Phytochemical Study, Antimicrobial and Antiangiogenic Activities of the Leaf Extracts of Gynura Segetum
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PHYTOCHEMICAL STUDY, ANTIMICROBIAL AND ANTIANGIOGENIC ACTIVITIES OF THE LEAF EXTRACTS OF GYNURA SEGETUM SEOW LAY JING UNIVERSITI SAINS MALAYSIA 2010 PHYTOCHEMICAL STUDY, ANTIMICROBIAL AND ANTIANGIOGENIC ACTIVITIES OF THE LEAF EXTRACTS OF GYNURA SEGETUM by SEOW LAY JING Thesis submitted in fulfillment of the requirements for the Degree of Master of Science August 2010 ACKNOWLEDGEMENT First of all, I would like to express my sincere gratitude to my main supervisor, Professor Mohd Zaini Asmawi for his invaluable guidance, patience and understanding, comments and suggestions throughout the study. My deepest gratitude also goes to my co-supervisors, Associate Professor Norhayati Ismail, Associate Professor Pazilah Ibrahim and Dr. Amin Malik Shah Abdul Majid for their support and guidance. I wish to extend my thanks the Dean of School of Pharmaceutical Sciences, USM for allowing me the full use of facilities and equipments during these years. I would also like to thank Mr. Adenan Jaafar for the taxonomic identification of the plant and Mr. V. Shanmugan (School of Biological Sciences, USM) for his kind help in preparing the plant sample for herbarium. Many thanks go to the academic staffs, laboratory assistants and technicians of the School of Pharmaceutical Sciences, USM for their cooperation and contribution to the success of this study. ii I appreciate the valuable help of Mr. Hilman bin Sulaiman (Center for drug research, USM) for recording GCMS spectra. My thanks go to Razak for his valuable comments in HPLC analysis. My sincere thanks also goes to my friends and labmates to support me especially Beh, Hwee Ching, Rosidah, Afrizal, Vuanghao, Yow Meng, Vicky and Bin Seng for giving hand in solving problem and inspiring working atmosphere during these years. I am indebted to my parents who provided me good education, very supportive and always being there for me. I am deeply grateful to my brothers for their kind support and encouragement. Lastly, I offer my regards and blessings to all of those who have supported me in any respect during these years. iii TABLE OF CONTENTS Page Acknowledgement ii Table of Contents iv List of Tables x List of Figures xi List of Abbreviations xiii List of Appendices xv Abstrak xvi Abstract xviii CHAPTER 1 – INTRODUCTION 1.1 General 1 1.2 Phytochemical and biological activities of genus Gynura 2 1.2.1 Gynura japonica 3 1.2.2 Gynura cusimbua 5 1.2.3 Gynura sarmentosa 7 1.2.4 Gynura bicolor 7 1.2.5 Gynura formosana 8 1.2.6 Gynura divaricata 9 1.2.7 Gynura scandens 13 1.2.8 Gynura elliptica 13 1.2.9 Gynura procumbens 14 iv 1.3 Plant Gynura segetum (Lour.) Merr. 15 1.3.1 Plant taxonomy 15 1.3.2 Plant morphology 17 1.3.3 Plant habitat 17 1.3.4 Plant growth habit/cultivation 18 1.3.5 Uses in traditional medicine 18 1.3.6 Previous work on Gynura segetum 19 1.4 Antimicrobial activity 21 1.4.1 Introduction 21 1.4.2 Kind of antibiotics and their actions 21 1.4.2.1 Gentamicin 22 1.4.2.2 Chloramphenicol 22 1.4.2.3 Penicillin 23 1.4.2.4 Tetracycline 23 1.4.2.5 Ampicillin 23 1.4.2.6 Amphotericin B 24 1.4.3 Antimicrobial susceptibility testing 24 1.5 Antiangiogenic activity 26 1.5.1 Introduction 26 1.5.2 The chick embryo chorioallantoic membrane (CAM) assay 27 1.6 Objectives of the present work 29 v CHAPTER 2 – MATERIALS AND METHODS 2.1 Materials and apparatus used for phytochemical screening, 30 extraction, column chromatography and thin layer chromatography 2.1.1 Chemicals 30 2.1.2 Reagent 30 2.1.2.1 Dragendorff’s reagent 30 2.1.2.2 Liebermann-Burchard reagent 31 2.1.2.3 Natural products reagent 31 2.1.3 Instruments 31 2.2 Materials and apparatus used for HPLC 32 2.3 Materials and apparatus used for GC-MS 32 2.4 Identification of the plant 33 2.4.1 Plant material 33 2.4.2 Phytochemical screening 33 2.4.2.1 Alkaloid test 33 2.4.2.2 Flavonoid test 34 2.4.2.3 Tannins test 34 2.4.2.4 Saponin test 34 2.4.2.5 Anthraquinone derivatives test 34 2.4.2.6 Liebermann-Burchard test for terpenes 35 2.5 Quality control parameter of leaves of Gynura segetum 35 2.5.1 Determination of ash 35 2.5.1.1 Total ash 35 vi 2.5.1.2 Acid-insoluble ash 36 2.5.1.3 Water-soluble ash 36 2.5.2 Determination of extractable matter 36 2.5.2.1 Hot extraction 36 2.5.2.2 Cold extraction 37 2.5.3 Determination of water and volatile matter 37 2.6 Method of extraction 38 2.6.1 Soxhlet extraction 38 