Ain Shams University Faculty of Pharmacy Pharmacognosy Dept. 2016 Phytochemical and Biological Studies of rivularis Family "Palmae"

A Thesis In partial fulfillment for the requirements of Master's Degree in Pharmaceutical Sciences (Pharmacognosy)

Submitted by Sherouk Hussein Abdullah Ali Sweilam B.Sc. of Pharmaceutical Sciences Helwan University, 2009

Supervised by Prof. Dr. Abdel Nasser B. Singab. Professor of Pharmacognosy, Dean of Faculty of Pharmacy, ASU Prof. Dr. Ibrahim I. Mahmoud. Professor of Pharmacognosy, Dean of Faculty of Pharmacy, ACU Associate Prof. Dr. Mohamed R. El-Gindi. Associate Professor of Pharmacognosy, Head of Pharmacognosy Department, Faculty of Pharmacy, ERU

َ َولَ ْو َأنََّما يِف اْ َْل ْر يض يمن ََش َرٍة َأ ْق ََل ٌم َوالْ َب ْح ُر يَ ُم ُّد ُه يمن بَ ْع يد يه س ب َع ُة َأ ْ ُْب ٍر َّما نَ يف َد ْت َيَكما ُت ا َّ يَّلل ا َّن ا َّ ََّلل َع يزي ٌز َح يك ٌي َ ْ َ ِ )سورة لقمان -اآلية 27(

وَما ُأويتي ُُت ي م َن الْ يع ْْل ا ََّّل قَ يلي ًَل َ ي ِ )سورة اإلسراء - اآلية 85(

Dedication

To …. My Father, my Mother and my brothers

Whom I owe them all that I have become and that

I will ever be.

Acknowledgment Acknowledgment

First and foremost, I feel always indebted to Allah, the most kind and the most Merciful thanks to who made me able to accomplish this work.

I would like to express my deepest gratitude and appreciation to Professor Dr. Abdel Nasser B. Singab, Dean of Faculty of Pharmacy, Ain-Shams University, for the continuous support and research, for his patience and for his supervision of this study.

I wish to express my deepest thanks, heartfelt appreciation and endless gratitude to Professor Dr. Ibrahim I. Mahmoud, Dean of Faculty of Pharmacy, Ahram Canadian University, for motivation, enthusiasm, and immense knowledge during this study.

I would like to express my deepest gratitude and sincere appreciation to Assoc. Prof. Dr. Mohamed R. El-gindi, Head of Pharmacognosy Department, Faculty of Pharmacy, Egyptian Russian University, for his guidance helped me in all the time of research and writing of this thesis.

I am thankful to Professor Dr. Ehab Fattoh, Dean of Faculty of Pharmacy, Egyptian Russian University, for the support and help along academic field.

Alongside, I would thank Professor Dr. Omayma Dawood El-gindi, Professor of Pharmacognosy, Faculty of Pharmacy, Egyptian Russian University.

Practically and personally, I couldn't imagine that I would have a better advisor and mentor for my life a godfather as Professor Dr. Fathy A. Ibrahim, Professor of Pharmaceutics and Pharmacokinetics, Faculty of pharmacy, Egyptian Russian University.

Gratifyingly, I would thank Assoc. Prof. Dr. Omayma Eldahshan, Acting Head of Pharmacognosy Department, Faculty of Pharmacy, Ain Shams University from my deepest heart for her gratitude.

I owe a deep sense of gratitude and thank to Dr. Mohamed Ibrahim, Lecturer of Pharmacognosy, Faculty of Pharmacy, Helwan University. Acknowledgment

The most thankful to the utmost respectful Dr. Mohamed Ashour, Assoc. Professor Doctor of Pharmacognosy, Faculty of Pharmacy, Ain Shams University.

I thank my workmates at Egyptian Russian University otherwise, my sincere and thanks to you Dr. Mohamed Al-Shazley, Lecturer of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, and Dr. Dina Al-Nagaar Lecturer of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University.

I am thankful to Madam Trease Labib, Taxonomist of the Orman Botanical Garden Herbarium, Giza, Egypt, for authenticating the identity of the . And it's my privilege to thank my household, the initial factor what's made me who I am today, and my close friend, Radwa Mouhammed.

And I quote: "Both living and dead, and how earnestly I must exert myself in order to give in return as much as I have received.” Albert Einstein. Thank you, and I do respect all of you. May Allah bless upon you.

