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

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

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-

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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 66 Reports on immunomodulatory and wound healing 11 67 activities of Clerodendrum species Geographical distribution, synonyms and common names 12 71 of the two Clerodendrum species under investigation Numerical values of the lower epidermises of the leaves 13 91 of C. inerme and C. splendens Results of Proximate analysis of the leaves of C. inerme 14 92 and C. splendens Results of preliminary phytochemical screening of the 15 93 leaves of C. inerme and C. splendens Percentage yield and organoleptic characters of the 16 94 extractives of the leaves of C. inerme and C. splendens Results of TLC fingerprinting of the n-hexane extracts of 17 95 the leaves Results of TLC fingerprinting of the EEs of the leaves 18 96 (using S8, p-anisaldehyde/H2SO4) Results of TLC fingerprinting of the EEs of the leaves 19 96 (using S12, AlCl3/UV) Results of TLC fingerprinting of the dichloromethane 20 97 fractions iv

Results of TLC fingerprinting of the ethyl acetate: n- 21 98 butanol (2:1) fractions Metabolites identified in the methanol extracts of the 22 leaves of C. inerme and C. splendens via UPLC-Orbitrap 103 HRMS in the negative ionization mode Absorbances of the different concentrations of standard 23 112 gallic acid Absorbances of the different concentrations of standard 24 114 quercetin Percentage of DPPH quenching of serial dilutions of the 25 117 EEs of the leaves of C. inerme and C. splendens Percentage of DPPH quenching of serial dilutions of the 26 118 reference antioxidant gallic acid DPPH quenching potency of the EEs of the leaves of C. 27 inerme and C. splendens, expressed as EC50 (µg/mL), 119 compared to standard gallic acid In-vitro cytotoxic activity of the EE of the leaves of C. 28 inerme and C. splendens on different cell lines compared 123 to standard doxorubicin In-vitro antibacterial activity of the EEs of the leaves of 29 C. inerme and C. splendens against MRSA compared to 126 standard vancomycin In-vitro antimycobacterial activity of the EEs of the 30 leaves of C. inerme and C. splendens compared to 128 standard isoniazide Weights and percentage yields of extractives of the 31 132 different fractions Total phenolic and total flavonoid contents (TPC and 32 133 TFC) of the different fractions Physicochemical, chromatographic and spectral data of 33 139 compound H2 Yields of the different fractions of the EE of the leaves of 34 142 C. inerme Results of TLC investigation of the dichloromethane 35 143 fraction Physicochemical, chromatographic and spectral data of 36 147 compound D1 Results of TLC investigation of the ethyl acetate/ n- 37 154 butanol fraction Physicochemical, chromatographic and spectral data of 38 159 compound E1 Physicochemical, chromatographic and spectral data of 39 163 compound E2 v

40 Results of TLC investigation of the methanol fraction 170 Physicochemical, chromatographic and spectral data of 41 174 compound M1 DPPH scavenging potency of the different fractions of the 42 EE of the leaves of C. inerme, expressed as EC50, 178 compared to standard gallic acid In-vitro cytotoxic activity of the n-hexane extract and 43 different fractions of the EE of the leaves of C. inerme, 179 expressed as IC50, compared to standard doxorubicin In-vitro cytotoxic activity of the compounds, isolated 44 from C. inerme extracts, expressed as IC50, compared to 180 standard doxorubicin Average peak areas of the different concentrations of 45 186 clerodermic acid Contents of clerodermic acid in the different extractives 46 187 of the leaves of C. inerme 47 Accuracy study of the proposed standardization method 189 Repeatability for retention time and peak areas expressed 48 189 as % RSD Intermediate precision for peak areas of two 49 190 concentrations of clerodermic acid over three days Results of robustness study for the proposed 50 standardization method for analysis of clerodermic acid 191 based on minor variation in flow rate Criteria for validation of the HPLC-based analytical 51 procedure proposed for standardization of the extractives 193 of the leaves of C. inerme Percentage yield and organoleptic characters of the 52 extracts of the leaves of C. inerme collected at different 194 stages of plant development Peak areas of the major constituents of the EEs of the 53 leaves of C. inerme collected at different stages of plant 196 development

List of Figures

No. Title Page 1 Flavonoid aglycones reported in Clerodendrum species 9 2 a Flavonoid glycosides reported in Clerodendrum species 9 2 b Flavonoid glycosides reported in Clerodendrum species 10 Phenylpropanoid glycosides reported in Clerodendrum 3 a 11 species Phenylpropanoid glycosides reported in Clerodendrum 3 b 12 species Phenylpropanoid glycosides reported in Clerodendrum 3 c 13 species Phenolic acids and phenolic acid derivatives reported in 4 14 Clerodendrum species Monoterpenoid iridoid glycosides reported in Clerodendrum 5 a 20 species Monoterpenoid iridoid glycosides reported in Clerodendrum 5 b 21 species 6 Megastigmane glycosides reported in Clerodendrum species 22 7 a Abietane diterpenoids reported in Clerodendrum species 23 7 b Abietane diterpenoids reported in Clerodendrum species 24 7 c Abietane diterpenoids reported in Clerodendrum species 25 7 d Abietane diterpenoids reported in Clerodendrum species 26 Neoclerodane diterpenoids reported in Clerodendrum 8 a 26 species Neoclerodane diterpenoids reported in Clerodendrum 8 b 27 species 9 Pimarane diterpenoid reported in Clerodendrum species 28 10 Acyclic diterpenoids reported in Clerodendrum species 28 11 Ursane triterpenoids reported in Clerodendrum species 28 12 Oleanane triterpenoids reported in Clerodendrum species 29 13 Lanostane triterpenoid reported in Clerodendrum species 29 14 Lupane triterpenoids reported in Clerodendrum species 30 15 Friedelane triterpenoid reported in Clerodendrum species 31

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16 Tetraterpenoid reported in Clerodendrum species 31 17 a Steroidal aglycones reported in Clerodendrum species 35 17 b Steroidal aglycones reported in Clerodendrum species 36 17 c Steroidal aglycones reported in Clerodendrum species 37 17 d Steroidal aglycones reported in Clerodendrum species 38 18 Steroidal glycosides reported in Clerodendrum species 39 19 a Other compounds reported in Clerodendrum species 40 19 b Other compounds reported in Clerodendrum species 41 20 Photographs of C. inerme shrub and C. splendens vine 82 21 Photographs of the leaves of C. inerme and C. splendens 83 22 Photographs of the flowers of C. inerme and C. splendens 84 23 Powdered leaves of C. inerme 86 24 Powdered leaves of C. splendens 89 Representative UPLC-Orbitrap HRMS total ion chromatograms (TIC) of the methanol extracts of the leaves 25 101 of C. inerme and C. splendens in the negative ionization mode Examples of identified natural products by UPLC-Orbitrap 26 102 HRMS analysis 27 MS/MS spectrum of peak 13 in the negative ionization mode 106 MS/MS spectrum of peak 5 (inerminoside A1) in the 28 107 negative ionization mode MS/MS spectrum of peak 30 (pictolinarigenin) in the 29 108 negative ionization mode 30 MS/MS spectrum of peak 34 in the negative ionization mode 108 MS/MS spectrum of peak 19 (rosmarinic acid) in the 31 109 negative ionization mode 32 Standard calibration curve of gallic acid 113 33 Standard calibration curve of quercetin 114 Line graph representing the concentration-dependent DPPH 34 quenching of the EE of the leaves of C. inerme and C. 118 splendens Line graph representing the concentration-dependent DPPH 35 119 quenching of standard gallic acid

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Bar chart representing the in-vitro anti-oxidant activity of the 36 EEs of the leaves of C. inerme and C. splendens compared to 120 standard gallic acid Bar chart representing the in-vitro cytotoxic activity of the 37 EEs of the leaves of C. inerme and C. splendens compared 123 to standard doxorubicin Bar chart representing the in-vitro antibacterial activity of 38 the EEs of the leaves of C. inerme and C. splendens against 127 MRSA compared to standard vancomycin Bar chart representing the in-vitro antimycobacterial activity 39 of the EEs of the leaves of C. inerme and C. splendens 129 compared to standard isoniazide 40 The proposed liquid-solid fractionation technique 132 Scheme for chromatographic fractionation of the n-hexane 41 136 extract of the leaves of C. inerme 42 UV-Visible spectra of standard and isolated β-carotene 137

1 43 H NMR spectrum of compound H2 140

13 44 C NMR spectrum of compound H2 141 Scheme for chromatographic fractionation of the 45 144 dichloromethane fraction of the leaves of C. inerme 1 46 H NMR spectrum of compound D1 148 13 47 C NMR spectrum of compound D1 149

48 HMQC spectrum of compound D1 150

49 Magnified part of HMQC spectrum of compound D1 151

50 HMBC spectrum of compound D1 152

51 Magnified part of HMBC spectrum of compound D1 153 HPLC fingerprint of the ethyl acetate/ n-butanol (2:1) 52 155 fraction Scheme for chromatographic fractionation of the ethyl 53 157 acetate/ n-butanol (2:1) fraction of the leaves of C. inerme 1 54 H NMR spectrum of compound E1 160 13 55 C NMR spectrum of compound E1 161

1 56 H NMR spectrum of compound E2 164

13 57 C NMR spectrum of compound E2 165

58 HSQC spectrum of compound E2 166

59 Magnified part of HSQC spectrum of compound E2 167

60 HMBC spectrum of compound E2 168

61 Magnified part of HMBC spectrum of compound E2 169

62 HPLC fingerprint of the methanol fraction 171 Scheme for chromatographic fractionation of the methanol 63 172 fraction of the leaves of C. inerme 1 64 H NMR spectrum of compound M1 175

13 65 C NMR spectrum of compound M1 176

66 HMBC spectrum of compound M1 177 Bar chart representing the in-vitro anti-oxidant activity of the 67 different fractions of the EE compared to standard gallic acid 178

Bar chart representing the in-vitro cytotoxic activity of the 68 different fractions of the EE of C. inerme compared to 180 standard doxorubicin Bar chart representing the in-vitro cytotoxic activity of the compounds, isolated from C. inerme extracts on lung 69 181 carcinoma cell line (H1299), compared to standard doxorubicin Bar chart representing the in-vitro cytotoxic activity of the compounds, isolated from C. inerme extracts on liver 70 181 carcinoma cell line (HepG-2), compared to standard doxorubicin Bar chart representing the in-vitro cytotoxic activity of the 71 compounds, isolated from C. inerme extracts on normal 182 fibroblasts (BHK), compared to standard doxorubicin HPLC fingerprints of the EE, its dichloromethane (M.C.) 72 185 and ethyl acetate/ n-butanol (2:1) (EtOAc) fractions 73 Standard calibration curve of clerodermic acid 186 HPLC chromatograms of the EE of the leaves of C. inerme, 74 188 isolated clerodermic acid & EE spiked with clerodermic acid HPLC chromatograms of isolated clerodermic acid at 75 192 different flow rates (1, 0.9 & 1.1 mL/min)

HPLC chromatograms of the EEs of the leaves of C. inerme 76 195 collected at different stages of plant development Effect of stage of development on the major constituents of 77 196 the EEs of the leaves of C. inerme

Introduction

Introduction Introduction Family Lamiaceae (the mint family) is the sixth largest family of flowering plants (Drew and Sytsma, 2012) and the largest family of the order Lamiales. It comprises about 236 genera and 7136 species (Trivellini et al., 2016). The largest genera in the family are Salvia, Clerodendrum, Scutellaria, Stachys, Plectranthus, Hyptis, Teucrium, Vitex, Thymus and Nepeta. The family members are distributed worldwide, many of them are aromatic and thus used as culinary herbs (Raja, 2012; Trivellini et al., 2016). Lamiaceae species are reported to contain several pharmacologically active compounds including, volatile constituents (Pelikan, 2006; Vani et al., 2009; Rodríguez-Solana et al., 2015; Méndez-Tovar et al., 2016), phenolics (Pedersen, 2000; Zgórka and Glowniak, 2001; Kivilompolo and Hyötyläinen, 2007; Trivellini et al., 2016) and iridoids (Pizza et al., 2005; Modaressi et al., 2009; Venditti et al., 2016). The Lamiaceae species are reported to exert a wide range of biological effects including antioxidant (Marinova and Yanishlieva, 1997; Wojdyło et al., 2007) cytotoxic (Berdowska et al., 2013), antimicrobial (Marino et al., 2001), antifungal (Waller et al., 2017) and anti-inflammatory (Çadirci et al., 2012) activities. Members of genus Clerodendrum grow mainly in tropical Africa and southern Asia (Singh et al., 2012). Among other species of the genus, Clerodendrum inerme L. Gaertn. and Clerodendrum splendens G. Don have been successfully introduced to Egypt as ornamentals (Hamdy et al., 2007). The objective of this work was targeted towards assessing the efficacy of the locally cultivated Clerodendrum inerme L. Gaertn. and Clerodendrum splendens G. Don, as a source of potential medicinals. To fulfil these goals, and following a comprehensive review of the available literature on the genus, a comparative study of the two cited species was planned to include the following: 1. Careful collection, identification and authentication of the plant material. 2. Establishment of useful criteria for taxonomical discrimination between the two selected species based on botanical profiling, phytochemical screening and determination of pharmacopoeial constants. 3. Comparative UPLC-Orbitrap HRMS metabolite profiling of the methanol extracts of the leaves of the selected species and chemometric analysis of the data. 4. Extraction and bioactivity-guided fractionation of selected extracts based on in- vitro evaluation of antioxidant, cytotoxic and antimicrobial activities.

Introduction 5. Qualitative and quantitative analyses of the constituents of the bioactive extractives via appropriate procedures (Spectrophotometric and HPLC). 6. Isolation, identification and structure elucidation of the main constituents using recent chromatographic and spectrometric techniques. 7. Correlation of recorded bioactivities to the isolated compounds. 8. Development and validation of an HPLC procedure for standardization of the bioactive extracts/fractions.

Review of Literature

Review of Literature Review of Literature The genus Clerodendrum L. (Lamiaceae) is very widely distributed in tropical and subtropical regions of the world. Around five hundred species of small trees, shrubs and herbs have been identified as members of the genus. Several of these are claimed as ethno-medicines especially in China, India, Japan, Korea and Thailand (Shrivastava and Patel, 2007; Kar et al., 2014). They are used for management of several ailments including: asthma and rheumatism (Hazekamp et al., 2001; Panthong et al., 2003; Shrivastava and Patel, 2007; Lee et al. 2011; Kar et al., 2014; Phosrithong and Nuchtavorn 2015); skin diseases, worms, fever and beriberi (Atta-Ur-Rahman et al., 1997; Kanchanapoom et al., 2001; Shrivastava and Patel, 2007; Kar et al., 2014), as well as hypertension (Kang et al., 2003; Shrivastava and Patel, 2007; Kar et al., 2014). Earlier investigations on Clerodendrum species led to the isolation of a variety of bioactive constituents. The majority of reports are concerned with phenolics that exert antihypertensive, anti-inflammatory, anti-asthmatic and antioxidant activities (Kang et al., 2003; Lee et al., 2011; Wahba et al., 2011; Ibrahim et al., 2014; Srisook et al., 2015) as well as cytotoxic terpenoids (Hosny et al., 2004; Sun et al., 2014) and steroids (Xu et al., 2013). Researches on the chemical composition and bioactivity of the most widespread Clerodendrum species are herein briefly surveyed to serve as guide during the current study. I. Major constituents

A. Phenolics Phenolics reported in Clerodendrum species include mainly flavonoid aglycones and glycosides, phenylpropanoid glycosides as well as phenolic acids and their derivatives. Data concerning either the detection and quantification or isolation and structure elucidation of these constituents are summarized in table 1. Structural formulae of representative examples of each group are displayed in figures 1-4.

B. Terpenoids

A large number of terpenoids have been isolated and/or identified in genus Clerodendrum. These comprised mono-, sesqui-, di-, tri- and tetraterpenoids; iridoid glycosides predominate the monoterpenoid group. Published data concerning the isolation, identification and/or structure elucidation of these components from the different species are summarized in table 2. Structural formulae of selected examples are represented in figures 5-16 as follows: iridoid glycosides (Figure 5), sesquiterpenoids (Figure 6), diterpenoids (Figures 7-10), triterpenoids (Figures 11-15) and tetraterpenoids (Figure 16). 3

Review of Literature C. Steroids Steroids constitute the third class of secondary metabolites frequently reported in the genus beside phenolics and terpenoids. Reports concerning the detection and/or isolation and structure elucidation of these constituents from different Clerodendrum species are summarized in table 3. The structural formulae of selected examples of the group are represented in figures 17 and 18. II. Minor constituents Alongside the aforementioned constituents, others were less frequently reported,in the genus including: benzofuranones as Rengyolone (Wahba et al., 2011); cyclohexyl ethanoids like Clerobungin A (Zhu et al., 2014), 5-O-butyl Cleroindin D, 1-Hydroxy-1-(8-palmitoyloxyethyl) cyclohexanone (Xu et al., 2014) and 2R- Acetoxymethyl - 1,3,3 - trimethyl - 4t - (3-methyl-2-buten-1-yl) -1c-cyclohexanol (Erukainure et al., 2016); anthraquinones such as Emodin; lignans as Spicatolignan B (Li et al., 2014) and triglycerides like Triacetin (Erukainure et al., 2016). Structural formulae of these constituents are represented in figure 19. III. Biological activities Scientifically-based bioactivities attributed to Clerodendrum species are numerous. Several members of the genus were claimed to exert anti-inflammatory, antioxidant, cytotoxic, hepatoprotective, antihyperglycemic, antihyperlipidemic, antihypertensive, antimicrobial, anti-malarial and immunomodulatory effects. Researches available in this concern are briefly reported in tables 4-11. Moreover, C. splendens and C. trichotomum leaves exhibited anti-ulcer (Tan et al., 1997) and anti-asthmatic (Lee et al., 2011) activities, respectively.

Review of Literature

Table 1: Reports on phenolics of Clerodendrum species

Reference Species Plant Part Summary The ethanol (90%) extracts of air-dried leaves and flowers of C. splendens were fractionated with petroleum ether, chloroform, ethyl C. splendens G. acetate and n-butanol in succession. Apigenin-7-O-β-D-glucoside, Leaves and (Shehata et al., 2001) apigenin and 3',4',7-trihydroxyflavone-7-O-β-D-glucoside were Don flowers isolated from the n-butanol fraction, crystallized from methanol and identified via UV spectral analysis in presence of different shift reagents. The aqueous fraction of the 90% ethanol extract of the air-dried leaves of C. splendens was chromatographed on a porous polymer gel column (Yousif and El-Regal, C. splendens G. followed by a sephadex LH 20 column to afford isovitexin-7-O-ß-D- Leaves 2004) Don glucoside and vitexin-7-O-ß-D-glucoside. Structures of the isolated compounds were established through H1NMR, UV and ESI/MS spectral data. The n-butanol fraction of the 95% ethanol extract of the aerial parts of C. inerme was subjected to CC on a macroporous resin to afford four phenylethanoid glycosides; 2-(3-methoxy-4-hydroxylphenyl) ethyl- C. inerme L. (Nan et al., 2005) Aerial parts O-2",3"-diacetyl-α-l-rhamnopyranosyl-(1-3)-4-O-(E) feruloyl-β-D- Gaertn. glucopyranoside, verbascoside, isoverbascoside and campneoside I. Structures of the isolated compounds were identified via H1 and C13 NMR, HSQC and HMBC spectroscopy. The dichloromethane extract of C. trichotomum stem was subjected to C. trichotomum sequential CC (SiO2) and medium pressure liquid chromatography to (Chae et al., 2006) Stem 1 Thunb. afford trichotomoside, whose structure was identified by MS, IR, H and C13NMR spectroscopy.

