A Comparative Pharmacognostical Study of Certain Clerodendrum Species (Family Lamiaceae) Cultivated in Egypt A Thesis Submitted By Asmaa Mohamed Ahmed Khalil For the Degree of Master in Pharmaceutical Sciences (Pharmacognosy) Under the Supervision of Prof. Dr. Prof. Dr. Soheir Mohamed El Zalabani Hesham Ibrahim El-Askary Professor of Pharmacognosy Professor of Pharmacognosy Faculty of Pharmacy Faculty of Pharmacy Cairo University Cairo University Assistant Prof. Dr. Omar Mohamed Sabry Assistant Professor of Pharmacognosy Faculty of Pharmacy Cairo University Pharmacognosy Department Faculty of Pharmacy Cairo University A.R.E. 2019 Abstract A Comparative Pharmacognostical Study of Certain Clerodendrum Species (Family Lamiaceae) Cultivated in Egypt Clerodendrum inerme L. Gaertn. and Clerodendrum splendens G. Don, two members of the cosmopolitan family Lamiaceae, are successfully acclimatized in Egypt. The current study aimed to evaluate the local plants as potential candidates for implementation in pharmaceutical industries, which necessitates an intensive investigation of safety and bioactivity of the cited species. To ensure quality and purity of the raw material, criteria for characterization of and/or discrimination between the two species were established via botanical profiling, proximate analysis, phytochemical screening and UPLC analysis. The leaves were subjected to comparative biological and chemical study to select the most suitable from the medicinal and economic standpoints. In this respect, the antioxidant cyotoxic and antimicrobial potentials of the defatted ethanol (70%) extracts of the tested samples were assessed in-vitro. Meanwhile, the chemical composition of the leaves was examined through qualitative and quantitative comparative analyses of the phenolic components. In this respect, The leaves of C. inerme were selected for more intensive both phytochemical and biological investigation. Moreover, a solid-liquid fractionation procedure (LSF) of the defatted ethanol (70%) extract of the leaves of C. inerme was proposed for optimization of phenolic extraction and compared to the conventional liquid-liquid fractionation method. To facilitate the incorporation of the extractives of the leaves of C. inerme in herbal formulations, a simple, valid and time saving RP-HPLC standardization procedure was developed for determination of its clerodermic acid content. Finally, to maximize the yield of bioactive constituents in the ethanol extract of the leaves of C. inerme, the effect of stage of plant development on its composition was assessed. Results revealed that the ethanol extract of the leaves of C. inerme exhibited broader cytotoxic effect and more pronounced antimicobial effect than that of C. splendens, in addition to apparently higher content of bioactive constituents. Repeated chromatographic fractionation of the different extracts of the plant allowed the isolation of six compounds. The isolated constituents included a tetraterpenoid (β-carotene), two phytosterols (22-dehydroclerosterol and 22- dehydroclerosterol -3-O-β-D-glucoside), a flavonoid (4’-methyl scutellarein), a diterpenoid (clerodermic acid) and a phenylpropanoid glycoside (verbascoside). Two of these (β-carotene and 22-dehydroclerosterol -3-O-β-D-glucoside) are for the first time reported in the species. Results of HPLC standardization of the ethanol extract of the leaves of C. inerme and its dichloromethane and ethyl acetate/ n-butanol (2:1) fractions, evaluated by the proposed method, were 9.76, 70.7 and 46.6 mg% (w/w), respectively. Finally, assessment of the effect of stage of plant development on the chemical composition of the leaves revealed that the highest contents of major constituents were recorded in leaf samples collected from plants during flowering stage, thus the leaves of C. inerme should preferably be collected at the flowering stage to ensure optimum yields of major bioactive metabolites. Key Words: Clerodendrum inerme L., Clerodendrum splendens G., botanical characteristics, bioactivities, phenolic profiling, HPLC standardization, validation, effect of stage of plant development. Acknowledgement My great thanks are, in the beginning and the last, to Allah, by the grace of whom, this work came to completion. I would like to express my deepest gratitude to my supervisors for their excellent guidance, caring, patience and providing me with an excellent atmosphere for doing this research. I am for ever indebted to them and hope that their good deeds return to them many folds. Neither words nor the available space can describe my greatest appreciation, deepest thanks and sincere gratitude to Prof. Dr. Soheir M. El Zalabani, Prof. of Pharmacognosy, Faculty of Pharmacy, Cairo University for supporting this work, by her