Phytochemical and Biological Studies of Ravenea Rivularis Family Arecaceae "Palmae"
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Ain Shams University Faculty of Pharmacy Pharmacognosy Dept. 2016 Phytochemical and Biological Studies of Ravenea rivularis Family Arecaceae "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 -اﻵية 27( وَما ُأويتي ُُت ي م َن الْ يع ْْل ا ََّّل قَ يلي ًَل َ ي ِ )سورة اﻹسراء - اﻵية 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 plant. 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. Taxonomy 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 i 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 ii 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 iii 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).