Review of Literature Table 1 (continued): Reports on phenolics of Clerodendrum species

Reference Species Plant Part Summary The 70% ethanol extract of C. trichotomum leaves was fractionated with ether and water successively. The aqueous fraction was subjected to CC on C. trichotomum (Lee et al., 2011) Leaves Diaion HP-20 followed by Sephadex LH-20 then ODS columns to yield Thunb. acteoside (verbascoside), which was identified by comparing its IR, MS, and NMR spectral data with those published. The 80% methanol extract of the dried powdered C. chinense leaves was fractionated between chloroform, ethyl acetate and n-butanol. The ethyl acetate fraction was successively subjected to CC on silica gel, polyamide C. chinense and RP-C18 to yield hispidulin, verbascoside and isoverbascoside. (Wahba et al., 2011) Leaves (Osbeck) Mabb. Meanwhile, cornoside and decaffeoylverbascoside were isolated from the n-butanol fraction by CC on polyamide and RP-18. Structures of the isolated compounds were identified by comparison of spectral data with those published. Powdered roots, leaves and flowers were defatted with petroleum ether Roots, (Gupta and Gupta C. infortunatum then extracted with methanol via continuous hot extraction. HPTLC was leaves and 2012) Linn. used for detection and quantification of quercetin in the methanol flowers extracts. The methanol (70%) extract of the air-dried aerial parts of C. inerme was C. inerme L. subjected to VLC, then SiO2 CC to yield five compounds including 5- (Ibrahim et al., 2014) Aerial parts 1 Gaertn. hydroxy-6,7,4'trimethoxyﬂavone whose structure was identified by H and C13NMR spectroscopy. The ethyl acetate and n-butanol fractions of the ethanol extract of the dried C. bungei roots of C. bungei were subjected to CC on MCI gel CHP 20P. The ethyl (Liu et al., 2014) Roots Steud. acetate fraction afforded 19 compounds including verbascoside, isoacteocoside, martynoside, isomartynoside, acetyl martynoside A and 6

Review of Literature Table 1 (continued): Reports on phenolics of Clerodendrum species

Reference Species Plant Part Summary B, leucosceptoside A, phlomisethanoside, trichotomoside, 3"-O-actyl martynoside and 2"- O-actyl martynoside. The n-butanol fraction afforded 2 compounds darendoside A and B. Structures of the isolated compounds were established based on HR-ESI-MS, H1 and C13 NMR and HMBC spectral data and acidic hydrolysis. The alcoholic extract of C. splendens leaves was fractionated with n-hexane, chloroform, ethyl acetate and n-butanol. The chloroform fraction yielded (Soliman et al., C. splendens G. quercetin. The ethyl acetate fraction afforded hispidulin 7-O-β-D-glucoside, Leaves 2014) Don hispidulin-7-O-(6''-O-trans-caffeoyl)-β-D-glucoside, kaempferol 3-O-β-D- glucoside, methyl rosmarinate and trans-caffeic acid. Meanwhile, neochlorogenic acid was isolated from the n-butanol fraction. HPLC-MS/NMR was used for qualitative and quantitative analysis of (Thitilertdecha et C. petasites phenolics in the ethanol extract of the dried aerial parts of C. petasites. The Aerial parts al., 2014) (Lour.) S.Moore identified compounds included verbascoside, luteolin, apigenin and hispidulin with the latter as major. The dichloromethane fraction of the 95% ethanol extract of the aerial parts of (Huang et al., C. inerme L. C. inerme was subjected to CC on silica gel to afford 2 pure compounds; Aerial parts 1 2015) Gaertn. hispidulin and acacetin. Their structures were established through their H and C13NMR, and HMBC spectra. The dichloromethane extracts of the dried powdered roots of C. indicum and C. indicum (L.) C. villosum were subjected to CC on silica gel and sephadex. C. indicum (Somwong et al,. Kuntze Roots extract yielded pectolinarigenin and hispidulin; while that of C. villosum 2015) C. villosum afforded verbascoside. Structures of the isolated compounds were Blume identified by MS, 1D and 2D NMR and by comparison with published data.

Review of Literature Table 1 (continued): Reports on phenolics of Clerodendrum species

Reference Species Plant Part Summary

The ethyl acetate fraction of the ethanol extract of the dried leaves of C. C. inerme L. inerme was subjected to repeated CC on silica gel to yield hispidulin, (Srisook et al., 2015) Leaves Gaertn. acacetin and diosmetin. Structures were identified by H1 and C13NMR spectroscopy. The dichloromethane fraction of the methanol extract of C. volubile leaves was fractionated using CC loaded with silica gel with gradual elution with (Erukainure et al., C. volubile P. Leaves mixtures of n-hexane and dichloromethane to afford pectolinarigenin. 2017) Beauv. The structure of the compound was established based on H1 and C13 NMR and MS spectral data. The ethyl acetate fraction of the 70% methanol extract of the powdered leaves of C. viscosum was subjected to CC on silica gel followed by C. viscosum (Shendge et al., 2018) Leaves preparative HPLC on C-18 column to afford ellagic acid. The structure of Vent. the isolated ellagic acid was identified by mass spectrometry, and FTIR and NMR spectroscopic techniques.

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R1 R2 R3 R4 R5 Compound Name

H OCH3 OH H OH Hispidulin H H OH H OCH3 Acacetin H H OH OH OCH3 Diosmetin H OCH3 OCH3 H OCH3 5-Hydroxy-6,7.4'-trimethoxy flavone H OCH3 OH H OCH3 Pectolinarigenin H H OH H OH Apigenin H H OH OH OH Luteolin OH H OH OH OH Quercetin

Figure 1: Flavonoid aglycones reported in Clerodendrum species

Kaempferol 3-O-β-D-glucoside

Figure 2 a: Flavonoid glycosides reported in Clerodendrum species

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R1 R2 R3 R4 Compound Name OH OCH₃ H H Hispidulin 7-O-β-D-glucoside trans- Hispidulin-7-O-(6''-O-trans-caffeoyl)-β-D- OH OCH₃ H caffeoyl glucoside OH H H H Apigenin-7-O-β-D-glucoside H H OH H 3',4',7-trihydroxyflavone-7-O-β-D –glucoside

R1 R2 Compound Name β-D-glucose H Isovitexin-7-O-β-D-glucoside H β-D-glucose Vitexin-7-O-β-D-glucoside

Figure 2 b: Flavonoid glycosides reported in Clerodendrum species

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R1 R2 R3 R4 R5 R6 R7 R8 Compound Name H H H H H H H H Acteoside (Verbascoside) CH3 H H H H H H H Leucosceptoside A CH3 H H CH3 H H H H Martynoside CH3 H H CH3 H Acetyl Acetyl H Acetyl martynoside A CH3 H H CH3 H Acetyl H Acetyl Acetyl martynoside B CH3 H H CH3 H Acetyl H H 2''-O-Acetyl martynoside CH3 H H CH3 H H Acetyl H 3''-O-Acetyl martynoside 2-(3-methoxy-4- hydroxylphenyl) ethyl-O- 2",3"-diacetyl-α-l- CH H CH H H Acetyl Acetyl H 3 3 rhamnopyranosyl-(1-3)-4- O-(E)-feruloyl-β-D- glucopyranoside H H H H OCH3 H H H Campneoside I

Figure 3 a: Phenylpropanoid glycosides reported in Clerodendrum species

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R Compound Name H Darendoside A 4-Hydroxy-3-methoxy benzoyl Phlomisethanoside

R Compound Name H Decaffeoyl verbascoside

CH3 Darendoside B

Figure 3 b: Phenylpropanoid glycosides reported in Clerodendrum species

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R Compound Name H Isoacteoside (Isoverbascoside)

CH3 Isomartynoside

Cornoside

Trichotomoside Figure 3 c: Phenylpropanoid glycosides reported in Clerodendrum species 13

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Trans-caffeic acid

Neochlorogenic acid

Methyl rosmarinate (phenolic ester)

Ellagic acid Figure 4: Phenolic acids and phenolic acid derivatives reported in Clerodendrum species

Review of Literature Table 2: Reports on terpenoids of Clerodendrum species

Reference Species Plant Part Summary C. splendens (Ohiri, 1987) Leaves Benzene extract of C. splendens leaves afforded α-amyrin. G. Don The dried aerial parts of C. inerme were extracted with hot methanol, the solvent evaporated and the residue suspended in water and defatted with diethyl ether. The remaining aqueous fraction was subjected to repeated CC (Kanchanapoom et C. inerme L. Aerial parts on porous polymer, silica gel and RP-C18 to afford 18 compounds among al., 2001) Gaertn. which were sammangaosides A and B (megastigmane glucosides). sammangaoside C (iridoid glucoside). Structure elucidation was through H1NMR and MS spectra. C. inerme L. Three iridoid glycosides, inerminosides A1, C and D were isolated. (Calis et al., 1993) Aerial parts Gaertn. Structure elucidation was through H1NMR and MS spectra. The n-hexane fraction of the ethanol extract of the dried aerial parts of C. C. inerme L. inerme was subjected to CC on silica gel and yielded clerodermic acid. (Pandey et al., 2003) Aerial parts Gaertn. Structure formula was established through comparison of its spectral data with those published. The diethyl ether fraction of the 70% ethanol extract of the dried powdered leaves of C. inerme was subjected to flash chromatography on silica gel C. inerme L. (Hosny et al., 2004) Leaves column to yield 3 diterpenoids; inermein A, inermein B and 3- Gaertn. epicaryoptin. Structures of the isolated compounds were established by H1, C13NMR and HR-ESI-MS analyses. The n-hexane fraction of the ethanol extract of the aerial parts of C. inerme was subjected to CC on silica gel followed by preparative TLC to yield C. inerme L. (Pandey et al., 2005) Aerial parts inermes A, inermes B, 15-Methoxy-14,15-dihydro-3-epicaryoptin and Gaertn. 15-Hydroxy-14,15-dihydro-3-epicaryoptin. Structure formulae of the isolated compounds were established through IR, H1 and C13NMR.

Review of Literature Table 2 (continued): Reports on terpenoids of Clerodendrum species

Reference Species Plant Part Summary The n-butanol fraction of the 95% ethanol extract of the aerial parts of C. inerme was subjected to CC on macroporous resin to afford 3 C. inerme L. (Nan et al., 2005) Aerial parts iridoid glycosides; monomelittoside, melittoside and inerminoside Gaertn. A1. H1 and C13 NMR, HSQC and HMBC spectral data were used for structure elucidation. The n-hexane fraction of the ethanol extract of the air-dried aerial parts C. inerme L. of C. inerme was subjected to repeated CC on silica gel to yield 3 (Pandey et al., 2007) Aerial parts Gaertn. compounds including betulinic acid, identified by IR, H1NMR and C13NMR spectroscopy. Three triterpenoids were isolated from the leaves of C. inerme viz., C. inerme L. lup-1,5,20(29)-trien-3-O-β-D-glucopyranoside, friedelin and β- (Parveen et al., 2010) Leaves Gaertn. amyrin. IR, H1NMR, C13NMR, DEPT and MS spectral data were used for their identification. Lupeol was isolated from the chloroform extract of the dried powdered C. chinense (Wahba et al., 2011) Leaves leaves of C. chinense by repeated CC on silica gel. Structure (Osbeck) Mabb. elucidation was by comparison of spectral data with those published. The chloroform fraction of the 95% ethanol extract of the dried leaves C. inerme L. of C. inerme was subjected to repeated CC on silica gel G to afford (Devi et al., 2012) Leaves Gaertn. ursolic acid that was identified by melting point determination, co- TLC and IR spectroscopy. The dried roots of C. trichotomum were defatted with petroleum ether then extracted with 90% ethanol. The petroleum ether extract upon C. trichotomum successive CC on silica gel yielded 12 diterpenoids including villosin (Wang et al., 2013 a) Roots Thunb. C and uncinatone. The 90% ethanol extract when applied to macroporous resin D101 followed by CC on silica gel afforded 2 other diterpenoids. Structure elucidation was via 1D and 2D NMR spectra. 16

Review of Literature Table 2 (continued): Reports on terpenoids of Clerodendrum species

Reference Species Plant Part Summary The petroleum ether extract of the dried roots of C. trichotomum was C. trichotomum subjected to repeated CC on silica gel to yield trichotomone whose (Wang et al., 2013 b) Roots Thunb. structure was identified by HR-ESI-MS, IR, UV, H1 and C13 NMR spectroscopy. The 70% methanol extract of air-dried, powdered aerial parts C. inerme was (Ibrahim et al., C. inerme L. subjected to VLC followed by CC on SiO2 to afford 5 compounds Aerial parts 2014) Gaertn. including: β-friedo-olean-5-ene-3β-ol and betulinic acid. Structures of the isolated compounds were identified by H1 and C13NMR spectroscopy.

The air-dried stems of C. trichotomum were extracted with 85% ethanol under reflux. The ethyl acetate fraction of the extract was subjected to CC C. trichotomum on silica gel and sephadex LH-20 to yield 11 compounds including: sugiol, (Li et al., 2014) Stems Thunb. uncinatone and crytophyllone B. Structures of the isolated compounds were determined by comparing spectroscopic data to those reported in the literature. The petroleum ether fraction of the ethanol extract of the dried roots of C. bungei was subjected to repeated silica gel column chromatography to C. bungei (Liu et al., 2014) Roots afford 9 compounds including: crytophyllone A, uncinatone and villosin Steud. C. Structures of the isolated compounds were established by HR-ESI-MS, H1, C13 NMR, HMBC spectroscopic analysis and acidic hydrolysis. The alcoholic extract of the leaves of C. splendens was fractionated with n- (Soliman et al., C. splendens G. hexane, chloroform, ethyl acetate and n-butanol. 15α-methoxy dihydro- Leaves 2014) Don epicaryoptin was isolated from the n-hexane fraction and lutein from the chloroform fraction.

Review of Literature Table 2 (continued): Reports on terpenoids of Clerodendrum species

Reference Species Plant Part Summary The ethyl acetate fraction of the 95% ethanol extract of the air-dried roots of C. bungei was subjected to CC on silica gel followed by sephadex LH 20 to C. bungei (Sun et al., 2014) Roots yield 3β-(β-D-glucopyranosyl) isopimara-7,15-dien-11α,12α-diol and 16- Steud. O-β-D-glucopyranosyl-3-β-20-epoxy-3-hydroxyabieta-8,11,13-triene. Structure formulae were established by 2D-NMR spectroscopy. The dichloromethane extracts of the dried powdered roots of C. indicum and C. indicum C. villosum were subjected to CC on silica gel and sephadex. Lupeol was (Somwong et al., (L.) Kuntze isolated from both C. indicum and C. villosum extracts. Oleanolic acid-3- Roots 2015) C. villosum acetate and taraxerol were obtained from that of C. indicum only and Blume betulinic acid from that of C. villosum. Structures of the isolated compounds were identified by MS, 1D and 2D NMR and by comparison with literature The ethyl acetate fraction of the 90% ethanol extract of the air-dried stems of C. kiangsiense was subjected to CC on silica gel and sephadex LH-20 to afford 8 diterpenoids; crypto japonol, 12-O-demethyl crypto japonol, C. kiangsiense 11,14-dihydroxy-8,11,13- abietatrien-7-one, mandarone A, fortunin E, (Xu et al., 2016) Stems Merr. fortunin F, taxusabietane A and 12-methoxy-6,11,14,16-tetrahydroxy- 17(15→16)-abeo-5,8,11,13-abietatetraen-3,7-dione. Structures of the isolated compounds were determined via 2D-NMR and comparison with published data. The methanol extract of the leaves of C. inerme was suspended in water, and extracted with dichloromethane. The organic fraction upon repeated CC on C. inerme L. silica gel and sephadex LH-20 afforded 8 compounds including a new (Vinh et al., 2017) Leaves Gaertn. rearranged abietane diterpene crolerodendrum B and the known compounds; crolerodendrum A, uncinatone and harwickiic acid. Structure formulae were identified by HR-ESI-MS, H1 and C13 NMR spectroscopy.

Review of Literature Table 2 (continued): Reports on terpenoids of Clerodendrum species

Reference Species Plant Part Summary

A lanostane-type triterpene carboxylic acid (3-hydroxylanost-7-en-29- carboxylic acid) and a lupane-type triterpenoid (betulin) were isolated from C. capitatum (Habila et al., the ethyl acetate-soluble fraction of the methanol extract of the leaves of C. Schumach. & Leaves 2018) capitatum. The structures of the compounds were elucidated based on analysis Thonn. of their spectral data, including H1, C13 and 2D NMR and by comparison with those reported in the literature.

