was observed at δ 5.46 (1H, d, J = 7.2 Hz) which may be assigned to anomeric proton H-1``. While four multiplets each of single H integration at δ 4.21, 4.24, 3.87 and 3.99 were assigned to the methine protons of sugar moiety. The sugar moiety was identified as β-D-glucose by means of its 1H- and 13C-NMR data (Table 3.3).

75 Chapter-3 Part- A Results and Discussion

The 13C-NMR spectrum (BB, DEPT) (Table3.3) of compound (S-3) showed nineteen signals, including two methyl, four methylene, ten methine and two quaternary carbons. The 1H- 13C- correlations were determined by HMQC spectral data, while the long range 1H- 13C- connectivities were established through HMBC technique.

In the 13C-NMR band, the downfield resonance at δ 157, 116.7 (2C), 126.2 (2C) and 135.5 were assigned to the aromatic carbons While the signals come into view at δ 101.1, 74.9, 77.9, 71.4, 77.8 and 62.8 were assigned to the carbons of sugar moiety.

In the HMBC spectrum (Fig.3.6) H-5` (δ 1.46) explained relationship with ` ` ` ` C-4 , C-6 and C-7 , While H-1 (δ 2.61) showed its correlation with C-4 (δ 135.5), C-1` (δ 35.5), and C-2` (δ 31.1). Similar H-1`` (δ 5.46) showed its correlation with C-2`` (δ 74.9) and C-1 (δ 157.2)

6` H H

1` 3` 5` OH 2 3 7` OH 4 2` 4` H 1 5 OH O O H 6 H H H H HO H

Fig.vi. Key HMBC interactions in (S-3)

Based on the above spectral data and other substantiation the structure of compound (S-3) was established as Indigofarine.

76 Chapter-3 Part- A Results and Discussion

Table 3.8 1H (400 MHz) and 13C NMR (100MHz) Data of (S-3) in MeOD

Entry C # 1H Δ (J HZ) 13C (Δ) Multiplicity HMBC Correlations

1 1 - 157.2 C - 2 2 7.20 116.7 CH - 3 3 7.08 126.3 CH 2 , 3 , 4 4 4 - 135.5 C - 5 5 7.08 126.3 CH - 6 6 7.20 116.7 CH 1 ,6 ,5

7 1` 2.61 , brm 35.5 CH2 4 ,1` ,2`

8 2` 1.63 , m 31.1 CH2 -

9 3` 1.19, m 28.2 CH2 -

10 4` 1.21 , m 39.3 CH2 -

11 5` 1.46 , m 27.8 CH2 4` , 5` ,6` ,7`

12 6` 0.87,d,J= 6.8 22.1 CH3 -

13 7` 0.91, d ,J= 6.8 22.0 CH3 - 14 1`` 5.46 , d, J= 7.2 101.1 CH 1 , 1`` , 2`` 15 2`` 4.21 , m 74.9 CH - 16 3`` 4.24 , m 77.9 CH - 17 4`` 3.87, m 71.4 CH - 18 5`` 3.99 , m 77.8 CH -

19 6`` 4.39 brd,11.89 62.8 CH2 - 4.52 , d d , 11.0, 4.8

3.2.2 Sructure elucidation of known compound 3.2.2.1 β-sitosterol:

The fourth compound 4 was isolated from the Indigofera gerardiana by column chromatography (silica- gel). It was eluted with solvent system in the ratio of methanol: chloroform (9:91). It was in the form of needle like crystals. With the help of spectroscopic techniques such as EI-MS of compound 4 illustrated the molecular ion at m/z 414.3855 relating to the molecular formula C29H50O (calcd. 414.3861) indicating five degree of un-saturation in the molecule.

77 Chapter-3 Part- A Results and Discussion

CH3

CH3 H3C

CH3 CH3

H CH3

H H

HO

Fig.vii. Structure of β-Sitosterol

The 1H-NMR of this compound exhibited close identity or characteristics of a steroids i.e. β-sitosterol that has been already reported.

Two 3H singlets at δ 0.65 and 0.91 may be assigned to the quaternary methyl (C-18 and C-19) correspondingly. The 3H doublets at δ 0.88 (d, J21, 20 =

6.4 Hz ), 0.81 (J26,25 = 6.4) and 0.77 (3H. t, J29,28 = 7.1 Hz) was assigned to the primary Me-29. The olefenic proton resonated at δ 5.31 (m) was assigned to H-6. The chemical shift and splitting pattern (multiplet) of the methine signals integrating for 1H at δ 3.36 were consistent with the H-3 α and 3β-hydroxyl group.

The identification of the isolated compound was based on no difference with an authentic sample of β-sitosterol and direct comparison on the TLC Plate with reference sample. Further more the spectroscopic data exhibited a good accord with the structure of β-sitosterol on comparison with the evidences reported in the literature. [Nukul., 1987 and Ullah., 1999].

78 Chapter-3 Part- A Results and Discussion

Table 3.9. 1H (400 MHz) and 13C NMR (100 MHz) Data of in β-sitosterol MeOD

1 13 Entry C # H Δ (J HZ) C (Δ) Multiplicity HMB Correlations

1 1 - - C - 2 2 - 106.5 C - 3 3 - 164.4 C - 4 4 6.15, d ,2.2 95.4 CH 3, 2, 4, 5 5 5 - 165.6 CH - 6 6 5.80, d ,2.2 95.5 CH 5, 6, 1 7 1` - 208.1 C - 2, 1`, 2`, 3` 8 2` 3.09, d d, 42.8 CH2 J=15.7,6.81 2.86, d d, J=15.7,7.4 - 9 3` 1.53, m 24.9 CH2 - 10 4` 1.48 , m 27.7 CH2 - 11 5` 1.52, m 34.5 CH2 5` ,6` , 7`, 8` 12 6` 1.89, m 38.0 CH2 - 13 7` 3.78,br,m 68.5 CH2 14 8` 1.09 , d, 6.6 17.2 CH3 -

79 Chapter-3 Part- A Results and Discussion

3.3 PHARMACOLGICAL STUDIES

3.3.1 PHARMACOLOGICAL STUDIES ON CRUDE EXTRACT AND FRACTIONS

3.3.1.1 Antibacterial Assay Tests were executed on 6 bacteria indicated strains normally were, Escherchia coli ATCC 25922, Bacillus subtilis ATCC 6633, Shigella flexeneri (clinical isolate), Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853 and Salmonella typhi ATCC 19430. Fungal strains included Trichophyton longifusus (clinical isolate), Candida albicans ATCC 2091, Aspergillus flavus ATCC 32611, Microspoum canis ATCC 11622, Fusarium solani ATCC 11712 and Candida glaberata ATCC 90030. They were maintained on agar slant at 4o C. The strains were activated at 37o C for 24 h on nutrient agar (NA) or Sabouraud glucose agar (SGA) respectively for bacteria, prior to screening. Crude methanolic extract and various other portions the exhibited zone of inhibition in millimeters against various bacterial strains percent comparison to standard drugs i.e. Imipenum in (Table3.5.). The dose 3mg/ml was given in single concentration. (Table1). Crude methanolic extract of Indigofera gerardiana. (SI- 1), n-hexane fraction (SI-2), butanol fraction (SI-5) and the aqueous fraction (SI- 6) showed good to significant action against , Staphylococcus aureus, Shigella flexeneri, Pseudomonas aeruginosa, Escherchia coli and Salmonella typhi.

Antibacterial activity was carried out against Escherichia coli (ATCC 25922), Bacillus subtilus (ATCC 6633), Shigella flexenari (clinical isolate), Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853) and Salmonella typhi (ATCC 19430). Zone of inhibition is presented in millimeters and in percent comparison to standard drugs i.e. Imipenum in Table (3.5). The dose (3 mg/ml) was given in single concentration.

80 Chapter-3 Part- A Results and Discussion

Staphylococcus aureus Escherichia coli

Pseudomonas aeruginosa Candida albicans

Fig. 3.1 Bacterial and fungal growth of some micro organisms on solid medium

The crude methanolic extract of indigofera gerardiana exhibited anti bacterial activity only against Staphylococcus aureus and Shigella flexenari and zone of inhibition observed was 11 mm and 11mm respectively and showed no

81 Chapter-3 Part- A Results and Discussion inhibitory activity against other test organisms. In other fractions, varied inhibitory activity was observed against different bacterial strains. n-Hexane showed the zone of inhibition against Escherchia col, Staphylococcus aureus and Salmonella typhi 10 mm, 13 mm and 15mm respectively. Chloroform fraction inhibited the activity of Escherichia coli and Salmonella typhi by 9 mm each. while fraction ethylacetate shows the inhibitory effect only against Shigella flexenari by 12 mm and butanol fraction only inhibited growth of Escherichia coli by 11 mm, Pseudomonas aeruginosa by 9 mm and Salmonella typhi by 12 mm respectively. While in case of aqueous fraction, inhibitory activity was observed against growth of Escherichia, coli, Shigella flexenari, Pseudomonas aeruginosa, and Salmonella typhi by 11 mm,9 mm and 12mm respectively (Fig.3.14-3.19 ).

In terms of susceptibility of organisms towards crude extract and n-hexane fraction remained the most active fraction against Salmonella typhi, Staphylococcus aureus and Escherichia coli respectively. (Fig.3.19-3.24).

.

82 Chapter-3 Part- A Results and Discussion

Table 3.10 Results of antibacterial assay of crude extract and fractions

Entry Sample E. Coli B. Subtilus S. flexenari S. Aureus P. Aeruginosa S. Typhi

1 2 1 2 1 2 1 2 1 2 1 2

1 SI-1 - - - - 11 39 11 39 - - 8 36 2 SI-2 10 41 - - - - 13 48 - - 15 57 3 SI-3 9 37 - - - - 9 37 - - - - 4 SI-4 - - - - 12 48 ------5 SI-5 - - - - 9 37 - - 11 39 7 26 6 SI-6 11 39 ------9 37 12 48 7 STD 24 - 23 - 28 - 27 - 20 - 26 -

. E. coli:Escherichia coli . S. aureus: Staphylococcus aureus . B. subtilus:Bacillus subtilus . P. aeruginosa: Pseudomonas aeruginosa . S. flexenari: Shigella flexenari . S. typhi : Salmonella typhi

. SI-1: Crude extract . SI-5: n-BuOH fraction . SI-2: n-hexane fraction . SI-6: H2O fraction . SI-3: CHCl3 fraction . STD: Imipenum. . SI-4: EtOAc fraction 1: Zone of inhibition (mm) 2: Percent inhibition to standard drug

83 Chapter-3 Part- A Results and Discussion

Zone of inhibition (mm) by crude extract and fractions against different bacteria.