continuous valuable supervision, kind, restful smile. She patiently corrected my writing and helped me to develop my scientific knowledgement. My great thanks are also due to Prof. Dr. Hesham I. El-Askary, Professor of Pharmacognosy, Faculty of Pharmacy, Cairo University, for his kind supervision, continuous guidance, education, encouragement, constructive comments, indispensable advice, backup throughout this study. Without his continuous support, this work could have never been accomplished. I wish to express my deep gratitude and appreciation to Assistant Prof. Dr. Omar M. Sabry, Assistant Professor of Pharmacognosy, Pharmacognosy Department, Faculty of Pharmacy, Cairo University for his kind supervision, guidance and encouragement throughout this study. I am grateful to all members of Pharmacognosy Department, Faculty of Pharmacy, Cairo University especially, Prof. Dr. Mohamed A. Farag, Assistant. Prof. Dr. Ali M. El-Halawany, Assistant. Prof. Dr. Engy A. Mahrous, Assistant. Prof. Dr. Mohammed Nabil, Lecturer Dr. Mohamed A. Salem, Lecturer Dr. Osama G. Mohamed and Lecturer Dr. Marwa Y. Issa for their everlasting concern, unlimited co-operation and great support with advices, time, needed chemicals and materials. My deep appreciation is extended to all my colleagues in the Pharmacognosy Department for their great help and support. They were and still are like my sisters and brothers. My intense and everlasting thanks, heartily gratitude and sincere love are devoted to my beloved mother and father to whome words are not enough to describe their care, tenderness, support and keen for seeing my success and prosperity. They were always there with a word of encouragement or just a listening ear, giving me a lot of love and support. From the deepest part of my heart I want to thank them for their enthusiasm, pride and curiosity to stand by me through all hard times. I owe them my life and success; they gave and still are giving me all what I need. Asmaa Mohamed Ahmed Khalil i Contents Subject Page Introduction 1 Review of Literature 3 Taxonomy 69 Material, Apparatus and Techniques 73 Part I: Comparative Study of Clerodendrum inerme L. Gaertn. and Clerodendrum splendens G. Don Chapter I: Botanical Characters 81 Chapter II: Proximate Analysis and Preliminary Phytochemical 92 Screening Chapter III: Extraction, Fractionation and Examination of the 94 Leaves Extractives Chapter IV: UPLC-Orbitrap HRMS Profiling 99 Chapter V: Quantitative Estimation of Phenolic Content 111 I. Determination of Total Phenolic Content (TPC) 112 II. Determination of Total Flavonoid Content (TFC) 113 Chapter VI: In-vitro Evaluation of the Biological Activities 116 I. Evaluation of the Antioxidant Activity 116 II. Evaluation of the Cytotoxic Activity 121 III. Evaluation of the Antibacterial Activity against Antibiotic Resistant Bacterial Strains 125 Part II: Phytochemical and Biological Investigation of Clerodendrum inerme L. Gaertn. Leaves Introduction 130 Chapter I: Optimization of Phenolic Extraction from the Leaves 131 of Clerodendrum inerme L. Gaertn. Chapter II: Investigation of the Different Extractives 134 I. Investigation of the n-Hexane Extract 134 A. Isolation of the constituents 134 B. Identification of the Isolated Compounds 137 II. Large Scale Fractionation and Investigation of the 70% Ethanol Extractives 142 A. Investigation of the Dichloromethane Fraction 142 B. Investigation of the Ethyl Acetate/ n-Butanol (2:1) Fraction 154 C. Investigation of the Methanol Fraction 170 ii Chapter III: Biological Evaluation of the Different Extractives and Isolated Compounds 178 I. Evaluation of the Antioxidant Activity 178 II. Evaluation of the Cytotoxic Activity 179 Chapter IV: Development and Validation of an HPLC Procedure for Standardization of the Extractives of the Leaves 184 Chapter V: Effect of Stage of Plant Development on the Chemical Composition of the Leaves 194 Summary 198 General Conclusion and Recommendation 208 References 210 Arabic Summary -1- Arabic Abstract -9- iii List of Tables No. Title Page 1 Reports on phenolics of Clerodendrum species 5 2 Reports on terpenoids of Clerodendrum species 15 3 Reports on steroids of Clerodendrum species 32 Reports on anti-inflammatory, antipyretic, analgesic and 4 42 antioxidant activities of Clerodendrum species Reports on cytotoxic, anti-cancer and anti-mutagenic 5 50 activities of Clerodendrum species Reports on hepatoprotective, anti-hyperglycemic, anti- 6 hyperlipidemic and anti-hypertensive activities of 54 Clerodendrum species Reports on cardiovascular activities of Clerodendrum 7 59 species Reports on central nervous system activities of 8 61 Clerodendrum species Reports on antimicrobial activity of Clerodendrum 9 63 species 10 Reports on antimalarial activity of Clerodendrum species