Review of Literature

R Compound Name H Monomelittoside β-D-glucose Melittoside β-D-glucosyl-(1-3)-β-D-glucose Sammangaoside C

R Compound Name H Inerminoside A1 Para-hydroxy benzoyl Inerminoside D Figure 5 a: Monoterpenoid iridoid glycosides reported in Clerodendrum species

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Inerminoside C

Figure 5 b: Monoterpenoid iridoid glycosides reported in Clerodendrum species

Review of Literature

Sammangaoside A

Sammangaoside B

Figure 6: Megastigmane glycosides reported in Clerodendrum species

Review of Literature

R1 R2 Compound Name H OH Mandarone A

OH OCH3 Taxusabietane A

R1 R2 R3 R4 R5 R6 Compound Name H OH H H H H Sugiol OH OH OH H H H Crytophyllone B OH OCH3 H H H H Crypto japonol OH OH H H H H 12-O-demethyl crypto japonol OH H H OH H H 11,14-dihydroxy-8,11,13- abietatrien-7-one H OH H H OH OH Fortunin E Figure 7 a: Abietane diterpenoids reported in Clerodendrum species

Review of Literature

R1 R2 Compound Name

CH3 CH3 Crytophyllone A

H CH2OH Villosin C

Fortunin F Uncinatone

Crolerodendrum A Crolerodendrum B Figure 7 b: Abietane diterpenoids reported in Clerodendrum species 24

Review of Literature

Trichotomone

12-methoxy-6,11,14,16-tetrahydroxy-17(15→16)-abeo-5,8,11,13-abietatetraen- 3,7-dione Figure 7 c: Abietane diterpenoids reported in Clerodendrum species

Review of Literature

16-O-β-D glucopyranosyl-3-β-20- epoxy-3- hydroxyabieta-8,11,13-triene Figure 7 d: Abietane diterpenoids reported in Clerodendrum species

R R’ Compound Name H H Inermes A

OCH3 H Inermes B Figure 8 a: Neoclerodane diterpenoids reported in Clerodendrum species

Review of Literature

3-Epicaryoptin Clerodermic acid

Harwickiic acid R Compound Name 15- Hydroxy-14,15- dihydro-3- H epicaryoptin 15- Methoxy-14,15- dihydro-3- CH 3 epicaryoptin Figure 8 b: Neoclerodane diterpenoids reported in Clerodendrum species

Review of Literature

3β-(β-D glucopyranosyl) isopimara-7,15- dien-11α,12α-diol Figure 9: Pimarane diterpenoid reported in Clerodendrum species

Inermein A

Inermein B Figure 10: Acyclic diterpenoids reported in Clerodendrum species

R Compound Name CH3 α-Amyrin COOH Ursolic acid Figure 11: Ursane triterpenoids reported in Clerodendrum species 28

Review of Literature

R1 R2 Compound Name

H CH3 β-Amyrin

COCH3 COOH Oleanolic acid -3- acetate

Taraxerol β-Friedo-olean-5-ene-3β-ol Figure 12: Oleanane triterpenoids reported in Clerodendrum species

3-Hydroxylanost-7-en-29-carboxylic acid Figure 13: Lanostane triterpenoid reported in Clerodendrum species

Review of Literature

R Compound Name

CH3 Lupeol COOH Betulinic acid

CH2OH Betulin

Lup-1,5,20(29)-trien-3-O-β-D-glucopyranoside

Figure 14: Lupane triterpenoids reported in Clerodendrum species

Review of Literature

Friedelin Figure 15: Friedelane triterpenoid reported in Clerodendrum species

Lutein Figure 16: Tetraterpenoid reported in Clerodendrum species

Review of Literature

Table 3: Reports on steroids of Clerodendrum species

Reference Species Plant Part Summary The ethanol extract of the air-dried aerial parts of C. inerme was fractionated between n-hexane, ethyl acetate and n-butanol. The unsaponifiable part of the n-hexane fraction was subjected to successive CC on silica gel to afford 2 compounds: 24β-ethyl (Atta-Ur-Rahman et C. inerme L. Aerial parts cholesta-5,22E,25(27)-trien-3-O-β-D-galactopyranoside and al., 1997) Gaertn. clerosterol. The structure of the first was established via interpretation of its IR, MS and H1 and C13NMR spectra. Meanwhile, clerosterol was identified by comparing its spectroscopic data with those reported. The 90% ethanol extracts of the air-dried leaves and flowers of C. splendens were separately fractionated with petroleum ether, C. splendens G. Leaves and chloroform, ethyl acetate and n-butanol. The unsaponifiable part of (Shehata et al., 2001) Don flowers the petroleum ether fraction of the leaves was subjected to vacuum liquid chromatography on silica gel to afford 22-dehydroclerosterol. Its structure was identified by H1NMR. The n-hexane fraction of the ethanol extract of the dried aerial parts of C. inerme was subjected to repeated CC on a silica gel column to C. inerme L. yield 5 compounds including: 4α-methyl-24β-ethyl-5α-cholesta- (Pandey et al., 2003) Aerial parts Gaertn. 14,25-dien-3β-ol and 24β-ethylcholesta-5,9(11),22E-trien-3β-ol. Structural formulae of the isolated sterols were established via IR, H1NMR and EI-MS spectroscopy. n-Hexane extract of the air-dried, powdered aerial parts of C. inerme was subjected to silica gel CC to afford stigmasterol glucoside, C. inerme L. (Pandey et al., 2006) Aerial parts which was identified by co-comparison with authentic standard. Upon Gaertn. acid hydrolysis, it yielded stigmasterol as aglycone and glucose as sugar moiety. 32

Review of Literature Table 3 (continued): Reports on steroids of Clerodendrum species

Reference Species Plant Part Summary The n-hexane fraction of the ethanol extract of the air-dried powdered aerial parts of C. inerme was subjected to CC on silica gel column to C. inerme L. yield 4α-methyl-24β-ethyl-5α-cholesta-14,25-dien-3β-ol and 24β- (Pandey et al., 2007) Aerial parts Gaertn. ethylcholesta-5,9(11),22E-trien-3β-ol. Structure elucidation of the isolated compounds was performed by IR, H1NMR and C13NMR spectroscopy. The chloroform extract of air-dried, powdered C. inerme leaves was (Shanthakumar et C. inerme L. subjected to CC on silica gel column followed by preparative TLC to Leaves al., 2013) Gaertn. afford stigmasterol. Its structure was identified via its H1 and C13 NMR spectra. The petroleum ether fraction of the 95% ethanol extract of the leaves of C. trichotomum was subjected to CC on silica gel to yield 7 steroidal compounds including: stigmasta-4,25-dien-3-one, 6β- C. trichotomum hydroxystigmasta-4,22E,25-trien-3-one, 3β-hydroxystigmasta- (Xu et al., 2013) Leaves Thunb. 5,22E,25-trien-7-one, stigmasta-22E,25-dien-3,6-dione, stigmasta- 5,22E,25-trien-3β,7β-diol, stigmasta-22E,25-dien-3β,6β,9α-triol. Structural formulae of the isolated compounds were established through IR, H1NMR and C13NMR spectroscopy. The 70% methanol extract of the air-dried aerial parts of C. inerme was C. inerme L. subjected to VLC followed by CC on SiO to afford β-sitosterol and (Ibrahim et al., 2014) Aerial parts 2 Gaertn. stigmasta-5,22,25-trien-3β-ol. Structures of the isolated compounds were elucidated via H1 and C13NMR spectral data. C. splendens G. The n-hexane fraction of the alcoholic extract of C. splendens leaves (Soliman et al., 2014) Leaves Don afforded β-sitoserol and β-sitoserol-3-O-β-D-glucoside.

Review of Literature Table 3 (continued): Reports on steroids of Clerodendrum species

Reference Species Plant Part Summary The dichloromethane extracts of the dried powdered roots of C. indicum and C. villosum were subjected to CC on silica gel and sephadex. Clerosterol, 22-dehydroclerosterol, clerosterol-3-O-β-D-glucoside and 22-dehydroclerosterol-3-O-β-D-glucoside were isolated from both C. indicum (L.) C. indicum and C. villosum roots. Whereas, (22E)-stigmasta-4,22,25- Kuntze (Somwong et al., 2015) Roots trien-3-one, stigmasta-4,25-dien-3-one, stigmasta-4,22-dien-3-one, C. villosum stigmasterol and stigmasterol-3-O-β-D-glucoside were obtained from Blume those of C. indicum only, and β-sitosterol and β-sitosterol-3-O-β-D- glucoside from those of C. villosum. Structural formulae of the isolated compounds were established through MS, 1D and 2D NMR spectroscopy and by comparison with published data. The finely blended fresh flowers of C. volubile were extracted with methanol. The methanol extract was fractionated with n-hexane, (Erukainure et al., C. volubile P. Flowers dichloromethane, ethyl acetate and n-butanol in succession. GC/MS 2016) Beauv. analysis of the dichloromethane fraction revealed the presence of 4 compounds with stigmastan-3,5-diene as major.

Review of Literature

R Double Bonds Compound Name

CH3 14-15, 25-27 4α-Methyl-24β-ethyl-5α-cholesta-14,25-dien-3β-ol H 5-6, 9-11, 22-23 24β-Ethyl cholesta-5,9(11),22E-trien-3β-ol H 5-6, 22-23,25-27 Stigmasta-5,22,25-trien-3β-ol H 5-6, 22-23 Stigmasterol H 5-6 β-Sitosterol

Double Bonds Compound Name 5-6, 25-27 Clerosterol 5-6, 22-23,25-27 22-Dehydro clerosterol

Figure 17 a: Steroidal aglycones reported in Clerodendrum species

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R Double Bonds Compound Name H 4-5, 25-27 Stigmasta-4,25-dien-3-one H 4-5, 22-23,25-27 Stigmasta-4,22E,25-trien-3-one H 4-5, 22-23 Stigmasta-4,22E-dien-3-one OH 4-5, 22-23,25-27 6β-Hydroxy stigmasta-4,22E,25-trien-3-one

3β-Hydroxy stigmasta-5,22E,25-trien-7-one

Figure 17 b: Steroidal aglycones reported in Clerodendrum species

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Stigmastan-3,5-diene

Stigmasta-22E,25-dien-3,6-dione

Figure 17 c: Steroidal aglycones reported in Clerodendrum species

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Stigmasta-5,22E,25-trien-3β,7β-diol

Stigmasta-22E,25-dien-3β,6β,9α-triol

Figure 17 d: Steroidal aglycones reported in Clerodendrum species

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Double Bonds Compound Name 5-6, 25-27 Clerosterol-3-O-β-D-glucoside 5-6, 22-23, 25-27 22-Dehydro clerosterol-3-O-β-D-glucoside

Double Bonds Compound Name 5-6 β-Sitosterol-3-O-β-D-glucoside 5-6, 22-23 Stigmasterol-3-O-β-D-glucoside

24 β-Ethyl cholesta-5,22E,25(27)-trien-3-O-β-D-galactopyranoside Figure 18: Steroidal glycosides reported in Clerodendrum species

Review of Literature

Rengyolone

Clerobungin A

5-O-butyl cleroindin D

1-Hydroxy-1-(8-palmitoyloxyethyl) cyclohexanone

Figure 19 a: Other compounds reported in Clerodendrum species

Review of Literature

2R- Acetoxy methyl-1,3,3- trimethyl-4t- (3-methyl-2-buten-1-yl)-1c-cyclohexanol

Emodin

Spicatolignan B

Triacetin

Figure 19 b: Other compounds reported in Clerodendrum species

Review of Literature

Table 4: Reports on anti-inflammatory, antipyretic, analgesic and antioxidant activities of Clerodendrum species

Reference Species Plant Part Summary

C. splendens leaf and flower extracts (aqueous, alcohol, petroleum ether and butanol) were subjected to determination of LD50, and evaluation of analgesic (writhing method, p-benzoquinone-induced pain), antipyretic (saline-induced hyperthermia), and anti-inflammatory (formalin-induced C. splendens G. Leaves and (Shehata et al., 2001) paw edema) activities in rats. The extracts were found safe and non- Don flowers toxic. The alcohol and petroleum ether extracts possessed a good analgesic activity (80% protection against p-benzoquinone-induced writhing as compared to paracetamol). All extracts exhibited significant antipyretic and anti-inflammatory effects. The anti-inflammatory and antipyretic activities of the methanol extract of C. petasites were assessed. The extract was found to possess moderate acute anti-inflammatory activity in ethyl phenylpropiolate- induced ear edema (ED50, 2.34 mg/ear) and carrageenan-induced hind paw edema (ED30, 420.41 mg/kg) in rats; but, had no effect on arachidonic acid-induced hind paw edema. In sub-chronic inflammatory model (cotton pellet-induced granuloma), the extract produced (Panthong et al., C. petasites ------significant reduction of transudation but had no effect on proliferative 2003) (Lour.) S.Moore phase; it also reduced serum ALP activity. Besides, the extract exerted an excellent antipyretic effect in yeast-induced hyperthermic rats. The anti-inflammatory and antipyretic effects of the extract were assumed to be due to inhibition of prostaglandin synthesis. However, the extract did not possess any analgesic activity in acetic acid-induced writhing in mice. Results showed that C. petasites has potent antipyretic and moderate anti-inflammatory properties without ulcerogenic effect.

Review of Literature Table 4 (continued): Reports on anti-inflammatory, antipyretic, analgesic and antioxidant activities of Clerodendrum species

Reference Species Plant Part Summary

The aqueous fraction of the ethanol 90% extract of the leaf of C. splendens and its flavone-containing fraction were found to possess (Yousif and El- C. splendens G. Leaves antioxidant activity in schistosome-infected mice. They restored the lipid Regal, 2004) Don peroxides value, reduced glutathione, levels of vitamins C and E and catalase activity to normal. The antioxidant properties of trichotomoside, a new natural glycoside isolated from the stem of C. trichotomum, were investigated. The C. trichotomum compound was active towards intracellular reactive oxygen species (ROS) (Chae et al., 2006) Stems Thunb. and exhibited DPPH-radical-scavenging effect. It protected the viability of Chinese hamster lung fibroblasts (V79-4 cells, sensitive to oxidative stress) exposed to H2O2 and -irradiation. The anti-arthritic effect of orally administered ethanol extract of C. phlomidis on Freund’s adjuvant-induced arthritis was studied in rats. The loss of body weight during the arthritic condition was corrected on (Kilimozhi et al., C. phlomidis Leaves treatment with the extract (250 and 500 mg/kg). Paws swelling during 2009) L.f. secondary lesions was also markedly reduced. Results were confirmed by radiographic analysis and changes in Hind Limb Bone Mass (HLBM) density measured using photodensitometer and aluminium step wedge. The anti-inflammatory and oxytocic properties were assessed. The leaf extract and aspirin were found to inhibit carrageenan-induced paw (Okwu and C. splendens G. Leaves oedema in albino rats and mice with a strong activity in case of aspirin Iroabuchi, 2009) Don (80.43% inhibition vs. 47.83% for C. splendens extract). The extract also exhibited uterine contraction activity on guinea pig.

Review of Literature Table 4 (continued): Reports on anti-inflammatory, antipyretic, analgesic and antioxidant activities of Clerodendrum species

Reference Species Plant Part Summary The antioxidant potential of the petroleum and methanol extracts of the leaves were in-vitro evaluated using different tests viz. determination of total polyphenolic content, as well as DPPH radical, NO, superoxide (Sannigrahi et al., C. infortunatum Leaves anion radical and hydroxyl radical scavenging, in addition to reductive 2009) L. power assay. The extract was found to contain a high amount of polyphenolics and to exert significant free radical scavenging activity in all assays. Three successive extracts of C. infortunatum leaves have been studied for their potential as antioxidants in DPPH model. The scavenging activity of the ethanol extract was found to be high compared to (Gouthamchandra C. infortunatum petroleum ether and chloroform ones and was selected to evaluate its Leaves et al., 2010) L. effects using in-vitro and in-vivo models. Its antioxidant and protective effects against CCl4-induced oxidative stress in rats were significantly high. Moreover, the petroleum ether and ethanol extracts exhibited a significant wound healing activity. The antioxidant effects of ethanol extract of the defatted leaves of C. infortunatum and aqueous extract of the leaves (obtained by maceration) C. infortunatum were evaluated, at various concentrations, by DPPH radical scavenging, (Modi et al., 2010) Leaves L. FRAP (Ferric Reducing Antioxidant Power) and hydrogen peroxide radical scavenging assays, as compared to vitamin C. Results revealed that the ethanol extract has significant antioxidant activity. The anti-inflammatory activity of C. phlomidis was investigated using carrageenan- and cotton pellet-induced inflammatory models. The (Babu et al., 2011) C. phlomidis L.f. Leaves antiarthritic activity was evaluated using Freund’s adjuvant-induced rat model; the crude extract was administered at different concentrations

Review of Literature Table 4 (continued): Reports on anti-inflammatory, antipyretic, analgesic and antioxidant activities of Clerodendrum species

Reference Species Plant Part Summary

(100, 200 and 400 mg/kg) and its effects on serum SGOT, SGPT, ALP, levels of pro-inflammatory cytokines, plasma lysosomal enzymes and protein bound carbohydrates of arthritic animals were studied. The anti-inflammatory, analgesic, and antipyretic effects of the methanol and C. chinense chloroform extracts of the leaves of C. chinense, C. indicum and C. glabrum (Osbeck) were evaluated. The acute anti-inflammatory effect was estimated by the Mabb. (Wahba et al., carrageenan-induced rat paw edema method, the antipyretic effect by the yeast- C. indicum Leaves 2011) induced hyperthermia method (compared to Paracetamol) and the analgesic (L.) Kuntze effect by using an electric current anxious stimulus (compared to Novalgin). C. glabrum The methanol extract of the leaves of C. chinense and verbascoside showed E. Mey. significant analgesic, anti-inflammatory and antipyretic effects. Egg-albumin-induced paw edema method was used for evaluation of the anti- (Adediwura and C. volubile P. inflammatory activity of the methanol extract of the leaves and its fractions Leaves Yewande, 2012) Beauv. (petroleum ether and ethyl acetate). All samples exhibited significant anti- inflammatory activities (compared to Diclofenac sodium). A preliminary study on the antioxidant activity of the alcohol extract of the leaf C. inerme L. and isolated Ursolic acid was evaluated by the DPPH free radical scavenging (Devi et al., 2012) Leaves Gaertn. assay. The extract gave very good radical scavenging activity as compared to Butylated Hydroxy Toluene (BHT). The total methanol extract (TME) and 5-Hydroxy-6,7,4’-trimethoxyflavone isolated therefrom showed scavenging activity with maximum inhibition of (Ibrahim et al., C. inerme L. Aerial parts 61.84% (100 μg/mL) and 37.19% (20 μM), respectively, using DPPH assay. In 2014) Gaertn. addition, TME exhibited anti-inflammatory activity more than Indomethacin at a dose of 200 mg/kg using the formalin-induced hind paw-edema method.