Fig. 3.2: Zone of inhibition by crude extract. Fig. 3.3: Zone of inhibition by n- hexane fraction

12 15 10 8 10 6

4 5 2 Zone of inhibition(mm) 0 (mm) Zone of inhibition 0 123456 123456

Fig. 3.4: Zone of inhibition by CHCl3 fraction Fig.3.5: Zone of inhibition by EtOAc fraction

10 12

8 10 8 6 6 4 4 2 2 Zone of inhibition (mm) Zone of inhibition (mm) Zone of inhibition 0 0 123456 123456

84 Chapter-3 Part- A Results and Discussion

12

10 8 6 4

2 Zone of inhibition (mm) Zone of inhibition 0 123456

Fig.3.6: Zone of inhibition by BuOH fraction

12 10 8 6 4 2 Zone of inhibition (mm) Zone of inhibition 0 123456

Fig.3.7: Zone of inhibition by aqueous fraction

30 25 20

15 10 5 Zone of inhbition (mm) 0 123456

Fig: 3.8 Zone of inhabition by standard drug

85 Chapter-3 Part- A Results and Discussion

Growth Inhibition (%) by crude extract and fractions against different bacteria

40 60 35 50 30 25 40 20 30 15 20 10 10

Growth (%) inhibition 5 Growth (%) Inhibition 0 0 123456 123456

Fig.3.9: Growth inhibition (%) by crude extract Fig.3.10: Growth inhibition (%) by n-hexane frction

40 50 35 30 40 25 30 20 15 20 10 10 Growth (%) Inhibition 5 (%)Growth inhibition 0 0 123456 123456

Fig. 3.11: Growth inhibition (%) by CHCl3 Fig.3.12: Growth inhibition (%) by EtOAc fraction fraction

50 40 35 40 30 30 25 20 20 15 10 10 Growth inhibition (%) Growth inhibition

Growth (%) Inhibition 5 0 0 123456 123456

Fig.3:13 Growth inhibition (%) by BuOH fraction Fig.3.14: Growth inhibition (%) byH2O fraction

1: E. coli 2: B. subtilus 3: S. flexinari 4: S. aureus 5: P. aurogenosa 6: S. typhi

86 Chapter-3 Part- A Results and Discussion

Zone of Inhibition (mm) by crude extract, fractions and imipenum (standard drug) against individual bacteria

25 25 20 20 15 15 10 10 5

Zone of inhbition (mm) Zoneof inhbition 5

0 Zone of inhbition(mm) 1234567 0 1234567

Fig.3.15: Zone of inhibition against E.Coli Fig. 3.16: Zone of inhibition against B.subtilus

30 30 25 25

20 20 15 15

10 10 5 5 Zone of inhbition (mm) Zone of inhbition Zone of inhibition (mm0 Zone of inhibition 0 0 1234567 1234567

Fig.3.17 : Zone of inhibition against S ..flexenari Fig.3.18: Zone of inhibition against S.aureus

20 30 25 15 20 10 15

10 5 5 Zone of inhibition (mm) Zone of inhibition Zone of inhibition (mm) Zone of inhibition 0 0 1234567 1234567

Fig.3.19: Zone of inhibition against P.aeruginosa Fig.3.20 : Zone of inhibition against S.typhi

1: Crude extract, 2: :n-hexane fraction , 3: CJCl3 fraction, 4: EtOAc fraction : 5: n-BuOH fraction, 6: H2O fraction, 7: Imipenum

87 Chapter-3 Part- A Results and Discussion

Growth Inhibition (%) by crude extract, fractions and imipenum (standard drug) against individual bacteria

100 25

80 20

60 15

40 10

20 5 Growth (%) Inhbition Zone of Inhibition (mm) Zone of Inhibition 0 0 1234567 1234567

Fig.3.21: Growth inhibition (%) against. E.coli Fig.3.22 :Growth inhibition (%) against B.subtilus

100 100

80 80

60 60

40 40

20 20 Growth (%) Inhbition Growth (%) Inhbition 0 0 1234567 1234567

Fig. 3.23 : Growth inhibition (%) against S.flexenari Fig.3.24: Growth inhibition (%) against S.aureus

100 100 80 80 60 60 40 40 20

20 Growth (%) inhbition

Growth (%) Inhibition 0 0 1234567 1234567

Fig.3.25 : Growth inhibition (%) against P.aeruginosa Fig.3.26 : Growth inhibition (%) against S.typhi

88 Chapter-3 Part- A Results and Discussion

3.3.1.2 Antifungal Assay Antifungal activity was performed against fungal strains include Trichophyton longifusus (clinical isolate), Candida albicans ATCC 2091, Aspergillus flavus ATCC 32611, Microspoum canis ATCC 11622, Fusarium solani ATCC 11712 and Candida glaberata ATCC 90030. They were maintained on agar slant at 4o C. The strains were triggered at 37o C for 24 hrs on nutrient agar (NA) or Sabouraud glucose agar (SGA) correspondingly for fungi, prior to any viewing. Growth inhibition is presented in percent in comparison to standard drugs. Amphotericin-B was used as standard drug against Aspergillus flavus while miconazole was used as standard drug against rest of fungal strains. The dose was given in a single concentration (400µg/ml).

The Indigofera gerardiana crude extract (SI-1), n-hexane (SI-2), chloroform (SI-3) and aqueous fraction exhibited the most interesting inhibitory activities against Trichophyton longifusus, Candida albicans, Aspergillus flavus, Microspoum canis and Fusarium solani. Crude extract and its fractionations exhibited region of reticence in mm alongsided of these strains (Table3.11 ) Crude extract of Indigofera gerardiana exhibited anti fungal activity against Trichophyton longifusus , Candida albicans , Microspoum canis and Fusarium solani by inhibiting 60 %,50 %,50 % and 40 % respectively. Similarly n-hexane fraction inhibited growth by 20 %, 90 % and 70 % of Aspergillus flavus , Mycosporum Fusarium solani Mycosporum canis respectively. While the chloroform fraction showed the inhibitory effect against Candida albicans, Aspergillus flavus, Fusarium solani and Fusarium solani by 10 %, 60 %, 50 % and 20 % respectively. Ethyl acetate produces inhibitory effect against Trichophyton longifusus, Candida albicans, Mycosporum canis and Fusarium solani of test organism in patron of 50 %, 80 %, 40 % and 40 % respectively. The butanol fraction exhibited the inhibition against Aspergillus flavus,Mycosporum canis and Fusarium solani. (Fig.3.27-3.32). 20 %, 90 % and 20 % respectively. While in case of aqueous fraction the % inhibition against the test organisms Trichophyton longifusus, Candida albicans, Aspergillus flavus, Mycosporum canis and Fusarium solani (Fig.3.33-3.38). However, all the fractions as well as crude methanolic extract demonstrated no activity against Candida glaberata.

89 Chapter-3 Part- A Results and Discussion

Table: 3.11 Result of antifungal assay of crude extract and fractions

% Inhibition Entry Fungal Standard Drug Strain SI-1 SI-2 SI-3 SI-4 SI-5 SI-6 1 T. longifusis 60 - - 50 - 30 Miconazole 2 C. albicans 50 - 10 80 - 10 Miconazole 3 A. flavus - 20 60 - 20 40 Amphotericin-B 4 M. canis 50 90 50 40 90 70 Miconazole 5 F. solani 40 70 20 40 20 40 Miconazole 6 C. glabarata ------Miconazole

. T. longifusis: Trichophyton longifusus . M. canis: Mycosporum canis . C.albicans: Candida albicans . F solani: Fusarium solani . A. flavus:Aspergillus flavus . C. glabarata: Candida glaberata

. SI-1: Crude extract . SI-5: n-BuOH fraction . SI-2: n-hexane fraction . SI-6: H2O fraction . SI-3: CHCl3 fraction . STD: Imipenum. . SI-4: EtOAc fraction linear growth in test (mm) % inhibition of fungal growth  100 - x 100 linear growth in control (mm)

90 Chapter-3 Part- A Results and Discussion

Growth Inhibition (%) by crude extract and fractions against different fungi

60 100

50 80 40 60 30 40 20 10 20 Growth(%) inhbition Growth(%) inhbition 0 0 123456 123456

Fig. 3.27: Percent growth inhibition by crude extract Fig. 3.28 : Percent growth inhibition by n-hexane

60 80 50 70 60 40 50 30 40 20 30 20 10 Growth inhbition(%) Growth (%) inhbition 10 0 0 123456 123456

Fig. 3.29: Percent growth inhibition by CHCl3 Fig.3.30: Percent growth inhibition by EtOAc fraction fraction

100 70 60 80 50 60 40

40 30 20 20

Growth (%) inhbition Growth (%) inhbition 10 0 0 123456 123456

Fig.3.31: Percent growth inhibition by n- BuOH Fig.3.32: Percent growth inhibition by H2O fraction fraction

1: T. longifusis 2: C. albicans 3: A. flavus 4: M. canis 5: F. solani 6: C. glabarata

91 Chapter-3 Part- A Results and Discussion

Growth Inhibition (%) by crude extract and fractions against individual fungi

60 80 50 70 60 40 50 30 40 20 30 20 10 Growth (%) inhbition Growth inhibition (%)Growth inhibition 10 0 0 123456 123456

Fig.3.33: Growth inhibition (%) against Fig.3.34: Growth inhibition (%) against T.longifusisI C.albicans

60 100

50 80 40 60 30 40 20 20 10 Growth (%) inhibtion Growth (%) inhbition 0 0 123456 123456

Fig.3.35: Growth inhibition (%) against Fig.3.36: Growth inhibition (%) against A.flavus M. canis

70 1

60 0.8 50 0.6 40 30 0.4

20 0.2 Growth (%) inhibition

Growth (%) inhibition 10 0 0 123456 123456

Fig.3.37: Growth inhibition (%) against Fig.3.38: Growth inhibition (%) against F.solani C.glabarata

92 Chapter-3 Part- A Results and Discussion

3.3.1.3 Brine Shrimp Bioassay The test was performed to evaluate the toxicity of medicinal plant and the drug was used in 10, 100 and 1000μg/ml dose and the lethality was observed in term of death of larvae and compared with standard drug i.e. Etoposide, which

exhibited total death of tested shrimps and LD50, was calculated as 7.4625µg/mL.

This study is useful in evaluating the acute cytotoxicity of the crude methanolic extract and its various segments were found to be absent at the tested concentrations (10, 100 and 1000 µg/mL) in the brine shrimp lethality assay. When the crude extract was tested against shrimps, no significant cytotoxicity was recorded as 27 shrimps survived at dose of 10 μg/ml and 27 shrimps survived out of 30 experimental shrimps at doses of 100 μg/ml and 1000 μg/ml. Different fractions exhibited varied cytotoxic activity. Chloroform and butanol fractions produced cytotoxicity at the highest dose (1000 μg/ml) only as 28 and 26 shrimps were killed respectively by these fractions. LD50 was calculated as 7.4625 µg/mL for each fraction. Aqueous and n-hexane, the survival rate was 30 and28 at doses of 1000 μg/ml. While 24, 27, 27, 23 larvae survived at 100μg/ml by chloroform, butanol, aqueous and n- hexane fractions respectively. While the survival rate for dose of 10ug/ml was 22, 28, 29 and 20 chloroform, butanol, aqueous and n-hexane fractions respectively. Thus all fractions failed to produce cytotoxicity. Graph showing activity at different doses. Maximum cytotoxicity was exhibited by n-hexane fraction i.e. 30 larvae survived at the dose of 10ug/ml. No cytotoxicity was exhibited by crude extract as well as, n-hexane, chloroform, ethyl acetate, butanol and aqueous fractions at all doses. The results are recorded in tabular form (Table.3.7).

93 Chapter-3 Part- A Results and Discussion

Table 3.12 Results of brine shrimp bioassay of crude extract and fractions

Entry Dose No. of No. of No. of dead LD50 Std. Drug LD50 (µg/ml) (µg/ml) shrimps survivors larvae (µg/ml) Crude extract (SI-1)

1 10 30 19 11 2 100 30 27 03 _ Etoposide 7.4625 3 1000 30 27 03 n-Hexane fraction (SI-2)

1 10 30 30 0 2 100 30 23 07 _ Etoposide 7.4625 3 1000 30 20 10 Chloroform fraction (SI-3)

1 10 30 28 02 2 100 30 24 06 _ Etoposide 7.4625 3 1000 30 22 08 Butanol fraction (SI-4)

1 10 30 26 04 2 100 30 27 03 _ Etoposide 7.4625 3 1000 30 29 01 Ethyl acetate fraction (SI-5)

1 10 30 26 04 2 100 30 27 03 _ Etoposide 7.4625 3 1000 30 29 01 Aqueous fraction (SI-6)

1 10 30 28 02 2 100 30 29 01 _ Etoposide 7.4625 3 1000 30 29 01

94 Chapter-3 Part- A Results and Discussion

Comparison of larvae dead at various doses of crude extract and fractions

12 10 8 6 4

No of dead larvae 2 0 123456

Fig.3.39: Comparison of larvae dead at 10µg/ml

7 6 5 4 3 2

No of larvae dead 1 0 123456

Fig.3.40: Comparison of larvae dead at 100µg/ml

10

8

6

4

2 No of larvae dead 0 123456

Fig.3.41: Comparison of larvae dead at 1000µg/ml

95 Chapter-3 Part- A Results and Discussion

3.3.1.4 Insecticidal Assay

The insecticidal activity of the test compound was carried out to observe the toxicity towards the killing or paralyzing of insects viz. Tribolium castaneum, Sitophilus oryzae, Rhyzopertha dominica and Callosobruchus analis. Permethrin was used as standard drug at dose of 235.71µg/cm2, which has shown 100 % mortality against stated insects. Samples were tested at dose of 1019.10 µg/cm2.The method of experiment is given in experimental part.

The crude extract exhibited 20% mortality against 20%Rhyzopertha dominica. and Callosobruchus analis.

n-heaxne fraction and aueous has 20% mortality against Callosobruchus analis, which is a non-significant. Butanol fraction has no insecticidal activity against all insects. Chloroform fractions also showd non significant incecticidal activity.

