Pharmacognostical Study of Mimusops laurifolia Cultivated in Egypt (Family Sapotaceae) Thesis Submitted by Mai Mohamed Hasan Raslan Assistant lecturer of Pharmacognosy, Faculty of Pharmacy, Nahda University, Bani-Sweif (NUB). For the Partial Fulfillment of the Requirements for the Doctoral of Philosophy in Pharmaceutical Sciences (Pharmacognosy) Under the supervision of Prof. Dr. Mohamed S. Hifnawy Professor of Pharmacognosy, Dean, Faculty of Pharmacy, Nahda University, Bani-Sweif (NUB). Assoc. Prof. Dr. Dr. Nadia M. Sokkar Shahira M. Ezzat Assoc. Professor of Pharmacognosy Lecturer of Pharmacognosy, Faculty of Pharmacy, Faculty of Pharmacy, Cairo University. Cairo University. Pharmacognosy Department Faculty of Pharmacy Cairo University A.R.E. 2011 بِســ م ﷲ الرحمــن الرحيــــم ‘‘قَالُوا ُس َبحان َــ َك ﻻ ِع ْل َم لَنـ ا إﻻ َما َعلﱠ ْمت َـنــ َا إنﱠ َك َأنت َالعلِيــ ُم َالح ِكيــ ُم ’’ صدق ﷲ العظيــــم سورة البقرة ،اﻻية (٣٢) Acknowledgement First of all I would like to thank almighty God (Allah) for all his blessings and guidance at every stage of my life. Allah always blessed me with courage, support and strength to complete this work. Neither words nor the available space can describe my greatest appreciation and sincere gratitude to Prof. Dr. Mohamed Said Hifnawy, Professor of Pharmacognosy, Dean of Faculty of Pharmacy, Nahda University, Bani-Sweif (NUB), for his continuous support and valuable advice. I am grateful to Assoc. Prof. Dr. Nadia Mohamed Sokkar, Associate Professor of Pharmacognosy, Faculty of Pharmacy, Cairo University for her supervision, continuous encouragement and concern. My deep gratitude and thanks are devoted to Dr. Shahira Mohamed Ezzat, Lecturer of Pharmacognosy, Faculty of Pharmacy, Cairo University, for her supervision, extensive effort done during this study and continuous support by all means. My sincere love and everlasting gratitude are dedicated to Prof. Dr. Seham Salah El-Din El-Hawary, Professor of Pharmacognosy, Faculty of Pharmacy, Cairo University, to whom words are not enough to describe her limitless kindness, continuous encouragement and support. I would like to expresse my sincere thanks to everyone helped me and supplied me with the facilities during this work, in particular; Prof. Dr. Amany Sleem, Professor of Pharmacology, National Research Center, Dr. Josline Yehia Salib, Assistant Professor of Chemistry of Tanning Materials and Leather, Technology Department, National Research Center, Dr. Hosam Mokhtar, Lecturer of Pharmacognosy, Faculty of Pharmacy, Bani-Sweif University and Dr. Mohamed Elgebaly, Senior Taxonomist. Special thanks are owed to all my colleagues in Faculty of Pharmacy, Nahda University, Bani-Sweif (NUB), for their cooperation and encouragement during this study. Finally, I would like to convey my deepest gratitude to my beloved family and daughter; Nour, for their continuous love, support and encouragement. They always give me the pleasure to live. Mai Raslan TToo MMyy LLoovveellyy DDaauugghhtteerr NNOOUURR You grow more precious day by day and fill my heart with pride Contents Subject Page 1. Introduction 1 2. Taxonomy 3 3. Review of literature 3.1. Review on the chemical constituents 6 3.2. Review on the biological activity 19 4.Aim of the study 22 5. Materials, Apparatus & Techniques 5.1. Materials 23 5.2. Apparatus 28 5.3. Techniques 29 6. Botanical Study of Mimusops laurifolia 6.1. Macromorphology of Mimusops laurifolia 33 6.2. Micromorphology of Mimusops laurifolia 37 6.3. DNA fingerprinting of Mimusops laurifolia 76 7. Biological Study of Mimusops laurifolia 7.1. In-vivo biological studies of Mimusops laurifolia 83 7.2. In-vitro cytotoxic activity against liver cancer 90 8. Phytochemical Study of Mimusops laurifolia 8.1. Preliminary phytochemical screening 94 8.2. Investigation of the n-hexane fraction of Mimusops laurifolia leaves and fruits 96 i i Subject Page 8.3. Investigation of the ethyl acetate fraction of Mimusops laurifolia leaves 124 8.4. Chemical characterization and nutritive value of the leaves and fruits of Mimusops laurifolia 170 9. Summary 175 10. Recommendations 185 11. References 186 12. Arabic summary ii List of Tables no. Table Page Structures of triterpenes previously isolated 1 8 from genus Mimusops. Structures of sterols and their glycosides 2 11 previously isolated from genus Mimusops. Structures of flavonoids and their glycosides 3 13 previously isolated from genus Mimusops. Structures of phenolic acids and tannins 4 14 previously isolated from genus Mimusops. Name and sequences of the selected random 5 26 primers used in RAPD-PCR analysis. Conditions for GLC analysis of the fatty acid 6 30 methyl esters and unsaponifiable matter. Conditions for HPLC analysis of flavonoidal 7 31 compounds. Conditions for HPLC analysis of vitamin C and 8 32 vitamin E. Microscopical measurements of the different 9 elements of the organs of (Forssk.) Friis in 74 microns (μm). 10 Components of RAPD-PCR mixture. 