Review of Literature Table 4 (continued): Reports on anti-inflammatory, antipyretic, analgesic and antioxidant activities of Clerodendrum species

Reference Species Plant Part Summary The ethanol and n-hexane extracts of the leaves of C. disparifolium and C. laevifolium were assessed for antioxidant and anti-inflammatory activities. The in-vitro anti-oxidant activity was evaluated through determination of C. dispairifolium phenolic (Folin–Ciocalteu method) and flavonoid (AlCl method) contents, (Phosrithong and Blume 3 Leaves free-radical scavenging activity (DPPH assay), total reductive capability assay, Nuchtavorn, 2015) C. laevifolium total antioxidant activity (ferric thiocyanate, FTC assay) and thiobarbituric Blume acid (TBA) method. The lipid peroxidation inhibitory effect was evaluated by FTC and TBA methods. The anti-inflammatory activity was assayed against lipoxygenase using Indomethacin as standard. The anti-inflammatory effect of the n-hexane, ethyl acetate and water fractions of the ethanol extract of C. inerme leaves was determined by measuring NO C. inerme L. (Srisook et al., 2015) Leaves levels produced in RAW 264.7 macrophage stimulated with LPS. Levels of Gaertn.. RNA and protein were determined by real-time reverse transcription- polymerase chain reaction and western blot analysis. The analgesic property of C. volubile, commonly used among others for pain management in southwestern Nigeria, was investigated. The anti- inflammatory activity of the methanol extract of the leaf was evaluated using C. volubile P. Wistar Albino Mice (WAM) of both sexes as compared to Acetaminophen, (Senjobi et al., 2017) Leaves Beauv. aspirin and indomethacin. Results revealed that the extract demonstrated a dose-dependent activity on acetic acid-induced writhing thus supporting the reported analgesic and anti-inflammatory activities of extracts of other species related to the studied plant. The DPPH radical scavenging activity of 8 compounds isolated from the C. inerme L. leaves of C. inerme was evaluated. Crolerodendrum B, uncinatone and (Vinh et al., 2017) Leaves Gaertn.. harwickiic acid exhibited strong DPPH radical-scavenging effects (ED50 values of 17.6 ± 2.1, 10.1 ± 0.8 and 11.3 ± 0.3 μM, respectively). 46

Review of Literature

Table 4 (continued): Reports on anti-inflammatory, antipyretic, analgesic and antioxidant activities of Clerodendrum species

Reference Species Plant Part Summary The anti-nociceptive and anti-inflammatory activities of the hydro-ethanol extract of the leaf of C. polycephalum (HeCP) were investigated in animals. Different doses of HeCP (100, 200 or 400 mg/kg, p.o.) were given 1h before administration of either acetic acid, formalin or capsaicin for evaluation of nociceptive behavior in mice. Meanwhile, carrageenan or cotton pellet were used to induce acute or chronic inflammation in rats. HeCP (100-400 mg/kg) reduced mean writhes number, duration of paw licking or biting in the acetic acid, formalin and capsaicin models, respectively. However, initial treatment (Amole et al., C. polycephalum Leaves of mice with L-NNA (neuronal nitric oxide synthase inhibitor), Naloxone 2018) Baker (opioid receptor antagonist), or Glibenclamide (ATP-sensitive K+ channel blocker) prevented the HeCP induced anti-nociception in mice. In contrast, initial treatment of mice with Sulpiride (dopamine D2-receptor antagonist) failed to reverse HeCP-induced anti-nociception. Besides, HeCP caused significant but not dose-dependent edema inhibition in carrageenan-induced inflammation and cotton pellet-induced granuloma formation in rats. Results indicated that the extract exerted anti-nociceptive and anti-inflammatory effects possibly due to its polyphenolic constituents. The anti-inflammatory activity of various extracts of the root bark of C. infortunatum were screened by proteinase inhibitory and nitric oxide scavenging assays. The total phenolic content was evaluated by Folin- C. infortunatum Ciocalteu method, and was correlated to the anti-inflammatory activity. The (Helen et al., 2018) Root bark L. active extract was then tested in-vivo on carrageenan-induced paw edema. The aqueous acetone extract possessed maximum anti-inflammatory activity in the in-vitro screening and highest phenolic content. In the carrageenan- induced acute inflammatory study, administration of this extract significantly 47

Review of Literature

Table 4 (continued): Reports on anti-inflammatory, antipyretic, analgesic and antioxidant activities of Clerodendrum species

Reference Species Plant Part Summary

reduced the paw volume in rats. The haematological, biochemical and histopathological findings also point to the anti-inflammatory activity of the extract. The phenolic compounds present in the root bark were suggested to be responsible for the activities exhibited by the plant and hence could be used against inflammatory diseases. A novel approach was employed for the synthesis of un-doped tinoxide and Cobalt-doped tinoxide (Co-doped SnO ) nanoparticles (NAPs) by using aqueous (Khan et al., C. inerme L. 2 Leaves extract of C. inerme with the help of eco-friendly superficial solution combustion 2018) Gaertn. method. The green synthesized Co-doped SnO2 NAPs demonstrated a substantial antioxidant activity by scavenging DPPH free radical. The anti-asthmatic activity of the hydro-alcoholic extract of C. serratum leaf was assessed in rats where most characteristics features of human asthma including pathological changes can be duplicated. In this study, the total and differential cell count from BALF (Broncho alveolar lavage fluid) was increased two fold in ova treated animals and less significant increase was observed in treated groups. (Nageswari et C. serratum (L.) During asthmatic attack, because of antigenic response of the allergen, the WBC Leaves al., 2018) Moon. count is commonly increased. Treatment with the extract might have reduced the migration of inflammatory cells in the BALF and hence reduced total number and differential leukocyte count. The ability of the extract to reduce MPO (Myeloperoxidase) activity pointed to a significant reduction in neutrophils migration into the lung tissue and hence protection from tissue damage due to inflammatory mediators.

Review of Literature Table 4 (continued): Reports on anti-inflammatory, antipyretic, analgesic and antioxidant activities of Clerodendrum species

Reference Species Plant Part Summary

The antioxidant effect of the ethanol extract and fractions of C. splendens aerial parts was evaluated on testes of rats. Testicular antioxidant level was determined by measuring superoxide dismutase, reduced glutathione and (Victoria and C. splendens glutathione-S-transferase activities. Results revealed that the testicular GST, Aerial parts Akinseye, 2018) G. Don SOD and GSH activities were significantly decreased in rats receiving various doses of the extract, except at 1000 mg/g b. wt. revealing that the extract and fractions had deleterious effects on the testes and accessory organs of the animals.

Review of Literature Table 5: Reports on cytotoxic, anti-cancer and anti-mutagenic activities of Clerodendrum species

Reference Species Plant Part Summary

The antitumor activity of the ethanol extracts of 12 medicinal plants of Bangladesh was studied using the potato disk bioassay as compared to C. viscosum Vent. (Haque et al., the vincristine/vinblastine producing Catharanthus roseus. Among these, (syn. Roots 2000) the extract of C. viscosum root (25.0 µg/disc) exhibited significant C. infortunatum L.) inhibition (33.31%) of crown gall tumors caused by Agrobacterium tumefaciens. The leaves of C. inerme afforded three diterpenoids; Inermein A and B and 3-Epicaryoptin. Among these, only Inermein B showed potent (Hosny et al., cytotoxicity (IC 7.34-12.30 µg/mL) against HL-60 (human acute C. inerme L. Gaertn. Leaves 50 2004) promyelocetic leukemia), U-937 (human hystocytic leukemia) and 3LL (human carcinoma) cell lines; while, 3-Epicaryoptin exhibited significant cytotoxicity against HL-60 (IC50 9.10 µg/mL). Dichloromethane and 90% methanol extracts of 42 South African plants were screened for mutagenicity and anti-mutagenicity using the Salmonella/microsome mutagenicity assay (Ames) against Salmonella typhimurium TA98 and TA100 bacterial strains in the presence and (Reid et al., C. myricoides Leaves absence of metabolic activator S9. Six species indicated anti-mutagenic 2006) (Hochst.) properties, all in the presence of S9 including the dichloromethane leaf extract of C. myricoides that indicated anti-mutagenicity against both TA98 at 5.0 and 0.5 mg/mL (91 and 40%) and TA100 at 5.0, 0.5 and 0.05 mg/mL (73, 66 and 42%). Among 14 rearranged abietane diterpenoids isolated from the roots of C. trichotomum, only 6 17(15→16), 18(4→3)- diabeo-abietane derivatives (Wang et al., C. trichotomum Roots showed cytotoxic effects against 5 human cancer cell lines (BGC-823, 2013 a) Thunb. Huh-7, KB, KE-97, and Jurkat; IC50= 0.83–50.99 µM). The CellTiter Glo™ Luminescent cell-viability assay method was used for assessment. 50

Review of Literature Table 5 (continued): Reports on cytotoxic, anti-cancer and anti-mutagenic activities of Clerodendrum species Reference Species Plant Part Summary

Three compounds were found more toxic indicating that introduction of a polar

group (e.g. keto or epoxy moiety) into A-ring could enhance the cytotoxicity. The CellTiter-Glo™ luminescent cell-viability assay was applied for estimation of the cytotoxic potential of a novel dimeric diterpene (trichotomone), isolated from (Wang et al., C. trichotomum Roots the roots of C. trichotomum. The tested compound exhibited significant 2013 b) Thunb. cytotoxicity against several human cancer cell lines (A549, Jurkat, BGC-823 and 293T WT; IC50 7.51-19.38 µM). The cytotoxicity of 6 out of 7 steroids isolated from C. trichotomum leaves were tested against HeLa cell line. Cell growth was estimated by MTT assay. Cell (Xu et al., C. trichotomum viability was obtained by scanning with a microplate reader at 570 nm and IC Leaves 50 2013) Thunb. values calculated. The new compound (20R,22E,24R)-3β-hydroxy-stigmasta- 5,22,25-trien-7-one and the previously isolated (20R,22E,24R)-stigmasta-5,22,25- trien-3β,7β-diol exhibited moderate cytotoxicity. The cytotoxicity of extracts of C. splendens aerial parts and compounds isolated therefrom was evaluated against different cell lines, using MTT assay. Results revealed that the ethyl acetate extract exerted high cytotoxic activity against HepG2, HEp-2, HCT-116 and MCF-7; while, the n-butanol extract was efficient (Soliman et al., C. splendens G. Aerial parts against HCT-116 only. Among compounds isolated from EtOAc extract, the 2014) Don following showed significantly high activity: Hispidulin-7-O-glucoside (against HepG2 and HEp-2), Methyl rosmarinate (against HCT-116) and caffeic acid (against HepG2, HEp-2 and HCT-116). While, Lutein isolated from the chloroform fraction exerted only moderate cytotoxic activity against MDA-MB-231. Two new diterpenoids [3β-(β-D-glucopyranosyl) isopimara-7,15-diene-11α,12 α- (Sun et al., C. bungei diol (1) and 16-O-β-D-glucopyranosyl-3β-20-epoxy-3-hydroxyabieta-8,11,13- Roots 2014) Steud. triene (2)], alongside 3 known ones, isolated from C. bungei roots were evaluated for cytotoxicity against several tumor cell lines. IC50s were determined using MTT

Review of Literature Table 5 (continued): Reports on cytotoxic, anti-cancer and anti-mutagenic activities of Clerodendrum species Reference Species Plant Part Summary

assay against B16 (murine melanoma), HGC-27 and BEL-7402 (human gastric and hepato-cellular carcinoma) cell lines as compared to Paclitaxel.

All compounds were inactive against the 3 cell lines (IC50>10µM), except (2) that showed moderate cytotoxicity (IC50=8.8, 9.8, and 7.1 µM). The cytotoxicity of 2 new cyclohexylethanoids [1-hydroxy-1-(8- palmitoyloxyethyl) cyclohexanone (1) and 5-O-butyl cleroindin D (2)] C. trichotomum (Xu et al., 2014) Leaves isolated from the leaves of C. trichotomum was evaluated (MTT assay) Thunb. against A549 human lung carcinoma cell line as compared to Fluorouracil (5-FU). Both compounds showed no obvious cytotoxicity. The cytotoxic activity of the ethanol extract of the leaves of C. inerme was (Kalavathi and C. inerme L. studied (MTT assay) against lung adenocarcinoma epithelial cell line Leaves Sagayagiri, 2016) Gaertn. (A549). The extract exhibited anticancer activity (IC50 15.6 μg/mL concentration). The cytotoxicity of 8 abietane diterpenoids, isolated from the hydroalcoholic extract of the herb was evaluated (MTT assay), against 4 human cell lines (HL-60 human myeloid leukemia, SMMC-7721 C. kiangsiense (Xu et al. 2016) Stems hepatocellular carcinoma, A-549 lung cancer and MCF-7 breast cancer). Merr. Cryptojaponol, fortunin E and the new compound 12-methoxy-6,11,14,16- tetrahydroxy-17(15→16)-abeo-5,8,11,13-abietatetraen-3,7-dione exhibited significant cytotoxicity against all cell lines. The cytotoxic activity of 8 compounds isolated from the leaves of C. inerme, including the new abietane diterpene, Crolerodendrum B, was C. inerme L. (Vinh et al., 2017) Leaves evaluated against MCF-7 (breast), HCT116 (colon) and B16F10 Gaertn. (melanoma) cancer cell lines. Harwickiic acid showed strong cytotoxicity against the HCT116 cell line (IC50 3.46 ± 0.01 μM).

Review of Literature Table 5 (continued): Reports on cytotoxic, anti-cancer and anti-mutagenic activities of Clerodendrum species Reference Species Plant Part Summary

A novel approach was employed for the synthesis of un-doped tinoxide and Cobalt-doped tinoxide (Co-doped SnO2) nanoparticles (NAPs) by using aqueous extract of C. inerme with the help of eco-friendly superficial solution combustion method. The green synthesized Co-doped SnO2 NAPs demonstrated significant in- (Khan et al., C. inerme L. vitro anticancer and in-vivo antitumor activity on breast carcinoma (MCF-7) and Leaves 2018) Gaertn. Ehrlich ascites carcinoma compared to standard. The hemolytic activity disclosed low cytotoxicity of fabricated NAPs (0.89 ± 0.05%) at 5 mg/mL, as indicated by their biocompatibility potential. Due to their multi-purpose properties, the NAPs synthesized in this study were suggested for pharmaceutical and nanomedicine applications. The cytotoxic and anti-proliferative potential of the hydroalcoholic extract of the aerial parts of C. inerme was evaluated. The extract showed cytotoxicity<100 ppm and LD50 at 30 ppm level on brine shrimp lethality assay and MTT assay. A decrease in cell viability was observed (at 213 μg/mL concentration). Antitumor (Kumar et al., C. inerme L. Aerial parts efficacy against Burkitt’s lymphoma cells and tumor model in female mice was 2018) Gaertn. revealed via the increased life span, as compared to control group. Treatment with C. inerme (200 and 400 mg/kg b. wt.) and Doxorubicin (2.5 mg/kg b. wt.) exhibited significant effect on tumor parameters. Hence, C. inerme was considered as potent antitumor. The extracts of both plants were screened for cytotoxicity on SW620 cell line. C. indicum Besides, the cytotoxic activity of the compounds isolated from the active extracts (Somwong and (L.) Kuntze Roots were evaluated against five human cancer cell lines (SW620, ChaGo-K-1, HepG2, Suttisri, 2018) C. villosum KATO-III and BT-474) using MTT assay. The dichloromethane extracts of both Blume C. indicum and C. villosum were active against SW620 cell line.

Review of Literature Table 6: Reports on hepatoprotective, anti-hyperglycemic, anti-hyperlipidemic and anti-hypertensive activities of Clerodendrum species Reference Species Plant Part Summary

The hepatoprotective activity of the ethanol extract of C. serratum root and isolated ursolic acid was evaluated against CCl4-induced toxicity in rats. C. serratum (L.) Serum markers (total bilirubin and protein levels, and ALT, AST and ALP (Vidya et al., 2007) Roots Moon. activities) were measured, as compared to Silymarin. Ursolic acid ameliorated all parameters better than the extract. This was confirmed through histological examination of liver tissue. Acute oral toxicity study revealed the safety of the aqueous extract of fresh leaves of C. capitatum (up to 5000 mg/kg b. wt.) although; transient somatomotor and behavioral toxicities were recorded. The antihyperglycemic and antihyperlipidemic effects of the extract were studied C. capitatum (Adeneye et al., in rats by oral administration of graded doses (100, 400 and 800 mg/kg/day) Schumach. & Leaves 2008) for 14 days. On the 15th day, blood samples were collected. The extract Thonn. exhibited significant dose-dependent antihyperglycemic and antihyperlipidemic activities but with no effect on the pattern of weight gain in the treated animals. These results supported the folk use of the plant in treatment of type 2 diabetes. The hepatoprotective activity of the ethanol extract of C. inerme was investigated against CCl4-induced hepatotoxicity in rats, at a dose of 200 mg/kg for seven days as compared to silymarin (200 mg/kg). At the end of (George and C. inerme L. treatment blood samples collected by direct cardiac puncture and serum were Leaves Joseph, 2008) Gaertn. used for the marker enzymes AST, ALT, alkaline phosphatase, as well as cholesterol and triglyceride contents. Rats treated with C. inerme extract showed a significant decrease in the markers levels indicating liver protection against carbon tetrachloride-induced hepatotoxicity.

Review of Literature Table 6 (continued): Reports on hepatoprotective, anti-hyperglycemic, anti-hyperlipidemic and anti-hypertensive activities of Clerodendrum species Reference Species Plant Part Summary

The protective role of the aqueous extract of the leaves C. glandulosum against high fat diet/fatty acid induced lipotoxicity was evaluated in models of non-alcoholic steatohepatitis (NASH). Supplementation of NASH mice with the extract (1% and 3% in high fat diet for 16 weeks) prevented the induced elevation in liver enzymes, plasma and hepatic lipids, mitochondrial C. glandulosum (Jadeja et al., 2010) Leaves oxidative stress and compromised enzymatic and non-enzymatic antioxidant Lindl. status and histopathological damage to hepatocytes. Results of in-vitro study indicated that addition of the extract (20–200 μg/mL for 24h) to HepG2 cells minimized oleic acid induced lipid accumulation, higher lipid peroxidation cytotoxicity and reduced cell viability. These studies suggested that the extract has the potential of preventing high fat/fatty acid induced NASH. The effect of the aqueous extract of the leaf C. glandulosum (CG) on expression of genes regulating visceral adiposity and in-vitro adipocyte differentiation and LEP release was explored. Body weight, lee index, plasma lipids and LEP, mRNA expression of PPAR-2, SREBP1c, FAS, C. glandulosum CPT-1 and LEP in epididymal adipose tissue of control and experimental (Jadeja et al., 2011) Leaves Lindl. groups were evaluated. The CG extract potential on adipocyte differentiation and LEP release was assessed in-vitro. Results revealed that CG extract prevents adipocyte differentiation and visceral adiposity through down regulation of PPAR-2 related genes and LEP expression thus validating its traditional use in controlling obesity. The methanol extract of the plant (400 mg/kg b. wt.) produced a significant (Panigrahi et al., C. inerme L. decrease in fasting blood glucose level (54.32%) after 10 hours of treatment. Aerial parts 2015) Gaertn. In the oral glucose tolerance test, the extract (400 mg/kg) decreased hyperglycemia and restored of the elevated glucose levels to normal.

