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Department of Botany University of Peshawar, Peshawar Pakistan Session 2012-2013

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Department of Botany University of Peshawar, Peshawar Pakistan Session 2012-2013 PHARMACOGNOSTIC AND PHARMACOLOGICAL EVALUATION OF MONOTHECA BUXIFOLIA (FALC.) A.DC. BY Maryam Ehsan DEPARTMENT OF BOTANY UNIVERSITY OF PESHAWAR, PESHAWAR PAKISTAN SESSION 2012-2013 PHARMACOGNOSTIC AND PHARMACOLOGICAL EVALUATION OF MONOTHECA BUXIFOLIA (FALC.) A.DC. A Thesis Submitted to the University of Peshawar in Partial Fulfilment of the Requirement for the Degree of Doctor of Philosophy In Botany By Maryam Ehsan DEPARTMENT OF BOTANY UNIVERSITY OF PESHAWAR, PESHAWAR PAKISTAN SESSION 2012-2013 In the name of Allah, the most Merciful and Compassionate, the most Gracious and Beneficent, whose help and guidance I always solicit at every step, at every moment. DECLARATION I Maryam Ehsan D/O Ehsan-ul-Haque hereby state that the PhD thesis titled “PHARMACOGNOSTIC AND PHARMACOLOGICAL EVALUATION OF MONOTHECA BUXIFOLIA (FALC.) A.DC.” is my own work and has not been submitted previously by me for taking any degree from this University (University of Peshawar, Peshawar, Pakistan) or anywhere else in the country/world. At any time if my statement is found to be incorrect even after my graduation, the university has the right to withdraw my PhD degree. Maryam Ehsan Signature: ______________ CERTIFICATE OF APPROVAL This is to certify that the research work presented in this thesis entitled ―PHARMACOGNOSTIC AND PHARMACOLOGICAL EVALUATION OF MONOTHECA BUXIFOLIA (FALC.) A.DC.” was conducted by Ms Maryam Ehsan under the supervision of Professor Dr. Muhammad Ibrar. No part of this thesis has been submitted anywhere else for any other degree. This thesis is submitted to the Department of Botany, University of Peshawar in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the field of Botany (Pharmacognosy). Department of Botany, University of Peshawar. MARYAM EHSAN _________________________ (Research Scholar) EXAMINATION COMMITTEE DR. BARKAT ULLAH __________________________ Assistant Professor Department of Botany, Islamia College University, Peshawar. DR. HASSAN SHER __________________________ Assistant Professor, Centre of Plant Sciences and Biodiversity, University of Swat. PROF. DR. MUHAMMAD SAEED __________________________ Chairman , Department of Pharmacy, University of Peshawar. PROF. DR. MUHAMMAD IBRAR __________________________ (Research Supervisor) Department of Botany, University of Peshawar. PROF. DR. GHULAM DASTAGIR __________________________ (HOD) Dedicated to my parents for making me whatever I am and to my husband and son for staying with me in hard times as a strong, sincere and reliable support. TABLE OF CONTENTS S. No. CONTENTS Page No. ACKNOWLEDGEMENT i-ii ABSTRACT iii-iv CHAPTER- 1 1 INTRODUCTION 1.1 Pharmacognosy 1 1.2 Medicinal plants 3 1.3 Ethnobotany 4 1.4 Pharmacognostic evaluation 4 1.4.1. Standardization 5 1.4.2. Authentic identification of plant material 6 1.4.3. Microscopic study/ Anatomy 6 1.4.4. Powder drug study 7 1.4.5. Phytochemical profile 7 1.4.6. Fluorescence study 7 1.4.7. Ash analysis 8 