2.6.2 Liquid-liquid extraction of methanol extract 40 2.7 Chemical identification of the leaves of Gynura segetum 41 2.7.1 TLC analysis 41 2.7.2 HPLC analysis 41 2.8 Antimicrobial activity 43 2.8.1 Materials and apparatus used for antimicrobial activity 43 2.8.1.1 Chemicals 43 2.8.1.2 Instruments 43 2.8.1.3 Culture medium 43 a) Nutrient agar 43 b) Nutrient broth 44 2.8.2 Microbial strains 44 2.8.3 Test samples 45 2.8.4 Antibiotics 45 2.8.5 Antimicrobial screening of the leaf extracts of Gynura segetum 45 vii 2.8.6 Antibiotics susceptibility 46 2.8.7 Fractionation of the active extract for antimicrobial activity 47 2.8.8 MIC determination of the active samples 47 2.8.9 GC-MS analysis of the active samples 49 2.9 Antiangiogenic screening 50 2.9.1 Materials and apparatus used for antiangiogenic screening 50 2.9.2 Fertilized chicken eggs 50 2.9.3 Sample preparation 50 2.9.4 Antiangiogenic activity of the leaf extracts of Gynura segetum 51 2.9.5 Statistical analysis 54 2.9.6 GC-MS analysis of the active samples 54 CHAPTER 3 – RESULTS AND DISCUSSIONS 3.1 Phytochemical screening 55 3.2 Quality control parameter of the leaves of Gynura segetum 56 3.2.1 Gravimetric study 56 3.2.2 Chemical identification of the leaves of Gynura segetum 57 3.2.2.1 TLC analysis 57 3.2.2.2 HPLC analysis 59 3.3 Antimicrobial activity 63 3.3.1 Antimicrobial screening of the leaf extracts of Gynura segetum 63 3.3.2 Antibiotics susceptibility 67 3.3.3 Antimicrobial activity of the active ethyl acetate fraction 68 viii 3.3.4 MIC determination of the active samples 73 3.3.5 GC-MS analysis of the active samples 76 3.3.6 Discussions 79 3.4 Antiangiogenic activity 83 3.4.1 Antiangiogenic effect of Gynura segetum leaf extracts 83 3.4.2 GC-MS analysis of the active samples 87 3.4.3 Discussions 90 CHAPTER 4 – CONCLUSIONS 95 SUGGESTION FOR FUTURE WORK 97 REFERENCES 98 APPENDICES 106 ix LIST OF TABLES Page Table 2.1 Sample preparation for MIC determination 48 Table 2.2 Score values for the evaluation of the antiangiogenic effect 53 Table 3.1 Results of phytochemical screening 55 Table 3.2 Antimicrobial activity of crude extracts of G. segetum leaves 64 Table 3.3 Antimicrobial activity of fractions from methanol extract of 65 G. segetum leaves Table 3.4 Antimicrobial activity of antibiotics 69 Table 3.5 Antimicrobial activity of ethyl acetate subfractions of 70 G. segetum leaves Table 3.6 Minimum inhibitory concentrations of active ethyl acetate 75 subfractions Table 3.7 The main compounds identified in ethyl acetate fraction 77 Table 3.8 The main compounds identified in subfraction E4 77 Table 3.9 Antiangiogenic effect of G. segetum leaf extracts and its 83 fractions on CAM assay Table 3.10 The main compounds identified in chloroform extract 87 Table 3.11 The main compounds identified in chloroform fraction 87 x LIST OF FIGURES Page Figure 1.1 Compounds from the rhizome of Gynura japonica 4 Figure 1.2 The structures of new cerebrosides from the rhizome 5 of G. japonica Figure 1.3 Major constituents of the oil from the aerial parts of 6 Gynura cusimbua Figure 1.4 Pyrrolizidine alkaloids of Gynura sarmentosa 7 Figure 1.5 The structures of four phenolics from Gynura formosana 8 Figure 1.6 Two alkaloids from Gynura divaricata 9 Figure 1.7 Two pheophorbides from G. divaricata subsp. Formosana 10 Figure 1.8 Ten compounds from above ground parts of G. divaricata 11 Figure 1.9 Compounds from G. divaricata 12 Figure 1.10 Pyrrolizidine alkaloids from Gynura scandens 13 Figure 1.11 Structure of 6-acetyl-2, 2-dimethylchroman-4-one 13 Figure 1.12 Plant Gynura segetum (Lour.) Merr. 16 Figure 1.13 Flower of Gynura segetum (Lour.) Merr. 16 Figure 1.14 Alkaloids from G. segetum 20 Figure 2.1 Schematic representation of extraction of leaves of G. segetum 39 Figure 2.2 Schematic representation of liquid-liquid extraction of 40 methanol extract of leaves of G. segetum Figure 2.3 CAM assay: A) Window made in the egg shell of a 3-day 53 old chick embryo; B) diagram of the technique; C) Filter paper discs with the test substances placed manually by microsurgical forceps onto the CAM of a 5-day old chick embryo xi Figure 3.1 TLC profiles of crude methanol extract of the leaves of 58 G. segetum and reference standard rutin (R) Figure 3.2 HPLC analysis of methanol extract 60 Figure 3.3 HPLC analysis of standard rutin 61 Figure 3.4 HPLC analysis of mixture of methanol extract and rutin 62 Figure 3.5 The chemical structure of identified compounds in ethyl acetate 78 fraction and subfraction E4 of G.