The effects of phenolic (free and bound) extracts of the leaves of C. volubile on enzymes [α-amylase, α-glucosidase, Angiotensin-I converting enzyme (ACE)], some pro-oxidant [Fe2+- and sodium nitroprusside-induced lipid peroxidation in the pancreas], and antioxidant properties were investigated in-vitro. The extracts (Adefegha and C. volubile P. Leaves inhibited α-amylase and α-glucosidase (key enzymes linked to non- insulin Oboh, 2016) Beauv. dependent diabetes, NIDDM), and ACE (key enzyme linked to hypertension). They also exhibited antioxidant and lipid peroxidation inhibitory activities. These findings could explain the biochemical rational behind the traditional use of the leaf in prevention and management of NIDDM and hypertension. This study aimed to screen α-amylase and α-glucosidase inhibitors from ethanol extracts of leaves and roots of C. inerme at different concentrations, in order to minimize side effects and toxicity of inhibitors like Acarbose. Restraint of these (Thirumal and C. inerme L. Leaves and enzymes proteins prevents increment of blood glucose level after a carbohydrate Muthusamy, 2016) Gaertn. roots diet and can be a critical technique in management of NIDDM. Results obtained indicated the potential of these extracts to manage hyperglycemia. IC50 values required to inhibit α-amylase and α-glucosidase were 65.64 and 54.00 μg/mL for leaf extract, and 76.98 and 46.70 μg/mL for that of the root. The protective effect of the methanol extract of the leaves of C. volubile against CCl4-induced hepatotoxicity in rats was investigated. The significant increase in the levels of ALT, AST, ALP enzymes, as well as hepatic degeneration, and (Molehin et al., C. volubile P. Leaves inflammation were attenuated by the extract. The extract significantly prevented 2017) Beauv. the decrease in HDL level and the increase in LDL in a dose-dependent manner. The decrease in total protein was moderately increased. Lipid peroxidation was significantly decreased; while glutathione level and hepatic antioxidant enzymes

(catalase, superoxide dismutase, and glutathione peroxidase) activities were significantly elevated. These findings indicated that the extract has a significant

protective effect against CCl4-induced hepatotoxicity in rats, which may be due to its antioxidant properties which was comparable to vitamin E. The protective effect of C. infortunatum (CI) was investigated by targeting its antioxidant properties against sub-acute arsenic-induced liver damage in rats. At the end of treatment period, liver weight were recorded and samples were C. infortunatum collected for examination of oxidative stress parameters (SOD and MDA levels) (Singh et al., 2017) Leaves L. and liver functioning enzyme markers (ALT and AST). Results suggested that CI extract might impede oxidative stress, lowered elevated liver enzyme levels and protected damaged cells. C. infortunatum played thus a protective role against arsenic-induced liver toxicity and might be used as a potential remedy. The anti-hyperglycemic, anti-hyperlipidemic and antioxidant effect of C. volubile leaves in (STZ)-induced diabetic rats was evaluated. The aqueous extract was prepared and its effect assessed on relevant enzymes associated with diabetes. Induction of diabetes in rats was by a single intraperitoneal injection of STZ (65 mg/kg b. wt.) while C. volubile extract (50, 100 and 200 mg/kg b. wt.) (Molehin et al., C. volubile P. was administered orally to diabetic and non-diabetic animals for 14 days. Leaves 2018) Beauv. Metformin (100 mg/kg b. wt.) served as positive control. The extract inhibited α-glucosidase (IC50 0.20 mg/mL) and α-amylase (IC50 0.58 mg/mL). The extract significantly reduced the elevated plasma glucose level and body weight, improved kidney functions, attenuated oxidative stress by decreasing MDA levels and enhancing activities of superoxide dismutase, catalase and glutathione peroxidase enzymes; it reinstated the lipid profile to a nearly normal

level and restored pancreatic histological integrity in diabetic rats. Results revealed that C. volubile represents a source of phytochemicals that exerts their

antidiabetic effects through modulation of glycemic and atherogenic indices as well as mitigation of free-radical mediated damage. Ellagic acid (EA) isolated from C. viscosum leaves is a natural antioxidant with previously reported pharmacological properties; yet, there is no significant evidence available on its protective potential against iron-overload induced hepatotoxicity. In this study, EA was evaluated for its in-vitro free radical scavenging and iron chelation potentials. EA was tested in-vivo for its protective activity against iron overload-induced hepatotoxicity in Swiss albino mice by evaluating liver iron content, reactive oxygen species (ROS), (Shendge et al., liver antioxidant enzymes, serum marker levels, liver damage and fibrosis, C. viscosum Vent. Leaves 2018) histopathological study and finally western blotting analysis. EA treatment significantly decreased liver iron and serum ferritin levels. Elevated ROS levels, decreased antioxidant parameters and elevated serum markers were normalized. Cellular morphology, iron-overload and liver fibrosis were effectively ameliorated. Finally, the protective effect of EA against iron overload-induced apoptosis was confirmed by western blotting. These results support the possible use of EA as a promising candidate against iron- overloaded diseases.

Review of Literature

Table 7: Reports on cardiovascular activities of Clerodendrum species Reference Species Plant Part Summary

C. trichotomum herb is used for treatment of hypertension in traditional Chinese medicine. To assess its action on blood pressure and renal function, the extract was administered to rats and dogs. Intravenous administration of C. trichotomum the extract elicited renal vasodilation and increased urine flow and urinary (Lu et al., 1994) Leaves Thunb. sodium excretion. Acute oral administration of the extract reduced blood pressure of spontaneously hypertensive rats but not of normotensive control rats. Chronic daily administration for 6 weeks prevented the increase in blood pressure of spontaneously hypertensive rats. Bioassay-guided fractionation and purification of the EtOAc-soluble extract afforded a number of phenylpropanoid glycosides (acteoside, C. trichotomum (Kang et al., 2003) Stems leucosceptoside A, martynoside, acteoside isomer, and isomartynoside), Thunb. which significantly inhibited angiotensin converting enzyme (ACE) activities. The protective effect of the aqueous extract of the leaf of C. colebrookianum against ischemic-reperfusion injury (IRI) and IRI-induced changes was examined in endogenous antioxidant enzyme activities of rat heart. Isolated hearts were perfused (Langendorff’s method), and 20 min of global ischemia followed by 40 min of reperfusion. Lipid peroxidation after IRI was significantly reduced in the treated heart compared to the control group and C. colebrookianum (Devi et al., 2005) Leaves leakage of lactate dehydrogenase (LDH) inhibited during reperfusion. Walp. Moreover, the extract diminished myocardial antioxidant enzymes (SOD, Catalase, GSH and GPx) depletion, and IRI-induced cellular damage was significantly less in treated myocytes. Results indicated that the extract protects against oxidative stress and cellular injury associated with IRI of rat heart; and suggested that these protective effects could be attributed to its antioxidant properties.

Review of Literature Table 7 (continued): Reports on cardiovascular activities of Clerodendrum species Reference Species Plant Part Summary

The aqueous extract of C inerme had no effect on blood pressure, at low doses (≤ 10-4 mg/mL). For doses ranging from 10-3 mg/mL to 10 mg/mL, it developed a gradual and reversible hypotension; a lowering of normal (N’Guessan, et C. inerme L. pressure level and a decrease in the power of systoles was observed. At 20 Leaves al., 2010) Gaertn. mg/mL, the hypotension remained steady. C inerme had no influence on blood pressure at doses ≤ 10-4 mg/mL. At higher doses (≥10-3 mg/mL), it lowered blood pressure. Hence, it was concluded that the aqueous extract of the leaves of C.inerme exerts a hypotensive effect. The antihypertensive potential of the aqueous extract of the leaves of C. colebrookianum, its n-butanol, ethyl acetate and chloroform fractions was evaluated in-vitro, in-vivo and ex-vivo, by using fructose-induced hypertensive rats and isolated frog heart. To investigate the mechanism of action, the ex-vivo muscarinic action (in isolated rat ileum), and in-vitro (Lokesh and C. colebrookianum assay for Rho-kinase (ROCK–II), phosphodiesterase-5 (PDE-5) and ACE Amitsankar, Leaves Walp. were carried out. The total phenolic and flavonoid contents were estimated 2012) in the samples to establish phyto-pharmacological relationship. It was found that the antihypertensive activity is mediated through cholinergic action and following ROCK-II and PDE-5 inhibition. The antioxidant properties of flavonoids present in the samples play a vital role in their antihypertensive effects.

(Adefegha and C. volubile P. Leaves See table (6) page (56). Oboh, 2016) Beauv.

Review of Literature Table 8: Reports on central nervous system activities of Clerodendrum species Reference Species Plant Part Summary

The psychopharmacological activities of the methanol extract of C. phlomidis leaves were evaluated. The extract was found to cause significant reduction in spontaneous activity, and decreases in exploratory behavioral (Murugesan et al., C. phlomidis L.f. Leaves profiles by the Y-maze and head dip tests. It also reduced muscle relaxant 2001) activity by rotarod, 30° inclined screen and traction tests, and potentiated the phenobarbitone sodium-induced sleeping time at doses of 200, 400 and 600 mg/kg body wt., as compared to standard controls. The nootropic potential (memory enhancing effects) of the methanol extracts of the leaves of Alternanthera sessilis (Amaranthaceae) and C. infortunatum was evaluated (at 100 and 200 mg/kg) using rectangular maze and Y maze (interoceptive behavioral models). The effect on acquisition, retention and (Gupta and Singh C. infortunatum retrieval of spatial recognition memory was determined. Bacopania Leaves 2012) L. monniera extract was used as standard drug and Scopolamine hydrobromide as amnestic agent. C. infortunatum extract (200 mg/kg) gave maximal response with results close to the standard drug. It afforded great memory enhancing effects, evoking pronounced alteration behavior and better learning assessments. The ethanol extract of C. inerme leaves was found to ameliorate methamphetamine-induced hyperlocomotion (MIH) in mice that mimic the hyper-dopaminergic status of tic disorders, Tourette syndrome, schizophrenia, or obsessive-compulsive disorder. Bioactivity-guided C. inerme L. (Huang et al., 2015) Leaves fractionation of the extract with n-hexane, dichloromethane, n-butanol and Gaertn. water revealed that its dichloromethane and n-hexane fractions were active. The flavonoid hispidulin isolated from the dichloromethane fraction significantly alleviated MIH in mice at doses that did not affect their spontaneous locomotor activity or performance in rotarod test, a measure for

Review of Literature Table 8 (continued): Reports on central nervous system activities of Clerodendrum species Reference Species Plant Part Summary

motor coordination. These results suggested a therapeutic potential of hispidulin in hyper-dopaminergic disorders. The effect of the hydroalcoholic extract of C. glandulosum leaf on oxidant- antioxidant status in ischemia-hypoperfusion injury, in rat forebrain, was investigated. To assess its preventive and curative effects, the extract (200 and 400 mg/kg), was orally administered, once daily for 2 weeks after bilateral common carotid artery occlusion and compared to Quercetin (standard drug, 10 mg/kg). An animal group was used as normal control, (Surapaneni et al., C. glandulosum and another as hypoperfusion control. By the end of the experiment, rats Leaves 2016) Lindl. were subjected to behavioral studies, then sacrificed and brain removed and homogenized. Lipid peroxidation (LPO), Myeloperoxidase (MPO) and protein levels, as well as Superoxide dismutase (SOD) and Catalase (CAT) activities were estimated. In addition, infarct size and histopathological changes were observed. Based on behavioral studies, antioxidant effects, marked decrease in cerebral infract size and histopathological report, it was concluded that the tested extract exhibits cerebroprotective activity.

Review of Literature Table 9: Reports on antimicrobial activity of Clerodendrum species

Reference Species Plant Part Summary

The ethyl acetate and n-hexane extracts of leaves and stems of C. inerme and C. C. inerme L. phlomidis were screened for antifungal activity by poison plate technique as (Anitha and Gaertn. Leaves and compared to Ketaconazole (6 mg/mL) and Nystatin (200 µg/µl). Extracts of C. Kannan, 2006) C. phlomidis stems phlomidis stem and leaf appreciably inhibited all tested plant and human L.f. pathogenic fungi. Yet, leaf n-hexane extract (1 mg/mL) of C. inerme inhibited plant pathogenic fungi better than human dermatophytes. The antimicrobial activities of different extracts (ethanol, benzene and aqueous) of C. inerme leaves and roots were evaluated, by the disc diffusion method against Gram-positive (Bacillus subtilis, Staphylococcus aureus) and Gram- (Chahal et al., C. inerme L. Leaves and negative (Escherichia coli, Pseudomonas aeruginosa) bacterial and fungal 2010) Gaertn. roots (Aspergillus niger, Aspergillus flavus, Candida albicans and Candida glabrata) strains. The methanol extract of the leaves exhibited highest zone of inhibition against S. aureus and A. niger with low MIC values. Meanwhile, no activity was observed for the aqueous extract against the tested pathogenic strains. The antimicrobial activity of the ethanol and chloroform extracts of roots, stems and leaves was tested, using the disc diffusion method, against 6 Gram-positive Leaves, and 9 Gram-negative bacterial and 7 fungal strains. All the extracts showed (Waliullah et al., C. infortunatum stems and significant inhibitory activity over the bacteria and fungus comparable to the 2014) L. roots standard drug tetracycline and fluconazole. MIC of the most susceptible microorganisms were determined. The effect of the leaf extracts was the most potent (leaf>root>stem). The antimicrobial potential of the petroleum ether, chloroform, methanol and aqueous extracts of the leaf and bark of C. splendens was evaluated, by disc (Ajaib et al., C. splendens G. Leaves and diffusion method, against 4 bacterial strains the Gram-positive S. aureus and the 2014) Don bark Gram-negative P. aeruginosa, E. coli and K. pneumonia, and 2 fungal strains A. niger and A. oryzae. The petroleum ether extracts showed the highest resistance

Review of Literature Table 9 (continued): Reports on antimicrobial activity of Clerodendrum species Reference Species Plant Part Summary

against all tested bacterial strains; the zones measured against K. pneumoniae were greater than the standard disc. The chloroform extracts displayed maximum antibacterial potential as compared to the others especially against K. pneumonia. The lowest activity was recorded for water extracts against all bacterial strains. Concerning the antimycotic potential, the A. oryzae had offered more resistance to the tested extracts than A. niger; the respective effectiveness was petroleum ether>chloroform>methanol>aqueous. The antibacterial efficacy of 5 Indian medicinal plants including C. inerme was evaluated, by the disc diffusion method, against opportunistic bacterial (Mickymaray et C. inerme L. pathogens isolated from HIV infected patients viz., the Gram-negative Leaves al., 2016) Gaertn. Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi and Serratia marcescens and the Gram-positive Staphylococcus aureus. The alcohol extract showed significant activity against S. typhi only. The antimicrobial and anti-tubercular activities of a lanostane type triterpene carboxylic acid (1) and a lupane type triterpenoid (2) isolated from the ethyl C. capitatum acetate soluble fraction of the leaves of C. capitatum were evaluated. The (Habila et al., Schumach. & Leaves compounds were active against 9 out of 12 tested microorganisms including; 2018) Thonn. Bacillus subtillis, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans and two resistant strains (vancomycin resistant entrococci VRE, and methicillin resistant Staphylococcus aureus MRSA). A novel approach was employed for the synthesis of un-doped tinoxide and Cobalt-doped tinoxide (Co-doped SnO2) nanoparticles (NAPs) by using C. inerme L. aqueous extract of C. inerme with the help of eco-friendly superficial solution (Khan et al., 2018) Leaves Gaertn. combustion method. Antimicrobial propensity of the synthesized NAPs was determined by agar well assay, SEM, TEM and confocal laser scanning microscopic analysis against various bacterial and fungal strains. Synthesized

Review of Literature Table 9 (continued): Reports on antimicrobial activity of Clerodendrum species Reference Species Plant Part Summary

Co-doped SnO2 NAPs showed extraordinary antibacterial and antifungal activities against E. coli, B. subtilis, A. niger, A. flavus, and C. albicans with zone of inhibitions of 30 ± 0.08 mm and 26 ± 0.06 mm, 17 ± 0.04 mm, 23 ± 0.08 mm and 26 ± 0.06 mm, respectively which were also evidenced from SEM, TEM and confocal laser scanning microscopy. The success of chemotherapy lies in the continuous search for new drugs to counter the challenge posed by resistant bacterial strains. The antibacterial activity of the aqueous extract of the leaf of C. serratum was investigated, by the agar disc (Rashid et al., C. serratum (L.) diffusion method, against 5 bacterial pathogens namely Staphylococcus hominis, Leaves 2018) Moon. Pseudomonas putida, Proteus vulgaris, Bacillus subtilis and Escherichia coli. Among the various concentrations used, 100 mg/mL of aqueous leaf extract was found to be more effective against all tested microorganisms. The highest activity was recorded against E. coli. The study aimed to develop an eco-friendly herbal fungicide from chloroform extract of C. infortunatum leaves. Seven phenolic acids were identified by LC- MS/MS analysis in different fractions. Emulsifiable Concentrate (20%) formulation made with the extract and fractions was tested against Phomopsis (Saha et al., C. infortunatum Leaves vexans that causes fruit rot disease in brinjal (eggplant). Of the various fractions, 2018) L. F8 displayed highest antifungal activity. The antifungal activity of leaf extract/fractions was correlated with total phenol, total flavonoid and total phenolic acid contents. The relationship between phenolic composition and activity was also reported.

Review of Literature

Table 10: Reports on antimalarial activity of Clerodendrum species Reference Species Plant Part Summary

Methanol extracts from 15 medicinal plants (representing 11 families) used traditionally for malaria treatment in Kenya were screened for antimalarial (Muregi et al., C. myricoides Leaves and activity in-vivo (mice) against a chloroquine (CQ)-tolerant Plasmodium 2007) (Hochst.) root bark berghei NK65, either alone or in combination with CQ. C. myricoides (leaf and root bark) was among plants with considerable in-vivo chemo- suppression. The ethanol extracts of 32 plants were tested against chloroquine-resistant C. splendens G. FcB1/ Colombia strain of Plasmodium falciparum among which was C. (Guédé et al., 2010) Whole plant Don splendens (whole plant). Yet, IC50 of its ethanol extract against P. falciparum was >50 μg/mL and thus considered as inactive. The sun-dried leaf powder of C. inerme was extracted with methanol, chloroform, petroleum ether and n-hexane. Extracts were tested against third/fourth instar larvae of Aedes aegypti and Culex quinquefasciatus. C. inerme L. (Patil et al., 2014) Leaves (according to WHO). The n-hexane extract exhibited effective growth Gaertn. disruptive activity against Ae. aegypti and was tested against Cx. quinquefasciatus and non-target organism Gambusia affinis (bio-control agent), but its LD50 against the latter was much higher. Plant samples were tested for antiplasmodial activity against chloroquine sensitive (D6) and resistant (W2) Plasmodium falciparum using the ability of their extracts to inhibit the incorporation of [G-3H] hypoxanthine into the (Muthaura et al., C. myricoides Leaves and malaria parasites. The in-vitro antiplasmodial activity of 50 plant species 2015) (Hochst.) root bark were evaluated among which 12 showed antiplasmodial efficacy of IC50<5 μg/mL including C. myricoides (root bark: methanol, dichloromethane; Leaf: methanol).