1.4.8. Fixed oils 8 1.4.9. Analysis of elemental profile 8 1.5 Pharmacological Bioassays 9 1.5.1. Acute toxicity study 10 1.5.2. Cytotoxic activity 11 1.5.3. Spasmolytic activity 11 1.5.4 Antidiarrheal activity 12 1.5.5. Hepatoprotective activity 12 1.6 Family Sapotaceae 13 1.6.1. In Pakistan 13 1.7 Genus description 14 1.8 Plant description 14 1.9 Distribution in Pakistan 15 1.10 Ethnobotanical uses 16 CHAPTER- 2 18 REVIEW OF LITERATURE 2.1 Review of literature for Monotheca buxifolia (Falc.) A. DC. 18 2.2 Review of literature for other plants of family Sapotaceae 23 2.3 Ethnobotany 26 2.4 Pharmacognostic evaluation 27 2.4.1. Preliminary phytochemical analysis 29 2.4.2. Fluorescence study 30 2.4.3. Ashing 31 2.4.4. Fixed oil analysis 31 2.4.5. Detection of elemental profile 32 2.5 Pharmacological bioassays 33 2.5.1. Acute toxicity 34 2.5.2. Cytotoxicity 36 2.5.3. Spasmolytic activity 39 2.5.4. Antidiarrheal activity 43 2.5.5. Hepatoprotective activity 46 Objectives of study 49 Expected outcomes 50 CHAPTER-3 52 MATERIALS AND METHODS 3.1 Morphological description of the research plant 51 3.2 Ethnobotany 51 3.3 Pharmacognosy 51 3.3.1. Collection 51 3.3.2. Preservation 52 3.3.3. Morphological studies 54 3.3.4. Microscopic studies 54 3.3.4.a Anatomical studies 54 3.3.4.b Features of leaf surface 55 i Occurrence and type of stomata 55 ii Stomatal number and stomatal index 55 iii Palisade ratio 56 iv Vein islet number 57 v Vein termination 57 3.3.5. Powder drug study 58 3.3.6. Preliminary phytochemical tests 58 3.3.6.a. Tests for amino acids and proteins 58 3.3.6.b. Tests for carbohydrates 59 3.3.6.c. Detection of fixed and volatile oils 60 3.3.6.d. Tests for detecting glycosides 60 3.3.6.e. Detection of alkaloids 60 3.3.6.f. Detection tests for tannins 61 3.3.6.g. Tests for presence of phenolic compounds 62 3.3.6.h. Indication of saponin 62 3.3.6.i. Detection of anthocyanins 63 3.3.6.j. Tests for triterpenoids and phytosterols 63 3.3.6.k. Detection of flavonoids 63 3.3.7. Study of florescence characteristics 63 3.3.8. Determination of moisture contents 64 3.3.9. Ash analysis 65 3.3.9.a. Total ash 65 3.3.9.b. Acid insoluble ash 66 3.3.9.c. Water soluble ash 67 3.3.10 GC-MS analysis of fixed oils 68 3.3.11. Elemental profile analysis 69 3.4 Bioassays 73 3.4.1. Acute toxicity analysis 74 3.4.2. Cytotoxicity 75 3.4.3. Spasmolytic activity of Monotheca buxifolia fruit pulp 78 3.4.4. Antidiarrheal activity of leaf and fruit extract of Monotheca 82 buxifolia 3.4.5. Hepatoprotective activity 85 CHAPTER-4 93 RESULTS AND DISCUSSIONS 4.1 Morphological studies of Monotheca buxifolia (Falc.) A. DC. 93 4.2 Ethnobotany 95 4.3 Ethnobotany of Monotheca buxifolia (Falc.) A. DC. 96 4.4 Pharmacognosy 101 4.5 Pharmacognosy of Monotheca buxifolia (Falc.) A. DC. 102 4.5.1. Macroscopy 102 4.5.2. Microscopic studies 110 4.5.2. a. Anatomical studies 110 i. Anatomy of fruit 110 ii. Anatomy of seed 112 iii. Anatomy of leaf 115 iii. a. Anatomy of leaf lamina 115 iii. b. Midrib anatomy of Monotheca buxifolia leaf 116 iv. Anatomy of stem bark 118 v Anatomy of root bark 120 4.5.2. b. Leaf surface characteristics 121 i. Epidermal cells 122 ii. Stomatal occurrence and types 122 iii. Palisade ratio 122 iv. Vein islet