Chloroform fraction killed 20 % of Callosobruchus analis respectively. Butanol fraction showed 20% actvityainst Rhyzopertha dominicaThe results are given (Table.3.8) and presented in (Fig.3.42-3.50.).

96 Chapter-3 Part- A Results and Discussion

Table 3.13 Results of insecticidal assay of crude extract and fractions

Sample Code T. castaneum (1) S.oryzae (2) No. of No. of Mortality No. of No. of Mortality survivors dead survivors dead % % SI-1 10 0 0 10 0 0 SI -2 10 0 0 10 0 0 SI -3 10 0 0 10 0 0 SI -4 10 0 0 10 0 0 SI -5 10 0 0 10 0 0 SI -6 10 0 0 10 0 0 Sample Code R. dominica (3) C.analis (4)

No. of No. of Mortality No. of No. of Mortality survivors dead survivors dead % % SI-1 08 2 20 08 2 20 SI -2 10 0 0 08 2 20 SI -3 10 0 0 0 0 0 SI -4 10 0 0 10 0 0 SI -5 08 2 20 10 0 0 SI -6 10 0 0 08 02 20

. T.castaneum: Tribolium castaneum . R.dominica: Rhyzopertha dominica . S.oryzae: Sitophilus oryzae . C.analis: Callosobruchus analis

. SI-1: Crude extract . SI-4: EtOAc fraction . SI-2: n-hexane fraction . SI-5: n-BuOH fraction . SI-3: CHCl3 fraction . SI-6: H2O fraction

Name of Insects % Mortality

+ ve Control - ve Control

Tribolium castaneum 100 0 Sitophilus oryzae 100 0 Rhyzopertha dominica 100 0 Callosobruchus analis 100 0

Concentration of test sample = 1019.10 µg/cm2 Concentration of Standard Drug = 235.9 µg/cm2 +ve control (Standard drug) = Permethrin (copex) -ve control = solvent

97 Chapter-3 Part- A Results and Discussion

Percent mortality of insects by crude extract and fractions

20 20

15 15

10 10

Mortality (%) Mortality 5 5 Mortality (%) Mortality 0 0 1234 1234

Fig.3.42: Percent mortality of insects by crude Fig.: 3.43: Percent mortality of insects by n-hexane extract fraction. fraction

20 20

15 15

10 10

Mortality (%) Mortality 5 5 Mortality (%) Mortality

0 0 1234 1234

Fig.3.44: Percent mortality of insects by CHCl3 Fig.3.45 Percent mortality of insects by EtOAc fraction fraction

20 20

15 15

10 10

Mortality (%) Mortality 5 (%) Mortality 5

0 0 1234 1234

Fig.3.46: Percent mortality of insects by Fig.3.47: Percent mortality of insects by H2O BuOHfraction. fraction.

1: Tribolium castaneum 2: Sitophilus oryzae 3: Rhyzopertha dominica 4: Callosobruchus analis

98 Chapter-3 Part- A Results and Discussion

Comparison of percent mortality of insects by crude extract and fractions against individual insects

1 20

0.8 15 0.6 10 0.4

Mortaility (%) 0.2 Mortality (%) 5

0 0 123456 123456

Fig.3.48: Comparison of percent mortality of Fig.3.49 Comparison of percent mortality of T.castaneum S.oryzae

2 20

1.5 15

1 10 Mortality (%) 0.5 (%) Mortality 5

0 0 123456 123456

Fig.3.50: Comparison of percent mortality of Fig.3.51: Comparison of percent mortality of R. dominica C.analis

1: Crude extract 2: n-hexane fraction 3: CHCl3 fraction 4: EtOAc fraction

5: n-BuOH fraction 6: H2O fraction.

3.3.1.5 Phytotoxicity Assay

The test was carried out with objective to check the toxicity of the crude extract and fractions against the Lemna min towards vegetative part of a plant Phytotoxic assay is also called Lemna bioassay for inhibitors and promoters of plant growth. Paraquat was used as standard drug at dose of 0.015µg/mL. The experiments were performed under control conditions. The detail of experimental procedure is already described in experimental part.

Crude extract inhibited 4 % growth at 1000 μg/ml, 10 % at 100 μg/ml and 9 % at 10 μg/ml. n-hexane fraction 100 % growth inhibition was observed at the

99 Chapter-3 Part- A Results and Discussion highest dose. While inhibition pattern at dose of 10 μg/ml and 100 μg/ml was 8 % and 46 % inhibition at 10 μg/ml and 100 μg/ml correspondingly. It means that percent inhibition by n-hexane is dose dependent.

In case of n-hexane, chloroform,ethylactate butanol and aqueous fractions, growth inhibition was 100 % at the dose of 1000 ug/ml. At the dose of 100 ug/ml growth inhibition is in the order of 42 %, 45 %, 41 %, 37 % and 28 % for n- hexane, chloroform, ethyl acetate, butanol and aqueous fraction was observed respectively. Similarly at dose of 10 μg/ml, n-hexane, chloroform, ethyl acetate, butanol and aqueous fractions showed the rate of inhibition such as 8. 0%, 15 %, 0 %, 5% and 0 % respectively. Maximum growth inhibition at the highest dose (1000 μg/ml) was observed in all fractions except crude extract. Maximum growth inhibition at medium and minimum dose (100 μg/ml) was exhibited by aqueous fraction. The results are given (Ttable 3.9) and (Fig.3.51-3.53) Table 3.14 Pytotoxic assay of crude extract and fractions

Entry Sample Conc. of No. of fronds No. of fronds % Growth code sample (μg/ml) Survived dead Regulation 1 SI-1 10 20 04 09 100 22 02 10 1000 23 01 0 2 SI -2 10 22 02 08 100 11 13 42 1000 24 24 100 3 SI -3 10 21 03 15 100 14 10 45 1000 24 24 100 4 SI -4 10 24 00 00 100 14 10 41 1000 24 24 100 5 SI -5 10 23 01 5 100 15 09 37 1000 24 24 100 6 SI -6 10 24 00 00 100 18 06 28 1000 24 24 100

. Number of fronds in control: 24

. SI-1: Crude extract . SI-4: EtOAc fraction . SI-2: n-hexane fraction . SI-5: n-BuOH fraction . SI-3: CHCl3 fraction . SI-6: H2O fraction

100 Chapter-3 Part- A Results and Discussion

Comparison of percent growth regulation by crude extract and fractions at various doses

16 14 12 10 8 6 4

Growth (%) Inhbition 2 0 123456

Fig: 3.52: Comparison of growth regulation (%) at 10µg/mL

50

40

30

20

10 Growth (%) Inhbition 0 123456

Fig: 3.53 Comparison of growth regulation (%) at 100µg/mL

100

80

60

40

20 Growth (%) Inhbition 0 123456

Fig: 3.54: Comparison of growth regulation (%) at 1000µg/mL

SI-1: Crude ex tract, SI-2: n-hexane fraction , SI-3: CHCl3 fraction, SI-4: EtOAc fraction, SI-5: BuOH fraction, SI-6: H2O fraction

101 Chapter-3 Part- A Results and Discussion

3.3.1.6 Anti Leishmanial Assay

The methanolic crude extract and different fractions of Indigofera gerardiana were studied for their anti-leishmanial activity. The results are

expressed in IC50 values, which indicate the effective concentration of sample necessary to achieve 50 % growth inhibition. The crude extract and fractions are

considered active if IC50 is less than 50µg/ml, while a pure compound is

considered potent if IC50 is less than 12.5µg/mL.The crude methanolic extract and various fractions were unable to produce any significant leishmanicidal Results are shown in Table (3.10) Table 3.15 Results of anti leishmanial assay of crude extract and fractions

Entry Sample Code IC50 (µg/mL)+S.D 1 SI- 1 >100 2 SI- 2 >100 3 SI- 3 >100 4 SI- 4 >100 5 SI- 5 >100 6 SI- 6 >100 7 Standard Drug 0.50+0.02 Amphotericin B

. SI-1: Crude extract . SI-4: EtOAc fraction . SI-2: n-hexane fraction . SI-5: n-BuOH fraction . SI-3: CHCl3 fraction . SI-6: H2O fraction

3.3.1.7 Anti Malarial Assay

Crude extract and fractions of Indigofera gerardiana was evaluated for anti malarial assay against Plasmodium falciparum. Chloroquine Di phosphate

was used as standard drug. Results are expressed in IC50 (µg/mL).

The methanolic crude extract of the plant and its various fractions exhibited no anti malarial activity against test organism ranging from no activity to good activity. Moderate anti malarial activity was expressed by crude extract,

chloroform and ethyl acetate fractions having IC50 values equivalent to 7.99, 7.49 and 5.48 µg/mL respectively. Significant anti malarial activity was exhibited by

102 Chapter-3 Part- A Results and Discussion chloroform fraction with IC50 value of 2.63µg/mL. n-hexane and aqueous fraction failed to produce anti malarial activity with IC50 value greater than 25µg/mL. Results are presented in Table (30.11).

Table 3.16Anti malarial assay of crude extract and fractions

Entry Sample Code IC50 (µg/mL)

1 SI- 1 7.99 2 SI- 2 >25 3 SI- 3 7.49 4 SI- 4 2.63 5 SI- 5 5.48 6 SI- 6 >25 7 Chloroquine Di phosphate 0.025

. SI-1: Crude extract . SI-4: EtOAc fraction . SI-2: n-hexane fraction . SI-5: n-BuOH fraction . SI-3: CHCl3 fraction . SI-6: H2O fraction

103 Chapter-4 Part- B Introduction

4.0 INTODUCTION

4.1 GENOUS Crataegus

Hawthorn [Crataegus species], belonging to the Rosacease family, consists of small trees and shrubs. Common names for hawthorns may include, mayblossom, quick thorn, whitethorn, haw hazels, halves, hawthorn, and bread and cheese tree. They are usually multi-branched 2-5m shrubby trees that can reach a height of up to 10m. The hawthorn tree prefers the forest margins of lower and warmer areas [Brown, 1995; Grieve, 1982; Wichita, 1996]. The fruits of our native hawthorns are also edible but, as most people who have tried them will testify, there are much nicer fruits around. The colour of the ripe fruit ranges from yellow, through green to red and on to dark purple. Most of the species ripen their fruit in early to mid autumn [Brown, 1995].

The medicinal use of hawthorn [Crataegus specie.] has a long tradition. The medicinal use of extracts or tinctures prepared from the leaves, flowers and/or fruits of plants species from the genus Crataegus [Rosacea] or hawthorns is very old. [Barnes et al., 2002; Bahorun et al., 2003]. Today the plant is mainly used for treating cardiovascular diseases and is considered to be generally safe and well- tolerated [Rigelsky and Sweet, 2002]. The studies showed that the extracts increased the force of myocardial contraction [Popping et al., 1995; Muller et al., 1996; Schwinger et al., 2000], enhanced coronary flow [Schussler et al., 1995], improved oxygen utilization by cardiomyocytes [Muller et al., 1996] and reduced the occurrence of reperfusion-induced cardiac arrhythmias [Muller et al., 1996; Garjani et al., 2000].

Other studies demonstrated the extract could protect the myocardium from injury in animal models of coronary ischemia and reperfusion [Krzemisnki and Chatter jee, 1993; Makdessi et al., 1999]

Nowadays, Crataegus extracts are mainly utilized for the treatment of mild form of congestive heart failure according to the New York Heart Association [NYHA] functional class II. This indication is well substantiated by pharmacological and clinical studies [Upton, 1999; Rigelsky and Sweet, 2002; Chang et al., 2002] and extracts prepared with aqueous alcohols from leaves with

104 Chapter-4 Part- B Introduction flowers of selected Crataegus species are registered therapeutics in Germany [Blumenthal et al., 1998]. Some Crataegus constituents are predicted to be good antioxidants. The flower and fruit constituents responsible for free radical scavenging activity are epicatechim, hyperoside and chlorogenic acid. They are also among the best antiilipperoxidants [Bahorun & Greiser, 1996; Bahorun & Trotin, 1984; Rakotoarison & Greissier, 1993].

Traditionally, the fruits or the berries are used for their astringent properties in heavy menstrual bleeding and in diarrhea. Both the flowers and berries act as diuretics and can be used to treat kidney problems and dropsy. Apart from their delicious flavor, hawthorn, fruits have been shown to have a tonic effect on the heart. Fruits of our native species are often used in the treatment of weak heart conditions, especially if this is accompanied by high blood pressure [Baytop, 1984; Grieve, 1982; Schussler & Holzl, 1995; Wichtl, 1996].