Review of Literature Table 11: Reports on immunomodulatory and wound healing activities of Clerodendrum species Reference Species Plant Part Summary

Polysaccharides extracted from C. splendens leaves were fractionated by ion exchange and size-exclusion chromatography. Immunomodulatory activity of the fractions was evaluated through determination of their ability to induce monocyte/macrophage nitric oxide (NO), cytokine production, and mitogen- activated protein kinase (MAPK) phosphorylation in mice, and severity of EAE was monitored in mice treated with intraperitoneal injections of the most active polysaccharide fraction. Lymph nodes (LN) and spleen were harvested, and levels (Kouakou et al., C. splendens Leaves of cytokines in supernatants from LN cells and splenocytes challenged with myelin 2013) G. Don oligodendrocyte glycoprotein peptide were determined. Fractions containing type II arabinogalactan had potent immunomodulatory activity. Specifically, the high- molecular weight sub-fraction CSP-AU1 (average of 38.5 kDa) that induced NO and cytokine production by human peripheral blood mononuclear cells (PBMCs) and monocyte/macrophages. Results revealed that the polysaccharide CSP-AU1 is a potent natural innate immunomodulator with a broad spectrum of agonist activity in-vitro and immunosupressive properties after chronic administration in-vivo. The immunomodulatory potentials of the crude methanol extract and fractions [n- hexane (Hex), dichloromethane (DCM), ethyl acetate (EtOAc) and n-butanol (BuOH)] of C. volubile flowers were investigated on whole blood phagocytic oxidative burst (using Luminol-amplified Chemiluminescence Technique) and through determination of free radicals scavenging activities. The DCM fraction (Erukainure et al., C. volubile P. showed significant anti-oxidative burst and free radical scavenging activities Flowers 2016) Beauv. (indicating high immunomodulatory and antioxidant potencies) and exerted a cytotoxic effect on CC-1 normal cell line. Triacetin; 3,6-dimethyl-3-Octanol; 2R- Acetoxymethyl-1,3,3-trimethtyl-4t-(3-methyl-2-buten-1-yl)-1c-cyclohexanol and Stigmastan-3,5-diene, were identified by GCMS in DCM fraction and docked with the active sites of cyclooxygenase-2 (COX-2). All compounds except Stigmastan- 3, 5-diene docked comfortably with COX-2 with good scoring function (-CDocker 67

Review of Literature

Table 11 (continued): Reports on immunomodulatory and wound healing activities of Clerodendrum species Reference Species Plant Part Summary

energy) indicating their inhibitory potency against COX-2. 3,6-dimethyl-3-Octanol displayed the lowest predicted free energy of binding suggesting its stronger interaction with COX-2. The observed suppressive effect of the DCM fraction on phagocytic oxidative burst indicates an immunomodulatory potential, which was also reflected in its free scavenging activities and synergetic modulation of COX-2 activities by its identified compounds in-silico. The immunomodulatory potentials of the crude methanolic extract of C. volubile leaves and its fractions [n-hexane (Hex), dichloromethane (DCM), ethyl acetate (EtOAc) and n-butanol (BuOH)] were investigated on whole blood, neutrophil and macrophage phagocytic respiratory burst using luminol-amplified chemiluminescence technique. DCM fraction showed higher inhibitory activity on (Erukainure C. volubile P. respiratory burst, indicating high suppressive immunomodulatory potency. The Leaves et al., 2017) Beauv. isolated pictolinarigenin was investigated for its inhibitory activity of neutrophil phagocytes respiratory burst as well as T-Cell proliferation. The compound exhibited significant activities (at p<0.05) indicating high suppressive immunomodulatory potency. The potent suppressive effect of pectolinarigenin on polymorphonuclear neutrophils (PMNs) respiratory oxidative burst and T-cell proliferation suggested an immunomodulatory potential and pathway of the flavonoid. The wound healing activity of the hydro-alcoholic (70% ethanol) extract of the leaves of C. infortunatum (as 4% ointment) was evaluated in cutaneous wound (Panigrahi C. infortunatum Leaves model in diabetic rats. The % wound contraction, oxidative stress related parameter et al., 2018) L. (SOD, CAT, GSH and LPO), hydroxyproline content and histopathology were estimated. Results showed that treatment with C. infortunatum ointment showed faster and organized healing of cutaneous wounds in STZ-induced diabetic rats.

Taxonomy

Taxonomy Taxonomy The systematic classification of the plant species, under investigation in this study, according to (Chopra et al., 1956; Benson, 1957; Jones and Luchsinger, 1986) is as follows: Plant names: Clerodendrum inerme L. Gaertn. Clerodendrum splendens G. Don Kingdom: Plantae Division: Angiosperm (Seed Plants) Class: Magnoliopsida (Dicotyledons) Subclass: Asteridae Order: Lamiales Family: Verbenaceae Genus: Clerodendrum L. Species: inerme L. Gaertn. and splendens G. Don Recent researches and phylogenetic studies indicated that the genus Clerodendrum should be transferred from Verbenaceae to Lamiaceae (Latta, 2008; Li et al., 2014). Family Lamiaceae Family Lamiaceae (the mint family) is the largest family of the order Lamiales (Trivellini et al., 2016) and the sixth largest family of flowering plants (Drew and Sytsma, 2012). It comprises about 236 genera and 7136 species (Trivellini et al., 2016). The family is divided into seven subfamilies (among which is the Ajugoideae) (Grayer et al., 2003). The subfamily Ajugoideae is the third-largest subfamily of Lamiaceae with 23 genera, including genus Clerodendrum, and about 760 species (Li et al., 2016; Xiang et al., 2018). Members of family Lamiaceae are of cosmopolitan distribution but they are mostly found in the Mediterranean region to central Asia (Lawrence, 1958; Boulos, 2002). They occur as trees, shrubs, lianes or herbs (Li et al., 2016). Characteristic features of family Lamiaceae Members of family Lamiaceae are characterized by the following (Wallis, 1939; Youngken, 1948; Youngken, 1954; Täckholm, 1956; Lawrence, 1958; Boulos, 2002; Youngken, 2003): • Habit: Predominantly annual or perennial herbs, sometimes shrubs and rarely trees, which produce creeping runners that spread out and have roots at the nodes. • Stems: Mostly quadrangular or four-angled, rarely cylindrical in outline, characterized by the presence of glandular hairs containing aromatic essential

Taxonomy oils consisting of a short one-celled stalk and a head (gland) of six, eight or twelve cells. • Leaves: Opposite or whorled, simple to pinnately dissected and compound, mainly petiolate with nearly serrate, dentate or crenate margins. • Inflorescences: Usually axillary double dichasial cymes, forming whorls of verticillasters. • Flowers: Zygomorphic, bisexual, pentamerous, bracteolate or not, and each cyme usually subtended by a foliaceous bract that may exceed the cyme. • Calyx: Composed of five united persistent sepals, four to five-lobed, synsepalous, ribbed, often bilabiate whose upper lip is bifid and the lower trifid. • Corolla: Typically, five-lobed, usually bilabiate whose upper lip is two-lobed while the lower three-lobed and often concave. • Stamens: Two or four, epipetalous, usually four didynamous, with an anterior pair usually the longer. The anthers are two-celled and longitudinally dehiscing. • Gynaecium: Bicarpellate with superior ovary, gynobasic slender style, two- lobed stigma and basal placentation derived from axile type. • Fruit: Four, one-seeded, nutlets enclosed by the calyx. • Seeds: Exalbuminous, with scant fleshy endosperm, often absorbed by the developing embryo. Genus Clerodendrum Genus Clerodendrum L. is a pantropical to subtropical genus (Xiang et al., 2018). Most Clerodendrum species are distributed in warm regions of the world (Boulos, 2002) especially Africa, Asia, and Pacific Oceania, with fewer representatives in the New World (Xiang et al., 2018). The genus comprises about 400-500 species (Xiang et al., 2018). The name Clerodendrum is derived from the Greek words Klero, meaning chance, and dendron, meaning tree, i.e. chance tree or the tree which may or not bring good luck (Shrivastava and Patel, 2007). Characteristic features of genus Clerodendrum The characteristic features of plants belonging to genus Clerodendrum can be summarised as follows (Täckholm, 1956; Boulos, 2002) • Habit: Evergreen trees, shrubs or lianes. • Leaves: Simple, entire or toothed, opposite or in whorls of 3 or 4. • Inflorescences: Axillary or terminal, lax cymes, corymbs, panicles or heads. • Calyx: Bell-shaped or tubular, truncate or five-toothed. 70

Taxonomy

• Corolla: Tubular, white, blue, violet or red, straight or curved cylinders often widened towards the throat. • Stamens: Four, didynamous, inserted in the corolla tube. • Gynaecium: Tetralocular ovary, terminal elongated style and bifid stigma. • Fruit: Drupes, four-grooved, with fleshy mesocarp and bony smooth or wrinkled endocarp. • Seeds: Four or two pairs of pyrenes. Investigated Species A. Geographical Distribution, Synonyms and Common names The geographical distribution, synonyms and common names of the two Clerodendrum species selected for this study are displayed in table 12 (Starr et al., 2003; Rohitash and Jai, 2010). Table 12: Geographical distribution, synonyms and common names of the two Clerodendrum species under investigation Clerodendrum inerme L. Clerodendrum splendens G. Scientific names Gaertn. Don Clerodendrum aurantium G. Don Synonyms Volkameria inermis L. Siphonanthus splendens G. Don Glory Bower, Indian Privet, Glory Tree and Flaming Common Names Seaside Clerodendrum and Glorybower Embrert Native to tropical Asia and the Native to tropical Africa and Pacific, cultivated in Hawaii, Hawaii, now widely cultivated Geographical Australia, the West Indies, in temperate parts of Asia, distribution Egypt and probably elsewhere Africa, Australia, Europe, North in warm climates America and the West Indies

B. Anatomical Features Clerodendrum inerme L. Gaertn. and Clerodendrum splendens G. Don could be distinguished from other members of the genus by their characteristic anatomical features. (Starr et al., 2003; Rohitash and Jai, 2010). 1. Clerodendrum inerme L. Gaertn. The plant is an evergreen shrub, 1-1.8 m tall. Stems are woody and smooth. Leaves are opposite, simple, ovate to elliptical, 5-10 cm long with acute to acuminate apex, green, smooth, slightly shiny upper surface, pinnate venation and entire margins. The inflorescence is a cyme or umbel usually comprised of 3 flowers joined at a common base point; the corolla is white, fused, with 5 lobes. The flower is

Taxonomy hermaphrodite with four, reddish to purple and upwardly curved stamens. The fruit is green turning black, 1-1.5 cm long and obovoid. The fruit is drupe. 2. Clerodendrum splendens G. Don Clerodendrum splendens is an ornamental evergreen climbing shrub up to 3 meters high. Leaves are opposite, simple, ovate to cordate, 6-18 cm long with acuminate apex, green surface, pinnate venation and entire margins. Flowers are red, small complete pedicellate, pentamerous, zygomorphic, hermaphrodite and arranged in terminal clusters. The corolla is bright red, fused, with 5 lobes. Stamens are four and reddish. The fruit is a dark four-lobed drupe.

Material, Apparatus and Techniques

Material, Apparatus and Techniques Material I. Plant Material Leaves and flowers of Clerodendrum inerme L. Gaertn. and Clerodendrum splendens G. Don were collected, during the years 2016-2018, from plants growing in the Medicinal Aromatic and Poisonous Plants Experimental Station (MAPPES), Faculty of Pharmacy, Cairo University (Giza, Egypt). Samples of leaves were gathered at different time intervals, before, during and after flowering. Dr. Mohamed Gibali, senior botanist and consultant at Orman Botanic Garden (Giza, Egypt), kindly authenticated the plant material. Voucher specimens (No. 19-3-2018 I and 19-3-2018 II) were deposited in the Herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Cairo University. For botanical study, leaves and flowers of the two plants under investigation were examined while fresh and their macro-morphological characteristics described. Moreover, the leaves were microscopically examined after being air-dried in shade and reduced to fine powder. For phytochemical investigation, the air-dried powdered leaves were saved in tightly closed glass containers until use. For biological screening, specific extracts were prepared, in different solvents, from the air-dried powdered leaves of the two plants, and comparatively processed. II. Plant Extracts A. For Comparative Study 1. n-Hexane extracts The air-dried powdered leaves of C. inerme and C. splendens (100 g, each) were, separately, defatted using n-hexane (60-80 oC), and the solvent removed by vacuum distillation at a temperature not exceeding 55 oC. The resulting dried extractives were cautiously saved in a desiccator for further studies. 2. Ethanol 70 % extracts and fractions The defatted powdered leaves were extracted with ethanol (70%) by cold maceration until exhaustion. The dried residues obtained upon solvent removal under vacuum (at up to 55 oC) were saved for liquid/liquid fractionation. Solvents of increasing polarities viz., dichloromethane and ethyl acetate: n-butanol (2:1) mixture, were used in succession for extraction of the prepared ethanol (70%) extracts suspended in water. The solvent, in each case, was removed by vacuum distillation at a temperature not exceeding 60 oC; the resulting solvent-free extractives were then weighed, and saved for further examination. 73

Material, Apparatus and Techniques

B. For Phytochemical and Biological Investigation of C. inerme Large-scale preparation of n-hexane and 70% ethanol extracts of C. inerme leaves was processed as aforementioned in the comparative study. In this respect, 1 kg of the leaves were extracted using n-hexane (60-80 oC) and the defatted mark macerated with ethanol (70%) until exhaustion. The dried ethanol extract was then subjected to a proposed liquid-solid fractionation procedure using dichloromethane, ethyl acetate: n-butanol (2:1) mixture and methanol in succession. The resulting solvent-free extracts and fractions were weighed, and carefully saved for investigation. III. Material for Chromatographic Study A. Stationary Phases These were selected according to the purpose of use, and purchased from their respective sources as follows: 1. For thin layer chromatography (TLC):

Precoated TLC plates (20 × 20 cm), silica gel F254 and RP silica gel were obtained from Fluka (Sigma-Aldrich chemicals, Germany). 2. For column chromatography: • Silica gel H 60 and RP-C18 silica gel for vacuum liquid chromatography (VLC) were obtained from E. Merck (Darmstadt, Germany). • Silica gel 60 (0.2-0.5 mm) for normal phase chromatography was obtained from E. Merck (Darmstadt, Germany). B. Solvent Systems The following solvent systems were selected, based on several trials, for developing the chromatograms, and are cited here in ascending polarity:

S1: Hexane-Ethyl acetate (98:2 v/v) S2: Hexane-Ethyl acetate (96:4 v/v) S3: Hexane-Ethyl acetate (90:10 v/v) S4: Hexane-Ethyl acetate (85:15 v/v) S5: Hexane-Ethyl acetate (80:20 v/v) S6: Hexane-Ethyl acetate (70:30 v/v) S7: Dichloromethane-Methanol (98:2 v/v) S8: Dichloromethane-Methanol (95:5 v/v) S9: Dichloromethane-Methanol (90:10 v/v) S10: Dichloromethane-Methanol-Water (95:5:5 v/v) S11: Ethyl acetate-Methanol-Water (100:16:13 v/v) S12: Ethyl acetate-Formic acid-Acetic acid-Water (100:10:10:10 v/v)

Material, Apparatus and Techniques

C. Chromogenic Spray Reagents Spot visualization on the chromatograms was performed by means of the appropriate spray reagent (s) for each type of constituents as follows: 1. Aluminium chloride (Markham, 1982), 1% in ethanol 95%, was used for detection of phenolics, the chromatograms being examined in visible and long wave UV (=365 nm) lights. 2. Natural product/polyethylene glycol reagent (NP/PEG) (Wagner et al., 1983), prepared by mixing a 1% methanolic solution of diphenylboric acid-β- ethylaminoester (diphenylboryloxyethyl-amine, NP) with 5% ethanolic solution of polyethylene glycol-4000 (PEG), was utilized for detection of phenolics including flavonoids. 3. Para-anisaldehyde reagent (Stahl, 1969), for detection of organic compounds, prepared by mixing 0.5 ml p-anisaldehyde with 10 ml glacial acetic acid, 85 ml methanol and 5 ml concentrated H2SO4 was used for identification of steroids and terpenoids. D. Authentic Reference Material Sterols, triterpenes and phenolics used as reference standards in TLC co- chromatography were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Authentic samples used during performance of HPLC analyses of phenolics and flavonoids, supplied by the Food Technology Research Institute, Agricultural Research Center (Giza, Egypt), were acquired from Sigma Chemical Co. (St. Louis, MO, USA) IV. Reagents and Chemicals for UV Spectroscopy Shift reagents, prepared according to published procedures (Mabry et al., 1996), and chemicals used during performance of the UV spectroscopic analysis of flavonoids were: 1. Sodium methoxide solution: freshly cut metallic sodium (2.5 g) was added cautiously in small pieces to cooled spectroscopic methanol (100 ml). 2. Aluminium chloride solution: anhydrous aluminium chloride (5 g) was cautiously dissolved in 100 ml of spectroscopic methanol. 3. Hydrochloric acid: 50 ml concentrated hydrochloric acid was mixed with 100 ml distilled water. 4. Sodium acetate (anhydrous powder). 5. Boric acid (anhydrous powder).

Material, Apparatus and Techniques

V. Material for Spectrophotometric Determination 1. Folin-Ciocalteu reagent, used for determination of total polyphenolic content, was obtained from Loba-Chemie (Mumbai, India). 2. Sodium Carbonate: sodium carbonate (75 g) was dissolved in 1000 ml of distilled water with sonication. 3. Aluminium chloride solution (0.1 M): anhydrous aluminium chloride (1 g) was cautiously dissolved in 50 ml of spectroscopic methanol. 4. Quercetin and gallic acid (Sigma Chemical Co, St. Louis, MO, USA) were used as authentic reference materials in spectrophotometric determination of total flavonoid and phenolic contents, respectively. VI. Test Solutions and Solvents

Dilute acids (HCl, H2SO4), alkalies (KOH, T.S.), alcoholic α-naphthol, Fehling’s solution and ferric chloride (T.S.) were prepared according to E.P. (2005) monographs (Egyptian Pharmacopoeia, 2005). Solvents used in this work viz., n-hexane, ethanol, dichloromethane, ethyl acetate, n-butanol and methanol were purified adopting the procedures described by Vogel (Vogel, 1975). Methanol used for spectrophotometric analyses was purchased from E-Merck, (Darmstadt, Germany). Solvents used for HPLC viz., acetonitrile and methanol were of HPLC grade and purchased from Sigma-Aldrich (Steinheim, Germany). Distilled water was used after additional purification using a Milli-Q system (Millipore, MA, USA). Solvents used for UPLC-Orbitrap HRMS analysis viz., water, acetonitrile and formic acid were of LC-MS grade and acquired from Fluka (Sigma-Aldrich chemicals, Germany). VII. Material for Evaluation of the Biological Effects A. Cell lines Four different cell lines were used for assessment of the cytotoxic activity: 1. H-1299: Human non-small cell lung carcinoma cell line. 2. HEPG-2: Human hepatocellular carcinoma cell line. 3. MCF-7: Human breast adenocarcinoma cell line. 4. BHK: Baby hamster kidney normal fibroblasts. The cell lines were obtained from the American Type Culture Collection (ATCC) via the Tumor Biology Department, National Cancer Institute (Cairo, Egypt), and were maintained frozen in liquid nitrogen at -180°C.

Material, Apparatus and Techniques

B. Reagents and chemicals 1. For DPPH radical quenching activity • 2, 2-Diphenyl-1-picrylhydrazyl (DPPH): was purchased from Sigma-Aldrich Co (St. Louis, Mo, USA) and prepared in concentration of 0.004% w/v in methanol. • Gallic acid acquired from Sigma-Aldrich Co (St. Louis, Mo, USA) was used as positive control. 2. For cytotoxic activity • SRB (Sulphorhodamine-B) was purchased from Sigma Co., USA. • Doxorubicin (Adriamycin®), acquired from Sigma-Aldrich Co (St. Louis, Mo, USA), was used as reference anticancer. • Buffers and other chemicals were of analytical grade. 3. For antibacterial activity • MTT and Alamar Blue were purchased for in-vitro evaluation of antibacterial activity from Sigma Co., USA. • Vancomycin and Isoniazide (Sigma Aldrich Co., St. Louis, Mo, USA) were utilized as reference antibacterial.

