Pharmacognostical Study of laurifolia Cultivated in Egypt (Family )

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

ــ بِس م ﷲ الرحمــن الرحيــــم ‘‘قَالُوا ُس َبحان َــ َك ال ِع ْل َم لَنـ ا إال َما َعلﱠ ْمت َـنــ َا إنﱠ َك َأنت َالعلِيــ ُم َالح ِكيــ ُم ’’ صدق ﷲ العظيــــم سورة البقرة ،االية (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. 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

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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

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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.

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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. 113 13 29 C-NMR spectrum (CDCl3, δ ppm) of compound M3. 114

30 HMBC spectrum (CDCl3, δ ppm) of compound M3. 115 1 31 H-NMR spectrum (CDCl3, δ ppm) of compound M4. 118 13 32 C-NMR spectrum (CDCl3, δ ppm) of compound M4. 119 1 33 H-NMR spectrum (DMSO, δ ppm) of compound M5. 122 13 34 C-NMR spectrum (DMSO, δ ppm) of compound M5. 123 Scheme for the chromatographic fractionation of the ethyl 35 129 acetate fraction of Mimusops laurifolia leaves.

36 Positive EI/MS of compound M6. 132

vii no. Figure Page

37 Negative ESI/MS of compound M7. 135

38 Negative ESI/MS of compound M8. 138

39 Positive ESI/MS of compound M9. 142 1 40 H-NMR spectrum (DMSO, δ ppm) of compound M9. 143 13 41 C-NMR spectrum (DMSO, δ ppm) of compound M9. 144

42 Positive ESI/MS of compound M10. 148 1 43 H-NMR spectrum (DMSO, δ ppm) of compound M10. 149 13 44 C-NMR spectrum (DMSO, δ ppm) of compound M10. 150

45 Positive ESI/MS of compound M11. 154 1 46 H-NMR spectrum (CD3OD, δ ppm) of compound M11. 155 13 47 C-NMR spectrum (CD3OD, δ ppm) of compound M11. 156

48 Positive ESI/MS of compound M13. 159

49 Positive ESI/MS of compound M14. 163 1 50 H-NMR spectrum (DMSO, δ ppm) of compound M14. 164 13 51 C-NMR spectrum (DMSO, δ ppm) of compound M14. 165 HPLC chromatogram of total ethanolic extract of leaves of 52 167 Mimusops laurifolia. HPLC chromatogram of standard myricetin-3-O-α-L- 53 167 rhamnopyranoside (M10). Calibration curve of myricetin-3-O-α-L-rhamnopyranoside 54 168 (M10) at 366 nm.

viii

List of Abbreviations

Abbreviation b. wt. Body weight Ca. Calcium Cc Column chromatography 13C-NMR 13Carbon-nuclear magnetic resonance Conc. Concentration Cpd. Compound dec. Decrease DMSO Dimethyl sulphoxide d.wt. Dry weight EI/MS Electron impact/Mass spectroscopy ESI/MS Electrospray ionization/Mass spectroscopy Eth. ac. Ethyl acetate eV Electron volt fr. Fraction GLC Gas liquid chromatography 1H-NMR 1Proton nuclear magnetic resonance hr. Hour 2-Dimensional Heteronuclear Multiple Bond HMBC Correlations HPLC High-performance liquid chromatography λ max Maximum wavelength LD50 Lethal dose to kill 50 % of animals M+ Molecular ion peak m.p. Melting point meth. Methanol min. Minute m/z Mass/Charge No. Number PC Paper chromatography PCR Polymerase chain reaction Ppm Part per million Rf Relative factor Rt Retention time RDA Recommended dietary allowances S.E. Standard error sh. Shoulder TLC Thin Layer Chromatography UV Ultraviolet VLC Vacuum Liquid Chromatography

ix

Introduction

1. Introduction

Mimusops laurifolia (Forssk.) Friis (family Sapotaceae) is an evergreen tree about 15-20 meters high, with small white fragrant flowers and small rounded or ovoid green edible sweet fruits. Its common name .in Arabic البرساء is Persea or The Sapotaceae represents a well-marked family of flowering with a considerable economic importance as a source of latex which has been used as gutta-percha and chewing gum, hard and durable wood, and many edible fruits (Metcalfe and Chalk, 1950; Pennington, 1990). Although genus Mimusops is widely distributed in India, Mimusops laurifolia presence in nature is restricted to Gulf of Aden and countries around the Red Sea specially Eritrea, Ethiopia and Somalia, though, it was cultivated successfully in other countries as Egypt, Uganda and the Sudan (Friis, 1980). Mimusops laurifolia was imported to Egypt since Pharos times; it is frequently found in tombs in Egypt from the First Dynasty onwards. The largest find of Mimusops laurifolia from ancient Egypt consists of several very large funeral bouquets of leafy twigs from the tomb of Tut-ankh- Amen (Friis, 1980). It was also discovered in a garland at the Roman tomb in Abu Rawash (Hammouda and Fahd, 1988). This regained popularity since its extracts were patented for skin-conditioning and -moisturizing effects, as part of preparations used in cosmetics, bath formulations and detergents (Ohara et al., 2001). The primary compounds of interest in Sapotaceae family are the triterpenes and triterpene glycosides. Many isolated triterpenes from this family as; betulinic acid and ursolic acid (Misra and Mitra, 1968; Jahan et al., 1995 A) were reported as potent hepatoprotective and antitumour

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