Approved Crataegus extracts have been reported to increase force of myocardial contraction [Joseph et al., 1995; Pöpping et al., 1995; Müller et al., 1996; Schwinger et al., 2000; Schmidt Schweda et al., 2000], to enhance coronary flow [Joseph et al., 1995], to improve oxygen utilization in cardiomyocytes [Müller et al., 1996] and to prolong the refractory period as well as the action potential duration in isolated hearts and papillary muscles, respectively [Joseph et al., 1995; Pöpping et al., 1995; Müller et al., 1996]. Besides, Crataegus extract have cardio tonic effects [Krzeminski and Chatterjee, 1993; Al Makdessi et al., 1999]. It was shown that oral treatment of rats with the Caraegus special extract WS® 1442 [100 mg.kg -1. day -1] for 7 days significantly protected animals against arrhythmias, mortality, and hypotensive crisis.

Studies have confirmed the potential of hawthorn fruits as good source of anti-oxidants constituents [Bahorun & Griesser, 1996; Bahorun & Trotin, 1994; Kery & Verzarne, 1977; Rakotoarison & Greisier, 1997]. No work was made on the chemical and the physical properties of hawthorn fruits hitherto. Therefore, attempts were made in this study to determine the chemical composition of hawthorn fruits.

Independent studies have established extracts of Crataegus arte rich in proanthocyanidins and flavonoids [Bahorun et al., 1996; Petkov, 1979; Barnes et

105 Chapter-4 Part- B Introduction

al., 2002] and many of these phenolic compounds have been shown to be cytoprotective by reducing oxidative stress [Herrog et al., 1993; Zhang et al., 2001; Rice-Evans, 2004] thereby giving a solid basis to the proposal that the antioxidant content of Caraegus could account for its cardioprotective properties [Chatterjee et al., 1998].

4.1.1. Related Information

Synonym Crataegus oxycantha Family: Rosaceae[Pullaiah,2006] Genus: Crataegus Species: Songorica English name: Hawthorn [Anwar. et al.1979] Local Ban-sangli, [Hindi], Ring [Kashmiri],Ban-Sangli [Punjabi] [ Anwar. Et al.1979], Goni [Chitrali]. Trade name: English Hawthorn [Anwar. et al.1979]; Bansangli [Shinwari .et al2006]. Habit: Tree [Shinwari .et al. 2006] Status: Common [ Shinwari .et al. 2006]

4.1.2. Natural Habitat

The plant grows in river valleys and rivine slopes in the-mountain zone in India, found in the temperate Himmalayas of Kashmir and Himachal Pradesh, altitude of 1800-3000m. [Nadkarni, 1976].

4.1.3. Native Distribution

In Pakistan, the plant is found in mountains of Chitral [Boni], Swat, Chitral, Balochistan from 1300 to 2700 meters, [Shinwari .et al. 2006]. This plant is exceptionally common in Boni [Chitral], Swat, Astor Gilgit and Muree hilly areas with local names shetal or gain respectively; also distributed from Afghnistan to Uttarr Pardesh 1500-2700m[Puollin, 1990].

Crataegus Songarica has about 200 spices [Borys 1996; Hobb Foster, 1990]. It is cultivated in Asia, Australia [Phipps, 1983], Canada [Dickinsan, 1985].

106 Chapter-4 Part- B Introduction

4.1.4. Plant Description

It is an ornamental small hardy, spiny, deciduous [Mehra 1968] tree or shrub up to 9m tall, bark peeling off in long flakes [[Anwar. et al.1979], armed with straight axillary’s or terminal thorns; leaves deeply and sharply irregularly 4- 7 lobed; the small rose- like white flowers are beautiful and abundant [Wealth of India, 2003], in many flowered terminal corymbose cymes, emitting spicy odour [Anwar. et al.1979], the flowers have a foetid smell some what like decaying fish; fruit red, globose, ovoid or scarlet or some times yellow, blue or black in colour, containing 2-3 more or less united, bony Pyrenees[Nadkarnis, 1976].it is 16mmin diameter, the flesh is mealy and dry, like that of rosehips [Anwar. et al.1979; Shinwari .et al. 2006], the fruit ripen in early to mid autumn [Brown, 1995].

The plant prefers light, medium and heavy soils and can grow in heavy clay soil .The plant prefers acid, neutral and basic [alkaline] soil. It requires moist or wet soil and can tolerate drought. The plant can tolerate strong winds but not maritime exposure. It can tolerate atmospheric pollution.

4.1.5 Medicinal Uses The liquid extract of the flowers and fruits are used as heart tonic, improves blood flow, in the coronary arteries and used in functional diseases of the heart, such as dysproea, hypertrophy, vulvular insufficiency and heart oppression, arteriosclerosis and angina pectoris [Shinwari, 2006; Nadkarnis, 1976; Kiritikar, 1918]. The plant is reported to be astringent, sedative, stomachic, hypertensive and antispasmodic properties [Anwar, 1979; Nadkarnis, 1976]. The drug relaxes the uterus and intestine but constricts the bronchi and coronary vessels. The fruits are used as a popular remedy for diarrhea or slight phlegmasia [Mehra, 1976]. The macerated oil shows antifungal activity [Nadkarknis, 1976]. It may be used as tincture [Kirtikar, 1918]. Both flowers and berries are act as diuretic and can be used to treat kidney problems and dropsy [Grieve, 1982; Baytop, 1984; Schussler & Holzl, 1995; Wicht, 1996].

107 Chapter-5 Part- B Expermental

5.0 PHYTOCHEMICAL STUDIES

5.1 General Notes Major portion of the research was performed at the Department of Pharmacy, University of Peshawar, like soaking, fractionation of crude extract, isolation and purification of the natural active principles. Where as spectroscopic studies and screening for biological activities were executed at Hussain Ebrahim Jamal Research Institute of International Center for Chemical and Biological Sciences, University of Karachi.

5.1.1 General experimental conditions 5.1.1.1 Spectroscopy

Spectroscopic techniques like Infrared (IR) spectra in CH3OH on JASCO A-320 Infrared spectrophotometer and proton magnetic resonance (1H NMR) were documented in MeOD at 400 MHz on Bruker, AM- 300, AM-400 and AMX-500 nuclear magnetic resonance spectrometer with facet 3000 data systems at a digital resolution of 32 K correspondingly. While the 13C NMR spectrum were confirmed in MeOD on 100 MHz on the same instrument. The Distortionless Enhancement by Polarization Transfer (DEPT) experiments were o o o carried out with 45 , 90 , and 135 for determination of CH, CH2, and CH3 groups, while the quaternary carbons were determined by subtracting the signals of these spectra from broad band (BB) 13C NMR spectrum.

Low resolution electron impact mass spectra were recorded on a Finnegan MAT 311 with MASPEC Data system. Peak matching, field dsorptions (FD) and field ionization (FI) was carried out on the Finnegan MAT 312 mass spectrometer. High-resolution mass measurement and fast atom bombardment (FAB) mass measurements were documented on Jeol JMS HX 110 mass spectrometer. FAB source, using glycerol or thioglycerol as the matrix and cesium iodide (CsI) as internal standard were used for accurate mass measurement.

108 Chapter-5 Part- B Expermental

5.1.1.2 Chromatography

There are numbers of chromatographic techniques but column chromatography was used for the study of natural products. Column chromatography performed on silica gel (Si 60, 70-230 mesh, E. Merck).

Pre-coated silica gel Silica-G/UV-254 preparative plates (2020 cm, 0.5 mm thick, Macherey-Nagel) were used for preparative thick layer chromatography. The authenticity of the sample was identified on thin layer chromatography plates.

5.1.1.3 Locating Reagent

Locating agent, Cerric sulphate reagent was used for detection of the compounds

5.1.1.4 Collection and Preservation

The fresh specimens of the plant Crateagus songrica K. Koch was collected from Chitral (Bonni) during the month of October–November 2005. Berries were cleaned, washed, the seeds were separated and dried in air for three weeks at room temperature and were used for different tests and analysis. The dried material was ground to be mesh 60 and later on preserved in airtight bottles.

The dried berries (7 Kg) were percolated in 80% methanol at room temperature with occasional stirring. Fortnightly, the methanol soluble material was separated by filtration. The procedure was replicated for three times after that the residues obtained after filtration of the percolates were combined. Methanol was evaporated under reduced pressure at 40 OC under vecuo.

Distilled water (2 L) was added to the crude extract (183 g) and shaken stoutly and then added (2.5 L) of n-hexane. After shaking well the mixture was allowed to separate through a separating funnel. The process was replicate two times and the three n-hexane portions were combined. n-hexane was evaporated under reduced pressure at 40 OC and 40 g n-hexane fraction was collected. Chloroform (2 L) was added to the aqueous portion and shaken vigorously, and then kept for some time. Two layers were formed which were separated through a

109 Chapter-5 Part- B Expermental separating funnel. The process was repeated two times and the three chloroform layers were mixed together and the combined chloroform was evaporated under reduced pressure at 40 OC. After evaporation of solvent dried chloroform fraction (26.8 g) was collected. In aqueous portion, ethyl acetate (1.5 L) was added and shaken well. The ethyl acetate layer was separated. The process was repeated two times. After combining the EtOAc fractions, the EtOAc was evaporated at low pressure and 7.2 g fraction was obtained in this way. The last fraction was collected in n-butanol. n-Butanol (1L) was added to aqueous portion. After shaking well, n-butanol and water were allowed to form two separate layers, which were separated from each other and the solvents were evaporated under vacuo. The 36 g n-butanol and 73 g aqueous portions were collected.

The different fractions obtained were stored at ambient temperature for further investigations.

110 Chapter-5 Part- B Expermental

Crushed Berries of Crateagus songrica 7 kg + 80% MeOH (25 litrs)

Process was repeated PercolatedPercolate for for 15 15 daysat daysat room roomtemperature, temperature, filtered throughfilter through Whatman Whatman filter paper filter paper three times

Evaporate MeOH under reduced pressure at 40oC

Crude extract 183 g

20 g separated for actvities

163 g for Phytochemical studies

Sceme ....Extraction of Crateagus songrica

Scheme 5.1: Extraction of Crataegus songrica

111 Chapter-5 Part- B Expermental

Scheme 5.2: Fractionation of Crataegus songrica

112 Chapter-5 Part- B Expermental

Crude Methanolic Extract (183 g)

Extracted with n-hexane

Hexane fraction TA-2 (40 g) Aqueous fraction

Extracted withCHCl3

CHCl3 fraction Aqueous fraction TA-3(26.8g)

Extracted with EtOAc

EtOAc fraction Aqueous fraction TA-4(7.2 g)

Extracted with n-BuOH

n-BuOH fraction Aqueous fraction TA-5(36 g) TA-6 (73 g)

Scheme:... Fractionation of Crateagus songrica

113 Chapter-5 Part- B Expermental

EtOAc Fraction EtOAC Fraction

Subjected to C C eluted with n-hexane,EtOAc and MeOH

Fraction Fraction Fraction Fraction W X Y Z

one major one major spot spot two major spots cc eluted with ccelutedethyl acetate: with cc eluted with ethyl acetate: Hx cceluted with cc eluted with Hx (10:90) ethyl acetate: cceluted with (10: 90 ) ethyl acetate: Hx ethylethyl acetate: acetate: Hx Hx ( 5: (15:95) 95) Hx (15:85) ( 15: 85) T-III

T-IV

T-I T-II

Scheme.... Isolation of cemical constituents of Crateagus songrica

Scheme 5.3: Isolation of chemical constituents of Crataegus songrica

114 Chapter-5 Part- B Expermental

5.2 Isolation of chemical constituents

The ethyl acetate fraction was loaded on a column using flash silica gel

(1500 g, 70-230 mesh, Merck) and eluted with n-hexane: CHCl3, CHCl3: MeOHl and finally with MeOH. Through increasing order of polarity four fractions (W-Z) were obtained. The primary fractions significant in hexane, were steroids, while the EtOAc portion comprises of flavonids. The flavonoids were isolated by using frequent column chromatography (flash silica gel, 230-400 mesh), PTLC, silica gel (Silica-G/UV-254) using different EtOAc: n-Hx systems accordingly.

The schemetic representation of isolation of different chemical constituents is given in scheme.5.3.