Material, Apparatus and Techniques

Apparatus and Equipment The laboratory procedures performed in the course of this research were carried out by using the following equipment and apparatuses. I. For Botanical Investigation Leica DFC500 and Leica DM750 light microscopes (Wetzlar, Germany), were used for anatomical examination. II. For Phytochemical Investigation 1. Sensitive electric balance (Sartorius, Germany). 2. Rotatory evaporator (Büchi, G. Switzerland) for concentration of extracts. 3. Electrothermal 9100, (U.K.) for determination of melting points (uncorrected). 4. Elma Terrasonic TS 540 sonicator 5. Chromatographic equipment and apparatuses: • Glass columns, of different dimensions, for column chromatography. • Glass jars of different dimensions, for development of TLC chromatograms. • Micropipettes (0.1ml), for spot application. • Glass sprayers for visualization of TLC chromatograms. • Ultraviolet lamp (max 254 and 325 nm, Shimadzu), a product of Hanovia lamps, for localization of spots on chromatograms. • HPLC apparatus Agilent Technologies 1100 series HPLC system, equipped with a quaternary pump, degasser G1322A, a UV detector and a LiChrospher 100 RP-C18 column, preceded by RP-C18 guard column was used. Data acquisition and processing was carried out by the aid of an Agilent ChemStation software. • UPLC system UPLC-Orbitrap HRMS system was used for profiling of secondary metabolites. It consisted of an Orbitrap Elite mass spectrometer (Thermo Fischer Scientific, Darmstadt, Germany) coupled to an UHPLC system (Dionex UltiMate 3000, Thermo Fischer Scientific), equipped with RP-C18 column (Acquity HSS T3, Waters, USA) and a photodiode array detector (220–600 nm, Thermo Fischer Scientific) and operated in the negative ion mode. 6. Spectrometers • Schimadzu UV-Visible spectrophotometer, UV-1650 PC (Schimadzu, Japan), was used for running UV spectra of isolated flavonoids.

Material, Apparatus and Techniques

• Tecan Infinite® F50 absorbance microplate reader (USA), equipped with Magellan™ reader control and data analysis software, was utilized for spectrophotometric determination of total phenolic and total flavonoid contents and measurement of biological activities. • Bruker NMR-spectrometer (Japan): 1H-NMR, 400 MHz, 13C, 100 MHz spectra were recorded in suitable deuterated solventS (CDCl3, DMSO, CD3OD or Pyridine) using TMS as internal standard and chemical shift values expressed in δ ppm (Micro Analytical Unit, Faculty of Pharmacy, Cairo University).

Material, Apparatus and Techniques

Techniques The general chromatographic procedures adopted in the phytochemical investigation are described in this section. The methodology followed for evaluation of the different biological activities will be presented, later on, in their respective chapters. Chromatographic Techniques I. Thin layer chromatography (TLC) (Stahl, 1969) The chromatoplates were developed by applying the ascending technique. The resulting chromatograms were air-dried and the spots visualized by appropriate means. II. Column chromatography (CC) (Cannell, 1998) Glass columns of different sizes were packed with suitable stationary phases viz., silica gel and RP-C18 by adopting the dry method. The analyzed samples were applied on to the top of the column after solubilization in the least volume of the first eluting solvent. III. High performance liquid chromatography (HPLC) Qualitative and quantitative analyses of the constituents of the ethanol (70%) extract and its fractions were performed by HPLC on a LiChrospher 100 RP-C18 column (250 mm × 4.6 mm, particle size 5 μm). Gradient elution was carried out using mixtures of acetonitrile and 0.3% phosphoric acid as mobile phase at a flow rate of 1 ml/min. Eluted peaks were detected at =325 nm. Retention times were compared to those of authentic samples and relative area percentages computed by integration. IV. UPLC-Orbitrap HRMS analysis Qualitative analysis of the secondary metabolites was performed according to a published procedure (Farag et al., 2017) using gradient elution with A (water - formic acid, 99.9:0.1 v/v) and B (acetonitrile - formic acid, 99.9:0.1 v/v). The CID mass spectra (buffer gas; helium) were recorded using a normalized collision energy (NCE) of 35%. The instrument was equipped with a heated electronspray ion source (negative spray voltage at 3 kV, capillary temperature of 300 °C, source heater temperature of 250 °C, FTMS resolution of 30.000) and RP- C18 column (particle size 1.8 μm, pore size 100 Å, 150 mm × 1 mm i.d., Acquity HSS T3, Waters; column temperature 40 °C). It was externally calibrated by the Pierce ESI negative ion calibration solution (product No. 88324) from Thermo Fisher Scientific. Resulting data were evaluated using the Xcalibur 2.2 SP1 software.

Part I Comparative Study of Clerodendrum inerme L. Gaertn. and Clerodendrum splendens G. Don

Chapter I Botanical Characters

Botanical Characters Chapter I Botanical Characters I. Habit Clerodendrum inerme L. Gaertn. (Figure 20 A) is an evergreen straggling shrub, ranging from 1 to 1.8 m in length, very branched with pale brown bark. The terminal branches are green, twinging and slender in shape bearing shiny green ovate to elliptical leaves and white tubular flowers that are arranged in cymes consisting of 3 flowers. Flowers appear in summer (from July to the end of September). The plant is cultivated in Egypt as ornamental. Clerodendrum splendens G. Don (Figure 20 B) is a woody or semi-woody vine that climbs by twinging and spreads over about 2 m. It is 2 to 4 m tall, having large, glossy, dark green ovate leaves and attractive red tubular flowers that are borne in dense terminal clusters. Flowers appear in spring (from end of March to end of June). The plant is also cultivated in Egypt as ornamental. II. Macromorphological Characters A. Leaves Leaves of C. inerme (Figure 21 A) are petiolate, elliptical or obovate, with green upper surface and light green lower surface, both are glabrous to the naked eye. They have entire margins, emarginate, acute or rounded apices, symmetric bases and reticulate pinnate venation (anastomosing near the margin). The leaves range from 2.5 to 6.5 cm in length and from 1.5 to 3.5 cm in width. The petiole is cylindrical, light green in colour and ranging from 0.6 to 1.5 cm in length. Leaves of C. splendens (Figure 21 B) differ morphologically from those of C. inerme in that they are larger ranging from 5.5 to 18 cm in length and 5 to 8.5 cm in width, darker in color, ovate or cordate in shape, with acuminate or acute apices and dark purplish petiole that ranges from 0.7 to 1 cm in length. B. Flowers Flowers of C. inerme (Figure 22 A) are hermaphrodite, with green tubular calyx and white tubular corolla. The androecium consists of four violet stamens whereas the gynoecium is formed of a violet inferior ovary, a violet slender style and pointed stigma. The flowers range in length from 6.5 to 7.5 cm. The calyx is 5- toothed, about 0.5 cm in length and 0.2 to 0.3 cm in diameter. The corolla ranges from 2.7 to 3 cm in length, the tubular part of the corolla is about 0.1 cm in diameter while the open part is about 2 to 2.5 cm in diameter and consists of 5 lobes. The exposed parts of the androecium and gynoecium range in length from 3 to 3.5 cm and from 2.3 to 2.5 cm, respectively.

Botanical Characters Flowers of C. splendens (Figure 22 B) are noticeably different from those of C. inerme. They have red tubular calyx and corolla. They are shorter and wider ranging from 3 to 4.5 cm in length. The calyx is also about 0.5 cm in length and 0.3 to 0.4 cm in diameter. The corolla is about 2 to 2.5 cm in length with a lower tubular part, about 0.15 cm in diameter, and upper open part of about 2.5 to 2.7 cm in diameter. The androecium and gynoecium are similar to those of C. inerme except that they are red in color and a little bit shorter, the exposed parts being 2.1 to 2.5 cm and 1.7 to 1.9 cm, respectively.

A B Figure 20: Photographs of C. inerme shrub and C. splendens vine A. C. inerme shrub (X= 0.25) B. C. splendens vine (X= 0.08)

Botanical Characters

B Figure 21: Photographs of the leaves of C. inerme and C. splendens A. Leaves of C. inerme (X= 0.5) B. Leaf of C. splendens (X= 1)

Botanical Characters

B Figure 22: Photographs of the flowers of C. inerme and C. splendens A. Flowers of C. inerme (X= 1) B. Flowers of C. splendens (X= 0.8)

Botanical Characters III. Examination of the Powdered Leaves The powder of C. inerme leaf is light green in colour with faint characteristic odour and bitter taste, while that of C. splendens is dark green, odourless and bitter in taste. Microscopic examination of the powdered leaves of C. inerme revealed the presence of the following elements that are represented in figure 23; 1. Fragments of the upper epidermis showing cells (8-10 μm wide and 8-17 μm long) with slightly wavy beaded anticlinal walls and smooth cuticle, labiaceous glandular hairs (22-25 μm in diameter) and uni- and bicellular non-glandular hairs (around 40 μm long) and being devoid of stomata. 2. Fragments of the lower epidermis showing cells (5-10 μm wide and 10-17 μm long) with wavy beaded anticlinal walls and striated cuticle, labiaceous glandular hairs (16-18 μm in diameter) and anomocytic stomata (10-14 μm wide and 11-18 μm long). 3. Fragments of epidermis of the petiole showing cells (5-10 μm wide and 7-12 μm long) with thick, straight anticlinal walls and smooth cuticle, labiaceous glandular hairs (around 16 μm in diameter) and being devoid of stomata. 4. Fragments of tortuous fibers having one straight and another undulating side, wide lumina and tapering apices. 5. Fragments of lignified xylem vessels with spiral, reticulate or pitted thickenings. 6. Non-glandular, uni- or bicellular hairs with warty cuticle, blunt apex and conical base. 7. Scattered cluster crystals of calcium oxalate. 8. Numerous oil globules.

Botanical Characters

Figure 23: Powdered leaves of C. inerme A. Upper epidermis B. Lower epidermis C. Petiole epidermis an. v., annular vessel; anom. st., anomocytic stomata; b. ant. w., beaded anticlinal wall; ca. ox. cl., calcium oxalate cluster; lab. gl. hr., labiaceous glandular hair; non-gl. bicel. hr, non-glandular bicellular hair; non-gl. unicel. hr., non-glandular unicellular hair; oil glob., oil globule; pit. v., pitted vessel; ret. v., reticulate vessel; sp. v., spiral vessel; str. cu., striated cuticle; tort. f., tortuous fibers. X= 600

Botanical Characters

lab. gl. hr. str. cu lab. gl. hr. anom. st.

b. ant. w.

b. ant. wall str. cu. non-gl. non-gl. unicel.hr bicel.hr. . A B

oil. glob.

ca. ox. lab. gl. hr. cl.

sp. v. an. v. tort. f ret. v. pit. v.

Figure 23

Botanical Characters Microscopic examination of the powdered leaves of C. splendens revealed the presence of the following elements that are represented in figure 24; 1. Fragments of the upper epidermis showing cells (12-20 μm wide and 30-38 μm long) with strongly wavy anticlinal walls and smooth cuticle, labiaceous glandular hairs (around 17 μm in diameter); uni- and bicellular non-glandular hairs (25 and 37-58 μm long, respectively) are present and stomata absent. 2. Fragments of the lower epidermis showing cells (7-14 μm wide and 16-28 μm long) with strongly wavy anticlinal walls and smooth cuticle, labiaceous glandular hairs (16-20 μm in diameter) are present and diacytic and few anomocytic stomata (8-9 μm wide and 9-10 μm long). 3. Fragments of epidermis of the petiole showing cells (5-10 μm wide and 18-28 μm long) with straight, beaded anticlinal walls smooth cuticle and purple- coloured patches, labiaceous glandular hairs (around 20 μm in diameter) are present and stomata absent. 4. Fragments of fibers surrounded by crystal sheath. 5. Fragments of lignified xylem vessels with reticulate and spiral thickenings. 6. Non-glandular, uni- or bicellular hairs with warty cuticle and acute apex.

Botanical Characters

Figure 24: Powdered leaves of C. splendens A. Upper epidermis B. Lower epidermis C. Petiole epidermis anom. st., anomocytic stomata; b. ant. w., beaded anticlinal wall; cr. sh., crystal sheath; dia. st., diacytic stomata; lab. gl. hr., labiaceous glandular hair; non-gl. bicel. hr, non- glandular bicellular hair; non-gl. unicel. hr., non-glandular unicellular hair; ret. v., reticulate vessel; sp. v., spiral vessel; wavy ant. w., wavy anticlinal wall. X= 600

Botanical Characters

wavy ant. w. lab. gl. hr.

wavy ant. w.

dia. st.

lab. gl. hr.

anom. st. non-gl. non-gl. bicel.hr. unicel.hr B . A

b. ant. wall

cr. sh

ret. v. sp. v. C lab. gl. hr. Figure 24 90

Botanical Characters

IV. Numerical Values The stomatal number and stomatal index of the lower epidermises of the leaves of C. inerme and C. splendens were determined and recorded in table 13. Table 13: Numerical values of the lower epidermises of the leaves of C. inerme and C. splendens

Numerical value C. inerme C. splendens Stomatal Number* 95 80 Stomatal Index* 14 % 22 % *Average of three determinations Conclusion The leaves and flowers of C. inerme and C. splendens are morphologically different. The leaves of C. inerme are elliptical or obovate, with emarginate, acute or rounded apices, and light green petiole, while those of C. splendens are ovate or cordate, with acuminate apices, dark purplish petiole and are much larger than those of C. inerme. Flowers of C. inerme have white corolla and green calyx, while those of C. splendens have red corolla and calyx and are shorter and wider than those of C. inerme. Microscopically, the powdered leaf of C. inerme is characterized by the presence of tortuous fibers, calcium oxalate clusters and oil globules, and that of C. splendens by the presence of crystal sheath. The sizes of labiaceous hairs of upper epidermis and stomata are distinctly larger in C. inerme while that of the epidermal cells is larger in C. splendens. The epidermal cells of C. inerme have beaded anticlinal walls and are covered with striated cuticle on the lower surface, in contrast the cuticle covering those of C. splendens is smooth. The epidermal cells of the petiole are larger in size and showing larger labiaceous hairs in C. splendens compared to C. inerme. The epidermal cells of the petiole of C. inerme have thick walls while those of C. splendens have beaded walls and show purple-coloured patches. These data could be considered as useful criteria for discrimination between the two species under investigation, besides allowing their identification among other related members of the genus.

Chapter II Proximate Analysis and Preliminary Phytochemical Screening

Proximate Analysis and Preliminary Phytochemical Screening Chapter II Proximate Analysis and Preliminary Phytochemical Screening I. Proximate Analysis Proximate analysis of the leaves of C. inerme and C. splendens was performed by adopting the method of the Association of Official Analytical Chemists (A.O.A.C.), 2000 (Official Methods of Analysis of the Association of Official analytical Chemist. , Washington, D.C. 14th edition, 2000). The analytical standards determined included total ash, acid insoluble ash, water- soluble ash, and moisture and crude fibers contents. Results, recorded in table 14, were the average of three determinations and calculated on dry weight basis. Table 14: Results of proximate analysis of the leaves of C. inerme and C. splendens

Analytical standard C. inerme (g %) * C. splendens (g %) * Total ash 12.389 16.678 Acid insoluble ash 5.504 8.687 Water-soluble ash 5.151 2.045 Crude fiber 15.835 17.435 Moisture 5.989 7.682 *Average of three determinations Results and Discussion The ash values of the leaves of the two species are distinctly different. The total and acid insoluble ash values are about 1.5 times higher in C. splendens than in C. inerme, while that of water-soluble ash in the latter is almost double that in C. splendens. The crude fibers and moisture contents of C. splendens exceeded those of C. inerme. These data could be considered as quality control criteria for the leaves of the two species. II. Preliminary Phytochemical Screening The air-dried powdered leaves of C. inerme and C. splendens were subjected to preliminary phytochemical screening for the presence of different types of constituents. This included testing the samples for the presence of steam volatile substances, crystalline sublimates, carbohydrates and/or glycosides, tannins, flavonoids, saponins, sterols and/or triterpenes, alkaloids and/or nitrogenous bases,

Proximate Analysis and Preliminary Phytochemical Screening anthraquinones, cardiac glycosides and oxidase enzymes (Geissman, 1962; Claus et al., 1970; Coutts and Snail, 1973; Wagner et al., 1983; Evans et al., 2002). Results obtained are recorded in table 15. Table 15: Results of preliminary phytochemical screening of the leaves of C. inerme and C. splendens

Constituents C. inerme C. splendens Steam volatile substances − − Crystalline sublimates − − Carbohydrates and/or glycosides + + Tannins • Condensed ++ + • Hydrolysable - - Flavonoids • Free ++ + • Combined ++ + Saponins ++ + Sterols and/or triterpenes ++ ++ Alkaloids and/or nitrogenous bases − − Anthraquinones • Free − − • Combined − − Cardiac glycosides − − Oxidase enzymes − − (++) =strongly positive; (+) = positive; (−) = negative Results and Discussion In conclusion, the data displayed in table 15 reveal that an almost similar response to the applied tests is observed among the leaves of the two species suggesting that they are closely related in composition. Besides, the following could be deduced: 1. Carbohydrates and/or glycosides, free and/or combined flavonoids, sterols and/or triterpenes, saponins, as well as condensed tannins are identified in all the tested samples. 2. Finally, since all samples gave negative response when tested for crystalline sublimates, cardiac glycosides and alkaloids and/or nitrogenous bases, these constituents may be considered as absent.

Chapter III Extraction, Fractionation and Examination of the Leaves Extractives

Extraction, Fractionation and Examination of the Leaves Extractives Chapter III Extraction, Fractionation and Examination of the Leaves Extractives I. Extraction, Fractionation and Determination of Extractives Yeilds A. Defatting and Ethanol (70%) Extraction of the Leaves The air-dried powdered leaves of C. inerme and C. splendens (100 g, each) were, separately, defatted with n-hexane (60-80 oC) (4 × 250 mL) then extracted with ethanol (70%) (10 × 300 mL) with the aid of sonication. The solvents were evaporated under vacuum, at temperature not exceeding 55 oC, the residues weighed, and percentage yields calculated based on dried plant material (g/100g). B. Fractionation of the Ethanol (70%) Extracts The ethanol (70%) extracts (EEs) of the leaves of C. inerme and C. splendens (10 g, each) were separately suspended in distilled water and subjected to liquid-liquid fractionation using dichloromethane and ethyl acetate: n-butanol mixture (2:1) in succession. The solvents were removed by evaporation under vacuum, at a temperature not exceeding 60 oC, and the dried residues weighed and kept in a desiccator for further investigation. Yields and organoleptic characters of the extracts and fractions are recorded in table 16. Table 16: Percentage yield and organoleptic characters of the extractives of the leaves of C. inerme and C. splendens

Yield (g/100g dried leaves) Extractive Color Taste Odor C. inerme C. splendens

n-Hexane 1.63 1.02 Y. brown Bitter N.C. EE 28.8 10.1 D. brown Bitter N.C. Dichloromethane 2.6 0.85 D. brown Bitter N.C. Ethyl acetate/ n- 3.5 1.25 D. brown Bitter N.C. butanol 2:1 EE, ethanol (70%) extract; D., dark; Y., yellowish; N.C., not characteristic

Extraction, Fractionation and Examination of the Leaves Extractives

II. TLC Fingerprinting of the Different Extractives

A. TLC Fingerprinting of the n-Hexane Extracts This was performed on precoated silica gel plates using different solvent systems S1-S5 (see page 74) and p-anisaldehyde/H2SO4 as visualizing agent. Best resolution was obtained on developing the chromatograms with S4 (Hexane: Ethyl acetate 85:15 v/v). Results are recorded in table 17. Table 17: Results of TLC fingerprinting of the n-hexane extracts of the leaves

C. inerme C. splendens Spot R * N f Color with p- Color Color with p- Color anisaldehyde intensity anisaldehyde intensity 1 0.967 Orange →Violet ± Orange →Violet + 2 0.879 Orange →Violet + - - 3 0.681 Pink ± - - 4 0.615 - - Violet ± 5 0.571 Pink ± - - 6 0.396 Green ± - - 7 0.374 Blue ± Blue ± 8 0.286 Blue + Violet ± 9 0.044 Violet ± Violet ± Number of 8 5 spots detected

*Rf values in S4: Hexane-Ethyl acetate 85:15 v/v (+) = dark, (±) = faint, (-) = absent B. TLC Fingerprinting of the Ethanol (70%) Extracts

This was performed on precoated silica gel plates using S8 (Dichloromethane: Methanol 95:5 v/v) and S12 (Ethyl acetate: Formic acid: Acetic acid: Water 100:10:10:10 v/v) as solvent systems. Spots visualization was carried out by means of p-anisaldehyde/H2SO4 and AlCl3/UV (325 nm) (for S8 and S12, respectively). Results are represented in tables 18 and 19.