77 11 PCR program (temperature profile). 77 12 RAPD polymorphic bands of the marker; Axygen 80 RAPD polymorphic bands of Mimusops 13 81 laurifolia, produced by ten primers. Effect of ethanolic extracts of Mimusops laurifolia fruits, leaves and its different fractions 14 (100 mg/kg), and silymarin drug (25 mg/kg) on 86 Aspartate Amino Transferase (AST) in liver damaged rats. Effect of ethanolic extracts of Mimusops laurifolia fruits, leaves and its different fractions 15 (100 mg/kg), and silymarin drug (25 mg/kg) on 87 Alanine Amino Transferase (ALT) in liver damaged rats. iii no. Table Page Effect of ethanolic extracts of Mimusops laurifolia fruits, leaves and its different fractions 16 (100 mg/kg), and silymarin drug (25 mg/kg) on 88 Alkaline Phosphatase (ALP) in liver damaged rats. IC50 of the ethanolic extract of Mimusops 17 laurifolia leaves and its fractions on liver cancer 92 cell lines (HEPG-2) Results of preliminary phytochemical screening 18 95 of different organs of Mimusops laurifolia. Results of the GLC analysis of the 19 unsaponifiable matter of Mimusops laurifolia 98 leaves. Results of the GLC analysis of the 20 unsaponifiable matter of Mimusops laurifolia 99 fruits. Results of the GLC analysis of fatty acid methyl 21 100 esters of Mimusops laurifolia leaves. Results of the GLC analysis of fatty acid methyl 22 101 esters of Mimusops laurifolia fruits. Results of TLC investigation of the n-hexane fractions of Mimusops laurifolia leaves and 23 103 fruits, using solvent system; chloroform/methanol (9.5:0.5 v/v). 24 Data of compound M1. 107 25 Data of compound M2. 109 26 Data of compound M3. 111 27 Data of compound M4. 116 28 Data of compound M5. 120 Results of PC investigation of the ethyl acetate 29 fractions of Mimusops laurifolia leaves and 125 fruits, using solvent system; n-butanol/acetic acid/water (4:1:5 v/v/v). 30 Data of compound M6. 130 31 Data of compound M7. 133 iv no. Table Page 32 Data of compound M8. 136 33 Data of compound M9. 139 34 Data of compound M10. 145 35 Data of compound M11. 151 36 Data of compound M12. 157 37 Data of compound M13. 158 38 Data of compound M14. 160 Responses (peak area) of the different 39 concentrations of standard myricetin-3-O-α-L- 168 rhamnopyranoside (M10). Percentages of amino acids content in the leaves 40 172 and fruits of Mimusops laurifolia Vitamin content of the leaves and fruits of 41 174 Mimusops laurifolia. v v List of Figures no. Figure Page Structures of saponins previously isolated from leaves of 1 17 Mimusops laurifolia. Structures of saponins previously isolated from seeds of 2 18 Mimusops laurifolia. 3 Photographs of different parts of Mimusops laurifolia tree 36 Micromorphology of the old branch of Mimusops 4 42 laurifolia. Micromorphology of the young branch of Mimusops 5 44 laurifolia. Micromorphology of the powdered branch of Mimusops 6 46 laurifolia. Micromorphology of the leaf lamina of Mimusops 7 52 laurifolia. Micro morphology of the powdered lamina of Mimusops 8 54 laurifolia. 9 Micromorphology of the petiole of Mimusops laurifolia. 58 Micromorphology of the powdered petiole of Mimusops 10 60 laurifolia. Micromorphology of the fruit stalk of Mimusops 11 65 laurifolia. Micromorphology of the powdered fruit stalk of Mimusops 12 67 laurifolia. 13 Micromorphology of the pericarp of Mimusops laurifolia. 71 Micromorphology of the powdered pericarp of Mimusops 14 73 laurifolia. RAPD electrophoretic profile of Mimusops laurifolia 15 79 generated by the ten primers. 16 Standard molecular weight curve of Mimusops laurifolia. 80 Effect of ethanolic extracts of Mimusops laurifolia fruits, leaves and its different fractions (100 mg/kg), and 17 86 silymarin drug (25 mg/kg) on Aspartate Amino Transferase (AST) in liver damaged rats. vi no. Figure Page Effect of ethanolic extracts of Mimusops laurifolia fruits, leaves and its different fractions (100 mg/kg), and 18 87 silymarin drug (25 mg/kg) on Alanine Amino Transferase (ALT) in liver damaged rats. Effect of ethanolic extracts of Mimusops laurifolia fruits, leaves and its different fractions (100 mg/kg), and 19 88 silymarin drug (25 mg/kg) on Alkaline Phosphatase (ALP) in liver damaged rats. Cytotoxicity of the positive samples on liver cancer cell 20 92 line (HEPG-2). Chromatogram of the GLC analysis of the unsaponifiable 21 matter of the n-hexane fraction of Mimusops laurifolia 98 leaves. Chromatogram of the GLC analysis of the unsaponifiable 22 matter of the n-hexane fraction of Mimusops laurifolia 99 fruits. Chromatogram of the GLC analysis of the fatty acid 23 methyl esters of the n-hexane fraction of Mimusops 100 laurifolia leaves. Chromatogram of the GLC analysis of the fatty acid 24 methyl esters of the n-hexane fraction of Mimusops 101 laurifolia fruits. Scheme for the chromatographic fractionation of the n- 25 106 hexane extract of Mimusops laurifolia leaves 26 Positive EI/MS of compound (M1). 108 27 Positive EI/MS of compound (M2). 110 1 28 H-NMR spectrum (CDCl3, δ ppm) of compound M3.