The fraction no: 1 (W) obtained through the elution of first column loaded on silica gel (8g) with the solvent system ethyl acetate: hex (15:85). The mixture of different components was separated to the desired pure compound T-I (20 mg) and T-II (23 mg) respectively.

The fraction no: 2 (X) gave the compound T-III (14 mg) by loading the mixture from the first column on silica gel (7 g). The solvent system used was ethyl acetate: hexane (10:90).

The fraction no: 3 (Z) was re-chromatographed over silica gel (10 g). Which gave compounds and T-IV (11mg) on elution with the solvent system ethyl acetate: hexane (5: 95).

115 Chapter-5 Part- B Expermental

5.2.1 Physical and spectroscopic data of isolated chemical constituents from fruit of Crataegus songrica 5.2.2 Stigmasterol 3-O-β-D-Glucoside (T-I)

The sub-fraction W gave a crystalline compound (T-I) (Scheme.5.2) when eluted with ethyl acetate: hexane (15:85). Its structure was determined by spectroscopy comparison with an authentic that it was stigmasterol 3-O-D- Glucose.

Table: 5.1 Physical and spectroscopic data of (T-I) Entry Parameter Observation 1 Physical state Colourless needles 2 Melting point 142-147 OC 3 Rf 0.40 [ethyl acetate: hexane (15:85)] 4 Yield 3.6 x 10-3 % -1 5 IRmax cm (CHCl3) 3500-3400 (OH), 1590 and 1460 (C=C). 1 6 H-NMR (300 MHz, CHCl3)  0.68 (3H, s, H-18), 0.76 (3H, d, J = 6.4, H- 27) 0.78 (3H, t, J = 7.0, H-29′), 0.81 (3H, d, J= 6.3, H-26), 0.88 (3H, d, J = 6.2,H-21), 1.00 (1H, s, H-19), 4.96 (1H, dd,J = 15.2.8.2,H-23), 5.09(1H, dd, J = 15.2,8.4,H- 22),5.32(1H,br,s,H-5),3.41-.56and4.35(1H,d, J = 7.7,H-1) 13 7 C-NMR (CDCl3, 100 MHz) δ 37.8 (C-1),32.8(C-2),80.4 (C-3),43.9 (C- 4).141.5 (C-5), 122.1(C-6),31.9(C-7), 31.7 (C-8), 50.8 (C-9),36.9(C-10), 21.5(C- 11),39.9(C-12),43.1(C-13),57.0(C-14), 24.5(C-15),28.9(C-16),56.1 (C-17),12.6 (C- 18), 19.5 (C-19), 40.5 (C-20), 21.9(C- 21),138.9 (C-22), 139.1 (C-23),52.1(C-24), 32.9 (C-25), 19.1 (C-26),21.7 (C-27), 25.6 (C-28), 12.1 (C-29), 102.8 (C-1`),74.2 (C- 2`),76.9 (C-3`) , 76.7 (C-5`) and 62.2 (C-6`).

8 HREI-MS (m/z) 573.5110 (observed), C35H57O6, 573.4151 (calcd.)

116 Chapter-5 Part- B Expermental

5.2.3 Lupeol (T-II)

The sub-fraction W was further purified column chromatography (silica gel, 200 mg) and eluted by using EtOAc: n-Hx (15:85) to afford colour less crystals of compound (T-II) (23 mg) (Scheme.5.2).

Table :5.2 Physical and spectroscopic data of(T-1I) Entry Parameter Observation 1 Physical state Colourless crystals 2 Melting point 210-211 OC 3 Rf 0.38 [ethyl acetate: hexane (15:85)] 4 Yield 4.4 x 10-3 % -1 5 IRmax cm (CHCl3) 3450 (OH), 3070, 1650 and 880 (C=C). 1 6 H-NMR (300 MHz, CHCl3)  4.63 (2H, m, H2-29), 3.64 (1H, dd, Jax,ax = 10.68, Jax ,eq = 4.27, H-3), 1.65 (3H, br, s, Me-30′), 1.05 (3H, s, Me-26), 0.96 (6H, s,Me-25, Me-27), 0.90 (3H, s, Me-24), 0.85 (3H, s, Me-28), 0.76 (3H, s, Me-23). 13 7 C-NMR (CDCl3, 100 MHz) δ 150.61 (C-20), 109.21 (C-29), 78.81(C-3), 78.81 (C-3), 55.27 (C-5), 50.49 (C-9), 48.21 (C-18), 47.91 (C-19), 42.91 (C-17), 42.81 (C-14), 40.86 (C-8), 39.94 (C-22), 38.84 (C- 4), 38.71 (C-1), 38.03 (C-13), 37.19 (C-10), 35.51 (C-16),34.25 (C-7), 29.84 (C-21), 28.04 (C-23), 27.44 (C-15),27.43(C-2), 25.12 (C-12), 20.91 (C-11),19.30 (C- 30),18.36 (C-6), 18.09 (C-28), 16.11 (C-25), 15.93 (C-26), 15.41 (C-24), 14.50(C-27) .

8 HREI-MS (m/z) 426.7189 (observed), C30H50O, 426.7194 (calcd.)

117 Chapter-5 Part- B Expermental

5.2.4 3, 5, 7-Trihrdroxy -6, 4-dimethoxy flavone (T-II1)

The sub-fraction X was sanitized through column chromatography (silica gel, 250 mg) and eluted through ethyl acetate: n-hexane (10:90) to afford amorphous yellow powder of compound (T-III) (Scheme.5.3). It was visualized under UV light on the pre coated silica gel TLC plates for confirmation of purity.

Table:5.3 Physical and spectroscopic data of (T-III) Entry Parameter Observation 1 Physical state Amorphous yellow powder 2 Rf 0.38 [ethyl acetate: hexane; (10:90)] 3 Yield 2.7 x 10-3 % -1 4 IRmax cm (CHCl3) 3310, 1642, 1603, 1350 and 860. 1 5 H-NMR (300 MHz, CHCl3)  8.12 (2H, d, J=7.9, H-2` and H-6`), 6.95 (2H, d, J = 7.9 H-3`and H-5`), 6.69 (1H, s, H-8), 392 (3H, s, OMe-6) and3.75 (3H, s, OMe-4). 13 6 C-NMR (CDCl3, 75 MHz) δ175.9 (C-4), 158.9 (C-2), 157.5 (C-4`), 152.5 (C-5), 151.5 (C-7), 149.2 (C-9), 136.5 (C-3), 115.4 (C-5`), 105.1 (C-10), 91.8 (C- 8), 56.8 and 55.4 (MeO-3 and MeO-4`).

7 HREI-MS (m/z) 330.0755 (observed), C17H14O7, 330.0739 (calcd.)

5.2.5 5, 4,-dihydroxy-3, 6, 7-trimethyoxy flavone (T-IV)

The obtained sub-fraction Z was adsorbed on column and eluted with ethyl EtOAc: n-Hx (5:95) to afford yellow crystals of compound T-IV (Scheme.5.2). It was visualized in UV light on TLC plates for confirmation of purity.

118 Chapter-5 Part- B Expermental

Table: 5.4 Physical and spectroscopic data of (T-1V) Entry Parameter Observation 1 Physical state Yellow crystals 2 Melting point 220-221 OC 3 Rf 0.42 [ethyl acetate: hexane (5:95)] 4 Yield 2.3 x 10-3 % -1 5 IRmax cm (CHCl3) 3335, 1650, 1600, 1370, 888. 1 6 H-NMR (300 MHz, CHCl3)  8.03 (2H, d, J =8.7, H-2` and H-6`), 7.09 (2H, d, J=8.7, H-3` and H-5`), 6.49 (1H, s ,H-8), 3.87, 3.68 and 3.60 (3H each, 3×OMe). 13 7 C-NMR (CHCl3, 100 MHz) δ 178.7 (C-4), 157.8 (C-4`), 155.1 (C-5), 154.1 (C-7), 152.5 (C-9), 152.1 (C-2), 138.1 (C-3), 132.1 (C-6),131.1 (C-2` and 6`), 122.3 (C-1`), 116.4 (C-3` and 5`), 106.1 (C-10), 94.6 (C-8), 60.3,60.5 and 55.9 (3×OMe ).

8 HREI-MS (m/z) 344.0862 (observed), C18H16O7, 344.0896 (calcd.)

119 Chapter-6 Part- B Results and Discussion

6.1 PHARMACOGNOSTIC INVESTIGATIONS

6.1.1 Macroscopic Features of Crataegus songrica fruit

The colour of Crataegus sangrica pome is reddish black with yellow pulp, sparsely punctuate plobose, rearly ellipsid. 1.2-1. 6cm in diameter in fresh and dark red in dry state. Odour and taste are the same in both fresh and dry forms. Average length and diameter is 2.3 cm and 1.7 cm in fresh form and 2.0 cm and 1.5 cm in dry form, respectively. Surface is smooth and shiny in fresh form and fluffy in dry form.

Hashmi and Ahmad (1997) reported the morphology of Delphinium denudatum, Aiken et al. (1999) reported the morphology of Rumex arcticus.

Table: 6.1 Macroscopical features of the fruit of Crataegus songarica K. Koch

Colour Odour Size Taste Surface Material Ave. Length Ave. Width Reddish Pleasant 2.3cm 1.7cm Pleasant Smooth and Fresh Shiny

Reddish Pleasant 2.0cm 1.5cm Pleasant Fluffy Dried black

6.1.2 Anatomical studies of Crataegus songrica. 6.1.2.1 Fruit Exocap. The upper layer of fruit is epidermis (Plate 1) coated with thin cuticle layer intrudes partway between the anti-clinal walls of the cells. Thin walled parenchyma cells underlay of the epidermis of mature and ripened fruit. (Plate 2).

Crystal’s idioblasts are present in parenchymatus cells (Plate 3). Young parenchymaus cells contain chloroplasts which on maturity are converted in to chlromoplasts.

119 Chapter-6 Part- B Results and Discussion

Mesocorp.

It is purple in colour and is composed of largely arranged polygonal cells. The Section parallel with surface showing abaxial surface of the fruit (Plate 4).

Endocorp.

It is composed of polygonal cholenchymatus cells (Plate 5). The endocarp consists of sclerieds (Plate 6) and forms the stone of the fruit.

6.1.3 Microscopic Study of Crataegus songrica

The powder drug of crataegus sangrica contained brachysclereid (Plate 6), parachymatous cells (Plate 7), epidermal cells, endodermis, mesocorp (Plate 8) and pericycle. Xylem, phloem, sclerchymatous cells (sclereds) (Plate 9) and vessel elements (Plate 10) are also found. Murtaza et al. (2006) reported parenchymatous cells, sclerenchyma and vascular bundales in Woodwardia unigemmata.

6.1.4 Nutritional analysis of Crataegus songrica

The moisture content in Crataegus songrica fruit is 4.8% and ash value found to be 4.79%. Fats and oils contents present are 3.03%. Crude fiber is 17.8% while carbohydrate is 66.00%. Protein present in the fruit is 3.7%. In a study conducted by Read (1946), the percent contents in Crataegus songrica were ash (0.79%), protein (0.44%), fats (1.03%), carbohydrate (22.1%). Read (1946) also reported protein (5.65%), fats (1.23%), ash (1.72%), carbohydrate (11.0%) and water (74.5%) in Sanuisorba minor.

Table: 6.2 Nutritional analysis of the fruit of Crataegus songrica. Plant Part Moisture Ash Fats and Oils Proteins Fiber Carbohydrate Pome 4.8 4.79 3.03 3.7 17.8 66.0 (65.88)

Crataegus songrica have revealed that these plants are good source of nutrients (proteins, fats, carbohydrates and fibers) and can be used as substrates deficit in either of these nutrients

120 Chapter-6 Part- B Results and Discussion

6.1.5 Microschemical Tests of Crataegus songrica

Anthraquinones were present in the plants samples. Anket et al (2006) reported new paranthocyandins from Rumaex actosa. Saponins were found in Crateagus songrica. Calcium oxalates were absent that there was no any effervescence. Taninns were present and gave greenish colour. Protein was present in and gave yellowish colour. Udayakumar et al. (2005) reported protein from Tridax procumbens.

Celloluse was present in Crataegus sangrica and gave yellowish brown colour. Fat was found. Catechin and mucilage were found in Crataegus sangrica. Gurbuz et al. (2005) reported mucilage from the inflorescence of some Verbascum species.