number and vein termination number 123 4.5.2. c. Powder drug microscopy 128 i. Powder drug study of fruit pulp 128 ii. Powder drug study of seed 130 iii. Powder drug study of leaf 131 iv. Powder drug microscopy of stem bark 133 v. Powder drug microscopy of root bark 134 4.5.3. Phytochemical profile of Monotheca buxifolia (Falc.) A. DC. 135 4.5.4. Fluorescence studies 144 4.5.5. Moisture contents 148 4.5.6. Ash analysis 149 4.5.7. Fixed oil yield 151 4.5.8. Elemental profile 157 i. Nitrogen (N) 157 ii. Potassium (K) 158 iii. Phosphorus (P) 159 iv. Manganese (Mn) 159 v. Zinc (Zn) 160 vi. Copper (Cu) 161 vii. Iron (Fe) 161 viii. Cadmium (Cu) 162 ix. Lead (Pb) 163 x. Cobalt (Co) 163 xi. Chromium (Cr) 164 xii. Nickel (Ni) 165 4.6. Pharmacological bioassays 173 4.6.1. Acute toxicity 173 4.6.2. Cytotoxicity 175 4.6.3. Effect of Monotheca buxifolia on gut motility 179 4.6.4. Antidiarrheal activity 184 4.6.5. Hepatoprotective activity of Monotheca buxifolia (Falc.) A. 187 DC. CONCLUSIONS AND RECOMMENDATIONS 203 REFERENCES 205 LIST OF TABLES S. No. Tables P. No 3.1. Column oven programming for GC-MS 68 3.2. Standard instrumental conditions required for elemental analysis 72 3.3. Composition of Tyrode‘s solution, potassium-normal Tyrode‘s 78 solution and potassium-rich Tyrode‘s solution 3.4. Experimental design for evaluation of hepatoprotective activity of 87 Monotheca buxifolia 4.1. Altitude and dimensions of the areas where ethnobotanical studies 99 were conducted. 4.2. Ethnobotanical uses of Monotheca buxifolia at four study sites 100 4.3. Macroscopic features of Monotheca buxifolia Fruit 104 4.4. Macroscopic features of Monotheca buxifolia Seed 104 4.5. Characteristic Macroscopic features of Monotheca buxifolia Leaf. 105 4.6. Macroscopic features of Monotheca buxifolia Stem Bark 106 4.7. Macroscopic features of Monotheca buxifolia Root Bark 106 4.8. Leaf constants of Monotheca buxifolia 124 4.9. Preliminary phytochemical profile of Monotheca buxifolia Fruit 139 pulp. 4.10. Preliminary phytochemical profile of Monotheca buxifolia Seed. 140 4.11. Preliminary phytochemical profile of Monotheca buxifolia Leaf. 141 4.12. Preliminary phytochemical profile of Monotheca buxifolia Stem 142 bark 4.13. Preliminary phytochemical profile of Monotheca buxifolia Root 143 bark. 4.14. Fluorescence behaviour of powder drugs of Monotheca buxifolia 145 under different wavelengths of light and with different reagents 4.15. Moisture contents of powder drugs prepared from fruit pulp, leaf, 148 stem bark, root bark and seed of Monotheca buxifolia 4.16. Ash values of Monotheca buxifolia 150 4.17. Fixed oil yield from fruit pulp, seed and leaf of Monotheca 153 buxifolia 4.18. Depicting the concentrations of various nutrient and trace elements 166 in powder drugs prepared from different parts of Monotheca buxifolia 4.19. Acute toxicity of Monotheca buxifolia 175 4.20. Cytotoxic potential of Monotheca buxifolia 177 4.21. Antidiarrheal activity of Monotheca buxifolia fruit pulp and leaf. 185 4.22. Effect of Monotheca buxifolia fruit methanol extract on level of 193 different Biochemical markers against paracetamol (Acetamenophen) induced hepatic injury in rats.
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