Sherouk Hussein

Contents

Glossary Page no. I. Introduction 1 II. II.1. Taxonomical features of family Arecaceae 3 II.2. Taxonomical features of Genus Ravenea 3 II.3. Taxonomical features of Ravenea rivularis 3 III. Literature Review III.1. Reported Isolated Phytoconstituents from Family Arecaceae 7 III.2. Reprted Biological Activities of Family Arecaceae 114 IV. Material, Apparatus and Methods IV.1 Material IV.1.1. Material for DNA profiling 131 IV.1.2. Material for the phytochemical investigation 133 IV.1.3. Material for biological assays 136 IV.1.4 Material for investigation of lipoidal matter 137 IV.2 Apparatus IV.2.1. Apparatus for DNA profiling 138 IV.2.2. Apparatus for the phytochemical investigation 138 IV.2.3. Apparatus for biological assay 139 IV.2.4. Apparatus for investigation of lipoidal matter 140 IV.3 Methods IV.3.1. Method for DNA profiling 141 IV.3.2. Methods for the phytochemical investigation 143 IV.3.3. Methods for biological assay 149 IV.3.4 Method used for investigation of lipoidal matter 151 1. DNA Profiling 1.15.1 Introduction 153 1.2 Results and Discussions 153 2. Investigation of lipoidal matter 2.1. Identification of the unsaponifiable matters (USM) 158 2.2. Identification of saponifiable matter (fatty acid methyl esters) 160 3. Phytochemical Screening and Investigation of the leaves of Ravenea rivularis Jum. & H. Perrier 3.1 Preliminary Phytochemical Screening of the leaves of Ravenea 162 rivularis Jum. & H. Perrier: 3.2 Investigation of several extracts of the leaves of Ravenea rivularis 163 Jum. & H. Perrier 3.2(H) Phytochemical investigation of the n-hexane extract of the 165 leaves of Ravenea rivularis 3.2.E Phytochemical investigation of the ethyl acetate extract of the 180 leaves of Ravenea rivularis 3.2.B Phytochemical investigation of the n-butanol extract of leaves 218 of Ravenea rivularis

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Contents

Glossary Page no. 4. Biological Screening of Ravenea rivularis 4.1. Antioxidant activity 4.1.1. Scavenging of DPPH radicals. 225 4.1.2. Results of antioxidant activity 225 4.1.3. Discussion of the results of antioxidant activity 227 4.2. Anti- inflammatory Activity 4.2.1. Estimation of nitric oxide 228 4.2.2. Results of anti-inflammatory activity 228 4.2.3. Discussion of the results of cytotoxicity and nitric oxide index 231 4.3. Cytotoxic Activity against Tumor Cell Line 4.3.1. Results of Cytotoxic Activity 231 4.3.2. Discussion of the results of cytotoxic activity 233 Conclusions and Recommendations 234 General Summary 235 References 242 Arabic Summary

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List of Figures

No. Figure no. Figure name Page 1 Figure 1 Photograph of Ravenea rivularis Jum. & Perrier palm 6 2 Figure 2 Photograph of Ravenea rivularis Jum. & Perrier leaves 6 3 Figure 3 The obtained RAPD-PCR products for Ravenea rivularis using 155 ten decamer primers. 4 Figure 4 GLC of Unsaponifiable Matter (USM) of R. rivularis leaves 159 5 Figure 5 GLC of saponifiable matter (fatty acid methyl esters) of Ravenea 161 rivularis leaves 6 Figure 6 Flow chart of extraction and fractionation of Ravenea rivularis 164 leaves 7 Figure 7 Structure of Lup-20(29)-en-3β-yl acetate [Lupeol acetate] 169 1 8 Figure 8 H-NMR spectrum of compound (1) in CDCL3, 400 MHz 170 13 9 Figure 9 C-NMR spectrum of compound (1) in CDCL3, 100 MHz 171 10 Figure 10 EI-MS spectrum of compound (1) 172 11 Figure 11 Structure of 3β-hydroxy-lup-20-en-28-oic acid [Betulinic acid] 176 1 12 Figure 12 H NMR spectrum of compound (2) in CDCL3, 400 MHz 177 13 13 Figure 13 C-NMR spectrum of compound (2) in CDCL3, 100 MHz 178 14 Figure 14 EI-MS spectrum of compound (2) 179 15 Figure 15 Structure of Apigenin [5, 7, 4'-Trihydroxyflavone] 186 1 16 Figure 16 H NMR spectrum of compound (3) in DMSO-d6, 400 MHz 187 13 17 Figure 17 C NMR spectrum of compound (3) in DMSO-d6, 100 MHz 188 18 Figure 18 EI-MS spectrum of compound (3) 189 19 Figure 19 Structure of 4-hydroxy-3-methoxy Cinnamic acid [Ferulic acid] 192 1 20 Figure 20 H NMR spectrum of compound (4) in CD3OD, 400 MHz 193 13 21 Figure 21 C NMR spectrum of compound (4) in CD3OD, 100 MHZ 194 22 Figure 22 EI. MS spectrum of compound (4) in CD3OD, 100 MHz 195 23 Figure 23 Structure of Luteolin [5, 7, 3', 4'-Tetra hydroxyflavone] 199 1 24 Figure 24 H NMR spectrum of compound (5) in DMSO-d6, 400 MHz 200 13 25 Figure 25 C NMR spectrum of compound (5) in DMSO-d6, 100 MHz 201 26 Figure 26 EI-MS spectrum of compound (5) 202 27 Figure 27 Structure of Luteolin-7-O-β-D-glucopyranoside [Luteolin-7-O- 208 glucoside] 1 28 Figure 28 H NMR spectrum of compound (6) in DMSO-d6, 400 MHz 209 13 29 Figure 29 C NMR spectrum of compound (6) in DMSO-d6, 100 MHz 210 30 Figure 30 Structure of 3(3, 4-Dihydroxyphenyl)-prop-2-enoic acid [Caffeic 214 acid] 1 31 Figure 31 H NMR spectrum of compound (7) in CD3OD, 400 MHz 215 13 32 Figure 32 C-NMR spectrum of compound (7) in CD3OD, 100 MHz 216 33 Figure 33 EI-MS spectrum of compound (7) 217 34 Figure 34 Structure of 3-Caffeoylquinic acid [Chlorogenic acid] 222