Table: 6.3 Microchemical tests of Crataegus sangrica

Plant Anthr Sap Alk Cao Tan Sta Pro Lig Cell Fat Cat Muc part Fruit + + + + + + + + + + - +

. Anthr: Anthraquinone derivatives . Pro: Protein, . Sap: Saponins . Lig: Lignins, . Alk: Alkaloids, . Cell: Cellulose, . Cao: Ca-oxalate, . Cat: Catechin, . Tan: Tannins . Muc: Mucilage. . Sta: Starch, .

121 Chapter-6 Part- B Results and Discussion

6.2 PHYTOCHEMICAL STUDY

The isolation and structure elucidation of chemical constituents isolated from Crataegus songrica are discussed in this chapter. Ethyl acetate fraction of Crataegus songrica (7.2 g) was loaded on column using silica gel column and then preparative thin layer chromatography that yielded two steroids along with two flavonoids.

The structures and stereochemistry of the these chemical constituents were established by extensive NMR, 13C-NMR broad-band, distortionless enhancement polarization transfer and two dimensional NMR, COSY, NOESY, HMBC and HMQC studies. Moreover, these compounds were recognized by comparing their spectroscopic evidences with that accounted in the literature.

6.2.1 Structure elucidation of compounds isolated from berries of Crataegus songrica 6.2.1.1 Stigmasterol 3-O-β-D-Glucoside (T-I) Stigmasterol glycoside compound (T-I) was isolated as colourless needles from the ethyl acetate portion of the Crataegus songrica. Molecular formula + C35H58O6 was established through HR-FABMS (-ve), that exhibited [M-H] peak at m/z 573.5110 calcd.573.4151. The mass spectrum showed characteristic fragmentation pattern of sterol. [Bernard, 1977 and Lenfant.1970].

CH2OH

O O OH

OH OH

Fig.6.1: Structure of Stigmasterol 3-O-β-D-Glucoside (T-I)

122 Chapter-6 Part- B Results and Discussion

The 1H-NMR band of compound (T-I) was compared with the reported data of known compound that completely corresponds to the data for stigmasterol except additional signals at δ 5.23 (1H, d, J = 7.4 Hz) and the proton signals between δ 3.05-3.86 due to the sugar moiety. The spectrum showed signals for three olefinic protons at δ 4.96 (1H, d d, J = 15.2, 8.2 Hz of H-23), 5.09 (1H, d d, J = 15.2, 8.4 Hz, H-22) and 5.32 (br. S, H-6). The six methyl appeared at δ 0.68 (s, H-18), 1.00 (s, H19), 0.88 (d J = 6.4 Hz, H-21), 0.81 (d, J = 6.3 Hz, H-26), 0.76 (d, J = 6.4 Hz, H-27), and 0.78 (t, J = 7.0 Hz, H-29)

The 13C-NMR band (BB, DEPT) illustrated the existence of twenty five signals which comprise of 6 methyl, ten methylene, sixteen methine and three quaternary carbons.

On the basis of above spectral data Co-TLC profile and mixed mp with the authentic sample, the identity of compound (T-I) was conclusively proved that the isolated compound is a stigmasterol 3-O-β-D-glucoside [Rubinstein, 1976 and Ullah, 1999]

6.2.1.2 Lupeol (T-II)

The HRMS of isolated compound (T-II) from ethyl acetate fraction, exhibited molecular ion at m/z 426.3835 matched to the molecular formula

C30H50O (calc.426.3861).

-1 It showed characteristic band for hydroxyl group at vmax 3455 cm in the IR region. The existence of terminal double bond was established by absorption + bands at vmax 3075, 1645 and 880. Beside [M] , other fragments occurring at m/z + + + + 385 [M-41] , 220[M-C15H26] , 218[M-C14H20O] and 207[M-C16H27] were characteristic for lupene series [Budzikiewicz., 1963, Shamma., 1962, Budzikiewicz., 1964]

123 Chapter-6 Part- B Results and Discussion

Fig.6.2 Structure of Lupeol (T-II)

In the 1H-NMR band of the chemical constituent (T-II) exhibited signals for methyls, methyene and methane protons. In the upper field area of the band, seven singlets each of three H integration resonating at δ 0.77, 0.79, 0.84, 0.94, 0.97, 1.05 and 1.65 were assigned to the tertiary methyls. A double doublet (d d) of single H integration resonating at δ 3.21 (J ax, ax = 9.9, J ax, eq = 4.5) was assigned to carbonylic proton. Its chemical shifts and coupling constant revealed β and equatorial configuration of the hydroxyl group at C-3. Two wide singlet each of single H integration at δ 4.62 and 4.75 were assigned to the protons.

The physiochemical data on comparison with literature values showed that compound (T-II) was considered as lupeol [Lehn., 1962and Wenkert.,1978].

6.2.1.3 3, 5, 7-Trihrdroxy -6, 4`-dimethoxy flavone (T-III) Compound (T-III) was obtained from the EtOAc fraction of Crateagus songrica through column chromatography and was eluted with solvent system EtAOc: n-Hx (5:95).

The HREI-MS of compound (T-III) illustrated molecular ion at m/z

330.0785 relating to the molecular formula C17H14O7 (calcd. 330.0739). The UV spectrum displayed maxima at 271 and 341nm respectively. The IR band of

124 Chapter-6 Part- B Results and Discussion isolated chemical constituent (T-III) exhibited absorption bands at 3310 (OH) 1642 (α, β-unsaturated ketone), 1603 (aromatic).

OCH3 3` 4` 2` 1` 8 5` HO O 6` 7 9 2

10 3 6 4 OH H3CO 5

OH O

Fig.6.3 Structure of 3, 5, 7-trihrdroxy -6, 4`-dimethoxy flavone (T-III)

The 1H-NMR data of compound (T-III) confirmed the two singlets, each of three proton integration resonating at δ 3.75 and 3.92 and were assigned to the methoxyl proton present at C-6 and C-4. While, in the down field area of the spectrum, a singlet of one proton integration resonating at δ 6.69 was allocated to H-8. In the same way, two doublets, each of two protons integration resonating at δ 8.12 and 6.95, having the coupling constant 8.9 Hz were due to H-2`, H-6` and H-3`, H-5` respectively.

13C-NMR data attribured the signals at δ 158.9 (C-2), 136.5 (C-3), 175.9 (C = O). The chemical shifts of C-5 (δ 152.5) specified the existence of phenolic group [Agarwal, 1989].

On the basis of spectral data, it was therefore confirmed that the compound (T-III) was 3, 5, 7-Trihrdroxy -6, 4-dimethoxy flavone [Wolenweber., 1971& 1985].

6.2.1.4 5, 4`,-Dihydroxy-3, 6, 7-trimethyoxy flavone (T-IV)

Compound (T-1V) was secluded from the EtOAc portion of the crude methanolic extort of Crateagus songrica by column chromatography and was eluted with EtOAc: n-Hx (10: 90)

125 Chapter-6 Part- B Results and Discussion

OH

H3CO O

H3CO OCH3 OH O

Fig.6.4 Structure of 5, 4`,-dihydroxy-3, 6, 7-trimethoxy flavone (T-1V)

The HR-EIMS of the compound (T-1V) verified the molecular ion at m/z

344.0862 matching to molecular formula C18H16O7 (calcd.344.0896). The UV band displayed the maxima at 357 and 272 nm. While IR spectrum exhibits the absorption bands at 3335,1650,1600,1370 and 888 cm-1.

The 1H-NMR band of T-IV explained a singlet of one proton integration resonating at δ 6.49 corresponding to the C-8 position, while 2 doublets each of two protons integration resonating at 7.09 and 8.03 having the coupling constant 8.7 Hz (each) were assigned to the H-2`, H-6` and H-3` and H-5` correspondingly.

13C-NMR (BB, DEPT) band of compound (T-IV) corroborated the existence of 3 methyl, five methine and 10 quaternary carbons. The signal at δ 94.6 corresponded to C-8, while the signal at δ 138.1 was due to C-3. The signals appeared at δ 60.3, 60.5 and 55.9 were due to the three methoxyl groups.

On the basis of above spectroscopic data and assessment with reported prose [Flores., 1958] compound (T-IV) was identified as 5,4`-Dihydroxy -3,6,7- trimethoxy flavone, that was reported previously from Brickellia pendula.

126 Chapter-6 Part- B Results and Discussion

6.3 PHARMACOLOGICAL STUDIES

6.3.1 Antibacterial Assay The crude methanolic extract of Crataegus songrica and its different fractions there of were studied against human pathogens Escherchia coli ATCC 25922, Bacillus subtilis ATCC 6633, Shigella flexeneri (clinical isolate), Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853 and Salmonella typhi ATCC 19430. They were remained on agar skew at 4o C. The strains were make actived at 37o C for twenty four hours on nutrient agar (NA) or Sabouraud glucose agar (SGA) for bacteria, before screening.

Crude extract of Crataegus songrica and various segments revealed region of inhibition in millimeters alongside various bacterial strain percent comparisons to standard drugs i.e. Imipenum in table. The dose (3 mg/ml) was given in single concentration. Crude methanolic extract (SC-1), n-hexane fraction (SC-2) and ethyl acetate showed well to significant activity against Escherchia coli and the zone of inhibition were 14 mm, 15 mm and 9 mm respectively. (Table6.1) and showed no inhibitory activity against other test organisms. In other fractions, varied inhibitory activity was observed against different bacterial strains. The n- hexane showed the zone of inhibition only against Escherchia coli, with diameter of 14 mm. Chloroform fraction inhibited the activity of Bacillus subtilis and Shigella flexenari 12 mm and 16 mm each. While fraction ethyl acetate illustrated the inhibitory effect only against Escherchia col and Bacillus subtilis inhibited growth 17 mm and 13mm respectively. Butanol fraction inhibited the growth of Bacillus subtilis and Pseudomonas aeruginosa with the zone of inhibition 17 mm and 11mm respectively. While in case of aqueous fraction, inhibitory activity was observed against growth of Shigella flexenari and Pseudomonas aeruginosa by 12 mm and 9 mm each. (Fig.6.1-6.18).

Crude extract of plant exhibited potential antibacterial activity against E. coli, B. subtilis and S. flexener

127 Chapter-6 Part- B Results and Discussion

Table: 6.4 Results of antibacterial assay of crude extract and fractions

Entry Sample E. coli B. subtilus S. flexenari S. aureus P. aeruginosa S. typhi

1 2 1 2 1 2 1 2 1 2 1 2

1 SC-1 14 58 9 39 15 53 ------2 SC-2 14 58 ------3 SC-3 - - 12 52 16 57 ------4 SC-4 17 70 13 56 ------5 SC5 - - 17 70 - - 11 40 - - - - 6 SC-6 - - - - 12 52 - - 9 39 - - 7 STD 24 - 23 - 28 - 27 - 20 - 26 -

. E. coli:Escherichia coli . S. aureus: Staphylococcus aureus . B. subtilus:Bacillus subtilus . P. aeruginosa: Pseudomonas aeruginosa . S. flexenari: Shigella flexenari . S. typhi : Salmonella typhi

. SC-1: Crude extract . SC-5: n-BuOH fraction . SC-2: n-hexane fraction . SC-6: H2O fraction . SC-3: CHCl3 fraction . STD: Imipenum. . SC-4: EtOAc fraction

1) Zone of inhibition (mm) 2) Percent inhibition to standard drug

128 Chapter-6 Part- B Results and Discussion

Zone of inhibition (mm) by crude extract and fractions against different bacteria.

16 14 14 12 12 10 10 8 8 6 6 4 4 2 2 Zone of inhibition(mm) Zone of Inhibition (mm) Zone of Inhibition 0 0 123456 123456

Fig.6.1: Zone of inhibition by crude extract Fig.6.2: Zone of inhibition by n-hexane

16 20 14 12 15 10 8 10 6 4 5 2 Zone of Inhibition (mm) Zone of Inhibition Zone of inhibition (mm) Zone of inhibition 0 0 123456 123456

Fig.6.3 : Zone of inhibition by CHCl3 fraction Fig.6.4: Zone of inhibition by EtOAc fraction

20 12 10 15 8

10 6 4 5 2 Zone of Inhibition (mm) Zone of Inhibition Zone of inhbition (mm) Zone of inhbition 0 0 123456 123456

Fig.6.5 Zone of Inhibition by BuOH Fraction Fig.6.6:.Zone of Inhibition by H2O Fraction

1: E. coli 2: B. subtilus 3: S. flexinari 4: S. aureus 5: P. aurogenosa 6: S. typhi

129 Chapter-6 Part- B Results and Discussion

Growth inhibition (%) by crude extract and fractions against different bacteria

60 60 50 50 40 40 30 30 20 20 10 10 Growth (%) inhbition Growth (%) Inhbition 0 0 123456 123456

Fig.6.7: Growth inhibition (%) by crude extract Fig.6.8: Growth inhibition (%) by n- hexane fraction

70 60 60 50 50 40 40 30 30 20 20

Growth (%) inhbition 10 10 Growth inhibition(%) 0 0 123456 123456

Fig.6.9: Growth inhibition (%) by CHCl3 Fig.6.10: Growth inhibition (%) by EtOAC fraction fraction

70 60 60 50 50 40 40 30 30 20 20 10 Growth(%) inhbition Growth (%) inhbition 10 0 0 123456 123456

Fig.6.11: Growth inhibition (%) by BuOH fraction Fig.6.12: Growth inhibition (%) by H2O fraction

1: E. coli 2: B. subtilus 3: S. flexinari 4: S. aureus 5: P. aurogenosa 6: S. typhi

130 Chapter-6 Part- B Results and Discussion

Zone of inhibition (mm) by crude extract, fractions and imipenum (standard drug) against individual bacteria

25 25

20 20

15 15

10 10

5 5 Zone of inhibition(mm) Zone of inhibition(mm) 0 0 1234567 12345678

Fig.6.13: Zone of inhibition against E.Coli Fig.6.14: Zone of inhibition against B. subtilus

30 30 25 25

20 20 15 15 10 10

5 5 Zone of inhbition (mm) Zone of inhbition Zone of inhibition (mm) Zone of inhibition 0 0 1234567 1234567

Fig.6.15: Zone of inhibition against S. flexenari Fig.6.16: Zone of inhibition against S. aureus

20 30

25 15 20 10 15 10 5 5 Zone of inhibition (mm) Zone of inhibition Zone of inhibition (mm) Zone of inhibition 0 0 1234567 1234567

Fig.6.17: Zone of inhibition against P. aeruginosa Fig.6.18: Zone of inhibition against S. typhi

1: Crude extract 2: n-hexane fraction 3: ChCl3 fraction 4: EyOAc fraction 5: n-BuOH fraction 6: H2O fraction 7: Imipenum

131 Chapter-6 Part- B Results and Discussion

Growth inhibition (%) by crude extract, fractions and Imipenum (standard drug) against individual bacteria

100 100

80 80

60 60

40 40

20 20 Growth (%) inhbition Growth inhibition (%)Growth inhibition 0 0 1234567 1234567

Fig.6.20 : Growth inhibition (%) against E.coli Fig.6.21: Growth inhibition (%) against B.subtillus

100 100

80 80

60 60

40 40

20 20 Growth (%) inhbition Growth inhibition (%) Growth inhibition 0 0 1234567 1234567

Fig.6.22: Growth inhibition (%) against S.flexnari Fig.6.23 : Growth inhibition (%) against S.aureus

100 100

80 80

60 60

40 40

20 20 Growth (%) inhbition Growth (%) inhbition 0 0 1234567 1234567

Fig.6.24: Growth inhibition (%) against Fig.6.25: Growth inhibition (%) against S.typhi P.aeruginosa

1: Crude extract, 2: n-hexane fraction 3: Chloroform fraction 4: Ethylacetat fraction 5: Butanol, fraction 6: Aqueous fraction 7: Imipenum

132 Chapter-6 Part- B Results and Discussion

6.3.2 Antifungal Assay Antifungal activity of methanolic crude extract and different portions were evaluated against fungal stains included Trichophyton longifusus (clinical isolate), Candida albicans ATCC 2091, Aspergillus flavus ATCC 32611, Microspoum canis ATCC 11622, Fusarium solani ATCC 11712 and Candida glaberata ATCC 90030. They were maintained on agar slant at 4o C. The strains were activated at 37o C for 24 hrs on nutrient agar (NA) or Sabouraud glucose agar (SGA) respectively for fungi, prior to any screening. Growth inhibition was presented in percent in comparison to standard drugs. Amphotericin-B was employed as representative drug against Aspergillus flavus while miconazole was used as standard drug against rest of fungal strains table. The dose was given in a single concentration (400µg/ml).

The Crataegus songrica crude extract (SC-1), n-hexane (SC-2), Chloroform (SC-3), Butanol fractiobn (SC-5) and aqueous fraction (SC-6) exhibited the inhibitory activities having region of reticence in mm against these strains (Table.6.2).

Crude extract of Crataegus songrica exhibited antifungal activity against Trichophyton longifusus,, Aspergillus flavus, Microspoum canis and Fusarium solani by inhibiting 50 %,50 %,50 % and 40 % respectively. Similarly n-hexane fraction inhibited growth by 60%, 80% and 70% of Aspergillus flavus, Mycosporum canis and Fusarium solani respectively. While the chloroform fraction showed the inhibitory effect against Aspergillus flavus, Mycosporum canis and Fusarium solani by 60%, 80% and 70% respectively. Ethyl acetate produced inhibitory effect against Trichophyton longifusus, Mycosporum canis, Aspergillus flavus, and Fusarium solani of test organism in patron of 50 %,60 %, 10 % and 30 % respectively. The butanol fraction exhibit the inhibition against Aspergillus flavus, Mycosporum canis and Fusarium solani. 20 %, 70 %and 40 % respectively (Fig.6.26-6.32). While in case of aqueous fraction the % inhibition against the test organisms Trichophyton longifusus, Aspergillus flavus, Mycosporum canis and Fusarium solani was 30 %, 50 %, 50 % and 60 % each (Fig.6.32-37).. However, all the fractions as well as crude methanolic extract illustrated no activity against Candida albicans and Candida glaberata.

133 Chapter-6 Part- B Results and Discussion

In antifungal bioassay, the crude methanolic extract and succeeding segments of the plant offered excellent activity against tested fungi including T. longifusus, Candida albicans, Aspergillus flavus, Microspoum canis, Fusarium solani and Candida glaberata as presented in table (6.2). The crude extract and subsequent fractions of C. songrica showed marked antifungal activity against T. longifusus, A. flavus, M. canis and F. solani. On the other hand, both the Candida species; C. albicans and C. glaberata were neither inhibited by the crude extract nor by the fractions of the plant.

Table: 6.5 Result of antifungal assay of crude extract and fractions Entry Fungal % Inhibition Standard Drug Strain SC-1 SC-2 SC-3 SC-4 SC-5 SC-6 1 T. longifusis 50 - - 50 - 30 Miconazole 2 C. albicans - - - 60 - - Miconazole 3 A. flavus 50 60 40 - 20 50 Amphotericin-B 4 M. canis 50 80 40 10 70 50 Miconazole 5 F. solani 40 70 50 30 40 60 Miconazole 6 C. glabarata ------Miconazole

. T. longifusis: Trichophyton longifusus . M. canis: Mycosporum canis . C.albicans: Candida albicans . F solani: Fusarium solani . A. flavus:Aspergillus flavus . C. glabarata: Candida glaberata

. SC-1: Crude extract . SC-4: EtOAc fraction . SC-2: n-hexane fraction . SC-5: n-BuOH fraction . SC-3: CHCl3 fraction . SC-6: H2O fraction

linear growth in test (mm) % inhibition of fungal growth  100 - x 100 linear growth in control (mm)

134 Chapter-6 Part- B Results and Discussion

Growth inhibition (%) by crude extract and fractions against different fungi

50 80 70 40 60 30 50 40 20 30 10 20 Growth (%) Inhbition

Growth inhibition (%) Growth inhibition 10 0 0 123456 123456

Fig.6.26: Percent growth inhibition by crude extract. Fig.6.27: Percent growth inhibition by n-hexane fraction.

50 60

40 50 40 30 30 20 20

10 10 Growth inhibition (%) Growth inhibition Growth inhibition Growth(%) inhibition 0 0 123456 123456

Fig.6.28: Percent growth inhibition by CHl3 Fig.6.29 Percent growth inhibition by EtOAc fraction fraction

70 60 60 50 50 40 40 30 30 20 20 10

Growth (%) inhbition 10 Growth (%) inhbition 0 0 123456 123456

Fig.6.30:Percent growth inhibition by BuOH Fig.6.31:Percent growth inhibition by H2O fraction fraction

1: T. longifusis 2: C. albicans 3: A. flavus 4: M. canis 5: F. solani 6: C. glabarata

135 Chapter-6 Part- B Results and Discussion

Growth inhibition (%) by crude extract and fractions against individual fungi

50 60

40 50 40 30 30 20 20 10 10 Growth(%) inhbition Growth (%) inhbition 0 0 123456 123456

Fig. 6.32: Growth inhibition (%) against Fig. 6.33 Growth inhibition (%) against T.longifusis C.albicans,

60 80 70 50 60 40 50 30 40 30 20 20 10 Growth (%) inhbition

Growth (%) inhbition 10 0 0 123456 123456

Fig. 6.34 Growth inhibition (%) against A.flavus Fig. 6.35 Growth inhibition (%) against M.canis

70 1 60 0.8 50 40 0.6 30 0.4 20 0.2 Growth(%) inhbition

Growth (%) inhbition 10 0 0 123456 123456

Fig.6.36 Growth inhibition (%) against F.salani Fig. 6.37 Growth inhibition (%) against C. glabarata

1: Crude extract, 2: n-hexane fraction, 3: CHCl3 fraction

4: EtOAc fraction, 5: n-BuOH fraction, 6: H2O fraction

136 Chapter-6 Part- B Results and Discussion

6.3.3 Brine Shrimp Bioassay The test was executed to estimate the toxicity of medicinal plant using the drug in 10 μg/ml, 100 μg/ml and 1000 μg/ml dose and the lethality was observed in term of death of larvae and compared with standard drug (Etoposide), which exhibited total death of tested shrimps and LD50, was calculated as 7.4625µg/mL.

This study is useful to evaluate the acute cytotoxicity of the crude methanolic extract and its various segments. The activity was found to be absent at the tested concentrations (10 µg/mL, 100 µg/mL and 1000 µg/mL) in the brine shrimp lethality assay. When the crude extract was tested against shrimps, then no significant cytotoxicity was recorded as 27 shrimps survived at dose of 10μg/ml and 27 shrimps survived out of 30 experimental shrimps at doses of 100 and 1000μg/ml.

Different fractions exhibited varied cytotoxic activity. Chloroform and butanol fractions produced cytotoxicity at the highest dose (1000 μg/ml) only as

28 and 26 shrimps were killed respectively by these fractions. LD50 was calculated as 7.4625µg/mL for each fraction. In aqueous and n-hexane fraction the survival rate was 30 and28 at doses of 1000 μg/ml. While 24, 27, 27, 23 larvae survived at 100 μg/ml by chloroform, butanol, aqueous and n-hexane fractions respectively. The survival rate of larvae for chloroform, butanol, aqueous and n-hexane fractions at the dose of 10ug/ml was 22, 28, 29 and 20 respectively. Thus all fractions failed to produce cytotoxicity. Graph showing activity at different doses.