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List of Figures

No. Figure no. Figure name Page 1 35 Figure 35 H NMR spectrum of compound (8) in D2O, 400 MHz 223 13 36 Figure 36 C-NMR spectrum of compound (8) in D2O, 100 MHz 224 37 Figure 37 Histogram representing the antioxidant activity against DPPH 225 radicals (Calculated SC50 for ascorbic acid and the tested samples). 38 Figure 38 Linear graph representing the antioxidant activity through 226 absorbance of the ascorbic acid and individual extract vs. conc. showing different antioxidant activities. 39 Figure 39 Standard curve of NO. 228 40 Figure 40 Linear gragh representing the secreted concentration of NO 229 (µmole/ml) related to serial concentrations of the used samples µg/ml). 41 Figure 41 Histogram representing the anti-inflammatory activity through 230 the level of Nitric oxide in RAW 264.7 cells supernatant after the treatment with the samples (125 µg/ml) for 24 hours compared with LPS-treated cells, as measured by Griess assay. 42 Figure 42 Histogram representing the anti-inflammatory activity through 230 the percentage of inhibition of Nitric oxide in LPS-stimulated RAW 264.7 cells supernatant after the treatment with the different concentrations of methanol extract (62.5-250 µg/ml) for 24 hours compared with LPS-treated cells, as measured by Griess assay. 43 Figure 43 Linear graph representing the cytotoxic effect of Sample against 232 MCF-7 cells using MTT assay (n=4), data expressed as the mean value of cell viability (% of control) ± S.D 44 Figure 44 Linear graph representing the cytotoxic effect of sample against 233 Hep-G2 cells using MTT assay (n=4), data expressed as the mean value of cell viability (% of control) ± S.D. 45 Figure 45 Extraction and isolation scheme of several compounds from 240 Ravenea rivularis

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List of Tables No. Table no. Table name Page 1 Table 1 Taxonomical position of Ravenea rivularis 4 2 Table 2 Description of Ravenea rivularis (Majestic palm) 4 3 Table 3 Chemical names and structures of flavonoids and related 7 compounds isolated from family Arecaceae 4 Table 4 Chemical names and structures of catechins and related 28 compounds isolated from family Arecaceae 5 Table 5 Chemical names and structures of phenolic acids and related 39 compounds isolated from family Arecaceae 6 Table 6 Chemical names and structures of sterols and related compounds 47 isolated from family Arecaceae 7 Table 7 Chemical names and structures of alkaloids isolated from 72 Family Arecaceae 8 Table 8 Chemical names and structures of carbohydrates isolated from 74 family Arecaceae 9 Table 9 Chemical names and structures of vitamins, carotenoids and 77 related compounds isolated from family Arecaceae 10 Table 10 Chemical names and structures of the essential oils isolated 83 from family Arecaceae 11 Table 11 Chemical names and structures of fatty acids and related 90 compounds isolated from family Arecaceae 12 Table 12 Several reported biological activities from family Arecaceae 114 13 Table 13 Traditional uses reported from family Arecaceae 129 14 Table 14 Sequence of the ten decamer arbitrary primers assayed in RAPD- 132 PCR 15 Table 15 Solvent systems used in chromatography techniques 135 16 Table 16 Molecular sizes in base pairs of amplified DNA fragments 155 produced by ten decamer primers in Ravenea rivularis Jum. &H. Perrier 17 Table 17 Total numbers of RAPD-PCR fragments in Ravenea rivularis 157 Jum. & H. Perrier 18 Table 18 GLC analysis of hydrocarbons and sterols in USM of Ravenea 158 rivularis Jum. & H. Perrier 19 Table 19 GLC analysis of fatty acid esters of Ravenea rivularis 160 20 Table 20 Results of phytochemical screening of Ravenea rivularis Jum. & 162 H. Perrier leaves 21 Table 21 Weights of different extracts obtained from Ravenea rivularis 163 Jum. & H.Perrier for chemical and biological investigations 22 Table 22 Fractions eluted from silica gel column 1 and their weights 165 23 Table 23 Fractions eluted from sub-column 1 and their weights 166 24 Table 24 1H NMR and 13C NMR spectral data of Compound (1) 167 25 Table 25 Data of compound (1) 168 26 Table 26 Fractions eluted from sub-column 2 and their weights 173 27 Table 27 1H NMR and 13C NMR spectral data of Compound (2) 174