No cytotoxicity was exhibited by crude extract as well as n-hexane, chloroform, ethyl acetate, butanol and aqueous fractions at all doses. The results are recorded in tabular form table (Table.6.3) and graphs (Fig.6.38-6.40)

137 Chapter-6 Part- B Results and Discussion

Table: 6.6 Results of brine shrimp bioassay of crude extract and fractions

Entry Dose No. of No. of No. of dead LD50 Std. Drug LD50 (µg/ml) (µg/ml) shrimps survivors larvae (µg/ml) Crude extract (SI-1)

1 10 30 21 09 2 100 30 22 08 _ Etoposide 7.4625 3 1000 30 26 04 n-Hexane fraction (SI-2)

1 10 30 21 09 2 100 30 26 04 _ Etoposide 7.4625 3 1000 30 25 05 Chloroform fraction (SI-3)

1 10 30 21 09 2 100 30 25 05 _ Etoposide 7.4625 3 1000 30 23 07 Butanol fraction (SI-4)

1 10 30 26 04 2 100 30 27 03 _ Etoposide 7.4625 3 1000 30 29 01 Ethyl acetate fraction (SI-5)

1 10 30 21 09 2 100 30 27 03 _ Etoposide 7.4625 3 1000 30 29 01 Aqueous fraction (SI-6)

1 10 30 26 04 2 100 30 27 03 _ Etoposide 7.4625 3 1000 30 25 05

138 Chapter-6 Part- B Results and Discussion

Comparison of larvae dead at various doses of crude extract and fractions

10

8

6

4

2 Nonof Larvaedead 0 123456

Fig. 6.38: Comparison of larvae dead at 10 μg/ml

8

6

4

2 No of larvae dead

0 123456

Fig. 6.39: Comparison of larvae dead at 100 μg/ml

7 6 5 4 3 2

No of Larvae dead 1 0 123456

Fig. 6.40 : Comparison of larvae dead at 1000 μg/ml

1: Crude extract, 2: n-hexane fraction, 3: CHCl3 fraction, 4: EtOAc fraction, 5: n-BuOH fraction, 6: H2O fraction

139 Chapter-6 Part- B Results and Discussion

6.3.4 Insecticidal Assay

The insecticidal activity of the test compound was carried out to observe the toxicity towards the killing or paralyzing of insects viz. Tribolium castaneum, Sitophilus oryzae, Rhyzopertha dominica and Callosobruchus analis. Permethrin was used as standard drug at dose of 235.71µg/cm2, which has shown 100 % mortality against stated insects. Samples were tested at dose of 1019.10 µg/cm2. The crude extract was unable to exhibit any mortality against Tribolium castaneum, Sitophilus oryzae, Rhyzopertha dominica and Callosobruchus analis. The result revealed that neither crude extract nor any fraction was found to have any insecticidal activity. The results are given in table (Table.6.4).

Table: 6.7 Results of insecticidal assay of crude extract and fractions

Sample Code T. castaneum (1) S.oryzae (2)

No. of No. of Mortality No. of No. of Mortality survivors dead survivors dead % % SC-1 10 0 0 10 0 0 SC-2 10 0 0 08 2 20 SC -3 10 0 0 10 0 0 SC-4 10 0 0 10 0 0 SC-5 10 0 0 10 0 0 SC-6 10 0 0 10 0 0 Sample Code R. dominica (3) C.analis (4)

No. of No. of Mortality No. of No. of Mortality survivors dead survivors dead % % SC-1 0 0 0 0 0 0 SC-2 0 0 0 0 0 0 SC-3 0 0 0 0 0 0 SC-4 0 0 0 0 0 0 SC-5 0 0 0 0 0 0 SC-6 0 0 0 0 0 0

. T.castaneum: Tribolium castaneum . R.dominica: Rhyzopertha dominica . S.oryzae: Sitophilus oryzae . C.analis: Callosobruchus analis

. SC-1: Crude extract . SC-4: EtOAc fraction . SC-2: n-hexane fraction . SC-5: n-BuOH fraction . SC-3: CHCl3 fraction . SC-6: H2O fraction

140 Chapter-6 Part- B Results and Discussion

Name of Insects % Mortality

+ ve Control - ve Control

Tribolium castaneum 100 0 Sitophilus oryzae 100 0 Rhyzopertha dominica 100 0 Callosobruchus analis 100 0 Concentration of test sample = 1019.10 µg/cm2 Concentration of standard drug = 235.9 µg/cm2 +ve control (Standard drug) = Permethrin (copex) -ve control = solvent

6.3.5 Phytotoxicity Assay The test was carried out with objective to check the toxicity of the crude extract and fractions against the Lemna min towards vegetative part of a plant Phytotoxic assay is also called Lemna bioassay for inhibitors and promoters of plant growth. Paraquat was used as standard drug at dose of 0.015µg/mL. The experiments were performed under control conditions.

The crude methanolic extract and various fractions confirmed phytotoxic pursuit against Lemna plant. Crude extract inhibited 4.2 % growth at 1000 μg/ml, 10.1 % at 100 μg/ml and 19.7% at 10 μg/ml. n-hexane fraction 100 % growth inhibition was observed at the highest dose. While inhibition pattern at the doses of 10μg/ml and 100μg/ml is inversely proportional i.e. 56.98 and 13.9% respectively. It means that percent inhibition by crude extract is dose dependent.

In case of chloroform and ethylacetate fractions growth inhibition observed was 72.2 %, 44.44 % , 38.8 % and 61.1 %, 33.3 % and, 100 % at the doses of 1000μg/ml, 100μg/ml and 10μg/ml respectively. Similarly in case of butanol fraction percentage of growth inhibition was 44.4 %, 16.6 % and 5.5 % at the doses of 1000 μg/ml, 100μg/ml and 10μg/ml respectively. Aqueous fraction showed growth inhibition as 51.61%, 46.2% and 19.35%. Maximum growth inhibition at the dose (1000 μg/ml) was observed in crude extract (100 %) and chloroform fraction (72.2 %). Chloroform fraction exhibited maximum growth inhibition i.e. 51.61 % and 19.35 % at medium doses of (100 μg/ml) and (10 μg/ml) respectively.

141 Chapter-6 Part- B Results and Discussion

The result revealed that crude extract and chloroform fractions have good phytotoxic potential at higher dose. The results are given table (Table.6.6 and 6.7) and graph (Fig.6.41-6.43).

Table:6.8 Phytotoxicity bioassay of the crude extract and fractions of Crataegus songrica Conc. of sample No. of fronds % Growth Sample (μg/ml) Survived Regulation code SC-1 1000 0 100 100 16 4.2 10 15 10.1 SC -2 1000 0 100 100 10 56.98 10 11 13.9 SC -3 1000 5 100 100 10 51.61 10 11 19.35 SC -4 1000 7 100 100 12 46.2 10 19 2.15 SC -5 1000 10 100 100 15 40.8 10 17 8.60 SC-6 1000 10 51.61 100 12 46.2 10 15 19.35 . Number of fronds in control = 18.6 . SC-1: Crude extract . SC-4: EtOAc fraction . SC-2: n-hexane fraction . SC-5: n-BuOH fraction . SC-3: CHCl3 fraction . SC-6: H2O fraction

142 Chapter-6 Part- B Results and Discussion

Table: 6.9 Phytotoxic assay of crude extract and fractions Entry Sample Conc. of No. of fronds No. of fronds % Growth code sample Survived dead Regulation (μg/ml) 1 SC-1 10 15 03 16.66 100 16 02 11.11 1000 0 18 100 2 SC -2 10 11 08 38.8 100 10 08 44.44 1000 18 00 100 3 SC-3 10 05 13 38.8 100 10 08 44.44 1000 11 07 72.2 4 SC-4 10 18 00 100 100 12 06 33.3 1000 07 11 61.1 5 SC-5 10 17 01 5.5 100 15 03 16.6 1000 10 08 44.4 6 SC-6 10 15 03 16.6 100 12 06 33.3 1000 10 08 44.4 . Number of fronds in control: 18 . SC-1: Crude extract . SC-4: EtOAc fraction . SC-2: n-hexane fraction . SC-5: n-BuOH fraction . SC-3: CHCl3 fraction . SC-6: H2O fraction

143 Chapter-6 Part- B Results and Discussion

Comparison of percent growth regulation by crude extract and fractions at various doses

100

80

60

40

20 Growth (%) inhibition 0 123456

Fig. 6.41: Comparison of growth regulation (%) at 10 μg/ml

50

40

30

20

10 Growth inhibition (%) Growth inhibition 0 123456

Fig. 6.42: Comparison of growth regulation (%) at 100μg/ml

100

80

60

40

20 Growth inhibition (%) Growth inhibition 0 123456

Fig. 6.43: Comparison of growth regulation (%) at 1000 μg/ml

1: Crude extract, 2: n-hexane fraction 3: CHCl3 fraction, 4: EtOAc fraction, 5: n-BuOH fraction , 6: H2Ofraction

144 Chapter-6 Part- B Results and Discussion

6.3.6 Anti Leishmanial Assay

The crude methanolic extract and various fractions of Crataegus songrica were studied for their anti-leishmanial activity. The results are expressed in IC50 values, which indicate the effective concentration of sample necessary to achieve 50 % growth inhibition. The crude extract and fractions were considered active if

IC50 is less than 50µg/ml, while a pure compound is considered potent if IC50 is less than 12.5µg/mL

The methanolic extract and different fractions were unable to produce any significant leishmanicidal effects. Results are shown in table (Table.6.8)

Table :6.10 Results of anti leishmanial assay of crude extract and fractions

Entry Sample Code IC50 (µg/mL)+S.D

1 SC- 1 >100 2 SC- 2 >100 3 SC- 3 >100 4 SC- 4 >100 5 SC- 5 >100 6 SC- 6 >100 7 Standard Drug 2.53+0.09 Amphotericin B

. SC-1: Crude extract . SC-4: EtOAc fraction . SC-2: n-hexane fraction . SC-5: n-BuOH fraction . SC-3: CHCl3 fraction . SC-6: H2O fraction

6.3.7 Anti Malarial Assay

Crude extract and fractions of Crataegus songrica were evaluated for anti malarial assay against Plasmodium falciparum. Chloroquine Diphosphate was

used as standard drug. Results are expressed in IC50 (µg/mL).

The methanolic crude extract and various fractions exhibited no antimalarial activity against test organisms ranging from no activity to good activity. Results are presented in table (Table.6.9).

145 Chapter-6 Part- B Results and Discussion

Table: 6.11 Anti malarial assay of crude extract and fractions

Entry Sample Code IC50(µg/mL)

1 SC- 1 >25 2 SC- 2 >25 3 SC- 3 >25 4 SC- 4 >25 5 SC- 5 >25 6 SC- 6 >25 7 Chloroquine Diphosphate 0.025

. SC-1: Crude extract . SC-4: EtOAc fraction . SC-2: n-hexane fraction . SC-5: n-BuOH fraction . SC-3: CHCl3 fraction . SC-6: H2O fraction

146 Part- A Plates

Plate 1: Transverse section of root

PERIDERM

CORTEX

Plate.2: T.S. showing periderm and cork of root

147 Part- A Plates

Plate.3: Arrow indicating ray

STARCH

CORTEX

PHLOEMMM CAMBIUM

FIBRES

Plate.4: Transverse section showing different cells

148 Part- A Plates

a b

Plate. 5: Longitudinal section of secondary xylem of root. (a) Fibers showing nucleus (b) Fibers filled by starch

Plate. 6: Xylem vessels of root

149 Part- A Plates

Plate.6a : Sclerified parenchyma form the central region of root

CAMBIUM CRUSHED EPIDERMIS

CORTEX

CORK PHLOEM

PHLODERM

Plate.7: Stem transverse section

150 Part- A Plates

LENTICEL

Plate.7(a): T.S. of stem showing lentice(zoom in)

Plate. 8: Crystals of calcium oxalate stem

151 Part- A Plates

Plate.9: Sclrered found in stem

VESSELLS MEDULLARY RAY

PARENCHYMA FIBERS

Plate.9.a: Anatomy of stem

152 Part- A Plates

a

b

Plate.10: Secondary xylem fibers as seen in transverse section of stem (a) tyloses (b) fibres

PLISADE

SUNKEN CUTICLE

Plate.11: T.S. of leaf showing palisades (mesophyll)

153 Part- A Plates

Plate.12: Bundle of sheath extension

Plate.13: stomata of leaf

154 Part- A Plates

Plate.14: T-shaped hair of the leaf

Plate.15: Vascular supply to the leaf

155 Part- A Plates

CAMBIUM FIBERS

PHLOEMM

PITH

XYLEM

Plate. 16: Transverse section of rhizome

Plate. 17: Trichomes with broken tip

156 Part- A Plates

Plate 18: a single fiber

Plate 19: Hooked hair

157 Part- A Plates

Plate. 20: A single crystal of calcium oxalate

Plate.21: A vessel element of fruit.

158 Part- B Plates

Plate.1: Epidermis of the fruit

Plate.2: Exocarp (epiderm) of the fruit

159 Part- B Plates

Plate.3: Parenchymatus cells

Plate.4: Section parallel with surface showing abaxial surface of the fruit (mesocorp)

160 Part- B Plates

Plate.5: cholenchymatus cells

Plate. 6: Sclereids

161 Part- B Plates

Plate. 6 : Brachysclereids as seen in the fruit

Plate.7: Parenchymatus cells

162 Part- B Plates

Plate .8: Mesocorp

Plate. 9: sclerchymatous cells (sclered)

Plate 10: A single vessel element

163 References

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