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List of Tables

No. Table no. Table name Page 28 Table 28 Data of compound (2) 175 29 Table 29 Fractions eluted from silica gel column A and their weights 181 30 Table 30 Fractions eluted from sub-column 3 and their weights 182 31 Table 31 Fractions eluted from sub-column 4 and their weights 183 32 Table 32 1H NMR and 13C NMR spectral data of Compound (3) 184 33 Table 33 Data of compound (3) 184 34 Table 34 Fractions eluted from sub-column 5 and their weights 190 35 Table 35 1H NMR and 13C NMR spectral data of Compound (4) 190 36 Table 36 Data of compound (4) 191 37 Table 37 Fractions eluted from sub-column 6 and their weights 196 38 Table 38 Fractions eluted from sub-column 7 and their weights 196 1 13 39 Table 39) H NMR and C NMR spectral data of Compound (5) 197 40 Table 40 Data of compound (5) 197 41 Table 41 Fractions eluted from silica gel column B of the second portion 203 (B) and their weights 42 Table 42 Fractions eluted from sub-column 8 and their weights 204 1 13 43 Table 43 H NMR and C NMR spectral data of Compound (6) 205 44 Table 44 Data of compound (6) 206 45 Table 45 Fractions eluted from sub-column 9 and their weights 211 46 Table 46 Fractions eluted from sub-column 10 and their weights 211 47 Table 47 1H NMR and 13C NMR spectral data of Compound (7) 212 48 Table 48 Data of compound (7) 212 49 Table 49 Fractions eluted from silica gel column 2 and their weights 218 50 Table 50 Fractions eluted from sub-column 11 and their weights 219 51 Table 51 Fractions eluted from sub-column 12 and their weights 220 52 Table 52 1H NMR and 13C NMR spectral data of Compound (8) 220 53 Table 53 Data of compound (8) 221 54 Table 54 Cytotoxic effect of different concentrations of methanolic extract 231 against MCF-7 cells using MTT assay (n=4), data expressed as the mean value of cell viability (% of control) ± S.d. 55 Table 55 Cytotoxic effect of different concentrations of methanolic extract 232 against Hep-G2 cells using MTT assay (n=4), data expressed as the mean value of cell viability (% of control) ± S.d.

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Abbreviations

 Chemical Shift Relative to a standard (e.g. TMS) λ Wave length (nm) 1H NMR Proton Nuclear Magnetic Resonance 13C NMR Carbon 13 Nuclear Magnetic Resonance 2DPC Two Dimensional Paper Chromatography ↑ Increase α Alpha BAW n-Butanol: Acetic acid: Water BPH Benign Prostatic Hyperplasia CC Column Chromatography CDCL3 Deutrated Chloroform CD3OD Deutrated Methanol CH2Cl2 Dichloromethane CHCl3 Chloroform Co. Company Co-PC Co-Paper Chromatography C.V.D Cardiovascular Disease d Doublet dd Double Doublet D2O Deutrated water DL Day Light DMSO Dimethyl Sulphoxide DMSO-d6 Deturated Dimethyl Sulphoxide DNA Deoxyribonucleic acid DPPH 2, 2-Diphenyl-1-picrylhydrazine (1, 1-Diphenyl-2-picrylhydrazyl radical, 2, 2-Diphenyl-1-(2, 4, 6- trinitrophenyl) hydrazyl) EI-MS Electron Impact Mass Spectrum ESR Erythrocyte Sedimentation Rate FBS Fetal Bovine Serum FeCl3 Ferric Chloride FID Flame Ionization Detector Fr. No. Fraction Number GLC Gas Liquid Chromatography H3BO3 Boric Acid HCL Hydrochloric Acid HepG2 Human Hepatocarcinoma Cells Hz Hertz I2/KI Iodine/ Potassium Iodide IC50 Half Maximal Inhibitory Concentration J value Coupling Constant KCl Potassium Chloride LPS Lipopolysaccharide m Multiplet m/z Mass to Charge Ratio MCF-7 Human Breast Adenocarcinoma Cells MeOH Methanol

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