Extraction, Fractionation and Examination of the Leaves Extractives

Table 18: Results of TLC fingerprinting of the EEs of the leaves (using S8, p- anisaldehyde/H2SO4)

C. inerme C. splendens Spot Rf* N Color with p- Color Color with p- Color anisaldehyde intensity anisaldehyde intensity 1 0.95 Violet + Violet ± 2 0.88 Green + Green + 3 0.71 Violet ± Violet ± 4 0.61 Blue ± Blue ± 5 0.52 Yellow + Blue ± 6 0.36 Green ± Blue + 7 0.29 Green + Green + 8 0.16 Blue ± - - 9 0.07 Green ± - - Number of 9 7 spots detected

*Rf values in S8: Dichloromethane-Methanol 95:5 v/v (+) = dark, (±) = faint, (-) = absent

Table 19: Results of TLC fingerprinting of the EEs of the leaves (using S12, AlCl3/UV)

C. inerme C. splendens Spot R * N f Response to Intensity of Response to Intensity of AlCl3/UV response AlCl3/UV response 1 0.93 Yellow F. + - - 2 0.8 Blue F. + Yellow F. + 3 0.74 Violet F. ± - - 4 0.57 Yellow F. + - - 5 0.46 Blue F. + Yellow F. + 6 0.37 Yellow F. + - - 7 0.28 Yellow F. ± Yellow F. + 8 0.22 Yellow F. ± - - 9 0.17 Violet F. ± - - Number of 9 3 spots detected

*Rf values in S12: Ethyl acetate-Formic acid-Acetic acid-Water 100:10:10:10 v/v F= fluorescence (at UV 325 nm); (+) = high, (±) = low, (-) = absent C. TLC Fingerprinting of the Dichloromethane Fractions TLC examination of the dichloromethane extractives obtained upon fractionation the EEs of the leaves of C. inerme and C. splendens was performed on precoated

Extraction, Fractionation and Examination of the Leaves Extractives silica gel plates using different solvent systems, S7-S9 (see page 74) and p- anisaldehyde/H2SO4 as visualizing agent. Best resolution was achieved on using system S8 (Dichloromethane: Methanol 95:5 v/v) for development. Results are displayed in table 20. Table 20: Results of TLC fingerprinting of the dichloromethane fractions

C. inerme C. splendens Spot Rf* N Color with p- Color Color with p- Color anisaldehyde intensity anisaldehyde intensity 1 0.94 Violet + Violet ± 2 0.9 Green + Green + 3 0.78 Green ± - - 4 0.71 Violet + Violet ± 5 0.61 Blue ± Blue ± 6 0.54 Green ± - - 7 0.49 Yellow + Blue ± 8 0.41 Green ± Blue + 9 0.37 Green + Green + 10 0.31 Yellow ± - - 11 0.27 Blue ± Blue ± 12 0.16 Blue ± - - 13 0.08 Blue ± - - Number of 13 8 spots detected

*Rf values in S8: Dichloromethane-Methanol 95:5 v/v (+) = dark, (±) = faint, (-) = absent D. TLC Fingerprinting of the Ethyl acetate: n-Butanol (2:1) Fractions TLC of the ethyl acetate: n-butanol (2:1) fractions of the EEs was performed on precoated silica gel plates. S11 and S12 were tried as solvent systems for developing the chromatograms, and AlCl3/UV (325 nm) was used for spot location. Best resolution was obtained on using S12 (Ethyl acetate: Formic acid: Acetic acid: Water 100:10:10:10 v/v). Results are recorded in table 21.

Extraction, Fractionation and Examination of the Leaves Extractives Table 21: Results of TLC fingerprinting of the ethyl acetate: n-butanol (2:1) fractions

C. inerme C. splendens Spot R * N f Response to Intensity of Response to Intensity of AlCl3/UV response AlCl3/UV response 1 0.93 Yellow F. + - - 2 0.85 Violet F. ± - - 3 0.78 Blue F. + Blue F. ± 4 0.41 Blue F. + Blue F. ± 5 0.12 Blue F. ± Yellow F. + Number of 5 3 spots detected

*Rf values in S12: Ethyl acetate-Formic acid-Acetic acid-Water 100:10:10:10 v/v F= fluorescence (at UV 325 nm); (+) = high, (±) = low, (-) = absent

Results and Discussion The previous data (Table 16) revealed that the yield of leaf extractives of C. inerme were higher than their C. splendens analogues. Besides, the chromatographic profiles obtained upon TLC examination of the different extractives of the leaves of C. inerme showed greater number of spots with stronger response to visualizing agents than those detected in C. splendens (Tables 17-21). In conclusion, the leaves of C. inerme could be suggested as a better source of secondary metabolites.

Chapter IV UPLC-Orbitrap HRMS Profiling

UPLC-Orbitrap HRMS Profiling

Chapter IV UPLC-Orbitrap HRMS Profiling Recent studies on the identification of secondary plant metabolites in complex plant extracts have shown that high performance liquid chromatography (HPLC) coupled to photodiode array detection provides a powerful tool for determination and quantitation of constituents, especially phenolics (Mateos et al., 2001; Irakli et al., 2012). Compared to conventional HPLC techniques, ultra-performance liquid chromatography (UPLC) achieves rapid metabolite analysis and can obtain better peak separation than usually possible with standard LC methods (Farag and Wessjohann, 2012). UPLC brings dramatic improvements in sensitivity, resolution and speed to analytical procedures compared to HPLC. It relies on operating at a higher pressure than that required for operating HPLC, allowing for utilization of a more fine stationary phase (particle size less than 2.5 μm) and mobile phases at a higher linear velocities, hence, decreasing the length of column, reducing solvent consumption and saving time. The high sensitivity of MS detection provides an important tool for detection and measurement of minor metabolites in complex plant extract samples (Nováková et al., 2006; Allwood and Goodacre, 2010). Several studies have applied this technology to look at metabolite profiles in closely allied plant taxa, different cultivars of individual taxa, or plants at different stages of development (Fiehn, 2001; Okada et al., 2009; Cao et al., 2011; Farag et al., 2014). The aim of our work is to define and compare the phytochemical composition of the leaves of Clerodendrum species under investigation, C. inerme and C. splendens, via ultra-performance liquid chromatography coupled to photodiode array detector, and an Orbitrap Elite mass spectrometer equipped with an electrospray ionization source. UPLC coupled with high resolution Orbitrap Elite mass spectrometer, is a relatively new technology that can detect chemical compounds with high sensitivity and assist in the identification of peaks from high resolution MS data. A. Sample preparation for UPLC-Orbitrap HRMS analysis Samples (5 mg, each) of the air-dried powdered leaves were separately extracted with methanol containing 10 μg/mL umbelliferone (internal standard) by the aid of sonication for 20 minutes. The extract was then vortexed vigorously and centrifuged at 10,000 g for 5 min. An aliquot (1 mL) was then filtered through 22 μm Millipore filter and the filtrate was injected directly in the UPLC instrument.

UPLC-Orbitrap HRMS Profiling

B. High resolution UPLC-Orbitrap HRMS analysis Chromatographic separation of methanolic extracts of the leaves of the two Clerodendrum species under investigation was achieved under the following conditions: Stationary phase: HSS T3 column (100 × 1.0 mm, particle size 1.8 μm; Waters). Mobile phase: The following binary gradient was applied: 0 to 1 min: isocratic 95% A (water/formic acid, 99.9/0.1 v/v), 5% B (acetonitrile/formic acid, 99.9/0.1 v/v); 1 to 11 min: linear from 5 to 100% B; 11 to 19 min: isocratic 100% B and 19 to 30 min: isocratic 5% B. Flow rate: 150 μL/min. Injection volume: 2 μL The CID mass spectra (buffer gas; helium) were recorded using a normalized collision energy (NCE) of 35%. The instrument was equipped with a heated electronspray ion source (negative spray voltage at 3 kV, capillary temperature of 300 °C, source heater temperature of 250 °C, FTMS resolution of 30.000) and RP- C18 column (particle size 1.8 μm, pore size 100 Å, 150 mm × 1 mm i.d., Acquity HSS T3, Waters; column temperature of 40 °C). It was externally calibrated by the Pierce ESI negative ion calibration solution (product No. 88324) from Thermo Fisher Scientific. The data were evaluated using the software Xcalibur 2.2 SP1. Metabolites were characterized by their UV–VIS spectra (220–600 nm), mass spectra and comparison to phytochemical dictionary of natural products database (CRC) and reported literature. C. Identification of metabolites: To assess for differences in the metabolite composition of the leaves of C. inerme and C. splendens, a non-targeted metabolite profiling of extracts derived from leaf samples was conducted. Methanol soluble constituents from both species were analysed via a high resolution UPLC-Orbitrap HRMS. The identification of metabolites was performed in the negative electrospray ionization (ESI) mode, which is characterized by mass spectra with molecular ions corresponding to [M-H]- and other lower m/z fragment ions attributed to the identification of different metabolites. Also, the sequential loss of sugar moieties allowed the determination of the aglycone molecular weight and the identification of different glycosides. A total of 36 metabolites were detected in the two Clerodendrum species under investigation, and 32 were identified. Following the identification process, 28 chromatographic peaks, belonging to various metabolite classes, were assigned in C. inerme, among these 25 were identified, whereas 12 out of 14 chromatographic peaks assigned in C. splendens were identified (Figure 25).

100

UPLC-Orbitrap HRMS Profiling

Metabolite assignments were made by comparing retention times, UV-VIS spectra and MS data (accurate mass, isotopic distribution and fragmentation pattern in negative ion mode) of the compounds detected with reference literature and searching in the existing phytochemical dictionary of natural products database (compact reinforced composite (CRC), Wiley) as discussed below and summarized in table 22. Identified metabolites belonged to various classes including phenylpropanoid glycosides, iridoid glycosides, flavonoids, diterpenoids, phenolic acid and fatty acid derivatives. Chemical structures of some of the identified compounds are illustrated in figure 26.

C. inerme

C. splendens

Figure 25: Representative UPLC-Orbitrap HRMS total ion chromatograms (TIC) of the methanol extracts of the leaves of C. inerme and C. splendens in the negative ionization mode

101

UPLC-Orbitrap HRMS Profiling

A, Verbascoside (1) or its isomer, isoverbascoside (2) as an example of phenylpropanoid glycosides; B, inerminoside A1 as an example of iridoid glycosides; C, pictolinarigenin as an example of flavonoids; D, clerodermic acid (1) or its isomer, marrubiagenin (2) as an example of diterpenoids and E, rosmarinic acid as an example of phenolic acids idntified in methanol extracts of the leaves of C. inerme and C. splendens Figure 26: Examples of identified natural products by UPLC-Orbitrap HRMS analysis

102

UPLC-Orbitrap HRMS Profiling

Table 22: Metabolites identified in the methanol extracts of the leaves of C. inerme and C. splendens via UPLC-Orbitrap HRMS in the negative ionization mode

Peak Rt Mol. ion Error Mol. UV MSn ions m/z (-) Identification C. I. C. S. no. (min) m/z (-) (ppm) Formula

1 0.67 249 377.0853 -3.375 C18H18O9 341.1089, 215.0329 Unidentified + +

2 1.44 ND 523.1668 2.377 C21H32O15 523.1668 Melittoside + - 117.0541, 113.0235, 2-Hydroxy-2-methyl 3 1.95 ND 279.1080 2.888 C H O + - 11 20 81 101.0231, 89.0235 butyric acid hexoside 4 8.82 350, 369 325.0924 0.203 C15H16O8 163.0404, 119.0490 Coumaric acid hexoside + - 463.1819, 375.1295, 5 9.15 324, 369 507.1706 0.253 C H O Inerminoside A1 + - 21 32 14 331.1397 281.0621, 251.0523, 6 9.35 343, 369 489.1602 -0.546 C H O Teucardioside - + 21 30 13 179.0355, 161.0247 325.0929, 205.0514, Coumaric acid pentosyl 7 9.54 334, 369 457.1343 0.826 C H O + - 20 26 12 163.0404, 119.0494 hexoside 341.0878, 179.0351, Caffeic acid pentosyl 8 9.67 329, 369 473.1292 0.133 C H O + + 20 26 13 135.0454 hexoside 369.1558, 207.1028, 9 9.75 329, 369 387.1656 1.708 C H O Unidentified + + 18 28 9 163.1131 10 9.93 329, 369 335.0765 1.272 C16H16O8 179.0353, 135.0453 Caffeoyl shikimic acid - +

11 10.07 325 637.1763 0.124 C29H34O16 475.1462 Rhamnazin-3O-rutinoside - +

12 10.15 328 669.2020 0.708 C30H38O17 507.1720 p-Coumaroyl melittoside + - 623.1984, 593.2029, Markhamioside C or one 13 10.32 285, 329 755.2395 0.337 C H O - + 34 44 19 461.1668 of its isomers Verbascoside or 14 10.33 288, 332 623.1975 0.358 C H O 461.1669 + - 29 36 15 isoverbascoside 15 10.47 289, 327 623.1975 0.84 C29H36O15 461.1669 Magnoloside A or D + -

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UPLC-Orbitrap HRMS Profiling

Table 22 (continued): Metabolites identified in the methanol extracts of the leaves of C. inerme and C. splendens via UPLC-Orbitrap HRMS in the negative ionization mode

Peak Rt Mol. ion Error Mol. UV MSn ions m/z (-) Identification C. I. C. S. no. (min) m/z (-) (ppm) Formula Nitensoside A, flavosorbin 16 10.54 280, 331 607.1658 0.236 C H O 299.0563, 284.0329 - + 28 32 15 or semecapetin 17 10.58 268, 334 673.2704 0.354 C31H46O16 507.1730, 489.1624 Inerminoside B + -

18 10.68 320 675.2857 -0.772 C31H48O16 507.1720, 489.1615 Inerminoside A + - 197.0467, 179.0351, 19 10.7 288, 329 359.0766 1.354 C H O Rosmarinic acid - + 18 16 8 161.0246 651.2295, 607.2244, Markhamioside B or one 20 10.84 286, 328 783.2702 -0.518 C H O + - 36 48 19 589.2136 of its isomers 4'-Methyl scutellarein-7- 21 10.9 286, 331 475.0874 0.374 C H O 299.0560, 175.0248 + + 22 20 12 O- hexuronide 223.0247, 197.0455, 22 11.03 319 343.0814 0.731 C18H16O7 179.0350, 161.0244, Caffeic acid derivative - + 145.0296, 135.0453 23 11.13 269, 332 459.0925 1.138 C22H20O11 283.0610, 175.0249 Acacetin-7-O-hexuronide + - 269.0456, 225.0556, 24 11.47 293 269.0456 2.193 C H O Apigenin + - 15 10 5 201.0558, 149.0223 535.2559, 475.2331, 25 11.51 ND 581.2590 -0.28 C29H42O12 373.2021, 345.2106, Unidentified + - 331.1913 314.0428, 311.2227, Tri hydroxy octadecenoic 26 11.58 ND 329.2300 2.276 C H O 293.2120, 229.1445, + - 18 34 5 acid 213.1140, 171.1028

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Table 22 (continued): Metabolites identified in the methanol extracts of the leaves of C. inerme and C. splendens via UPLC-Orbitrap HRMS in the negative ionization mode

Peak Rt Mol. ion Error Mol. UV MSn ions m/z (-) Identification C. I. C. S. no. (min) m/z (-) (ppm) Formula

284.0328, 153.0196, Diosmetin, hispidulin or 27 11.69 297, 339 299.0558 2.693 C H O + - 16 12 6 119.505 4'-Methyl scutellrein

287.2226, 269.2126, Di hydroxy hexadecenoic 28 11.71 ND 287.2226 3.182 C H O + + 16 32 4 241.2175 acid 29 11.86 292 271.0606 2.762 C15H12O5 256.0379, 119.0488 Naringenin + -

30 11.88 292, 334 313.0713 2.093 C17H14O6 298.0485, 283.0249 Pictolinarigenin + - Hydroxy trimethoxy 31 11.99 293, 328 343.0816 1.168 C H O 328.0589 + - 18 16 7 flavone 303.1869, 259.2072, Trihydroxy abietatrienoic 32 12.04 293 347.1858 1.497 C H O + - 20 28 5 243.1760 acid

33 12.28 ND 837.4135 2.453 C39H66O19 ---- Monolinolein tri hexoside + - Clerodermic acid or 34 12.37 ND 331.1910 1.885 C H O 287.2018 + + 20 28 4 marrubiagenin 555.2835, 299.0443, 35 13.69 ND 555.2835 6.342 C H O Unidentified + - 28 43 11 255.2332, 225.0073 295.2274, 249.2228, Hydroxy octadecadienoic 36 13.88 ND 295.2274 2.197 C H O - + 18 32 3 155.1443, 141.1287 acid C. I., Clerodemdrum inerme; C. S., Clerodendrum splendens (+) and (-) indicate presence and absence of a metabolite, respectively; ND, not detected; Rt, retention time; Total number of metabolites identified include: 4 phenylpropanoid glycosides, 6 iridoid glycosides, 9 flavonoids, 2 diterpenoids, 6 phenolic acid derivatives, 5 fatty acid derivatives.

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UPLC-Orbitrap HRMS Profiling

Identification of Phenylpropanoid Glycosides Phenylpropanoid glycosides were identified, with the highest abundance, in methanol extracts of the leaves of C. inerme and C. splendens (peaks 13, 14, 15 and 20). MS signals at [M-H]- of 755.2395, 623.1975, 623.1975 and 783.2702 were assigned to phenylpropanoid glycosides. They were identified from their characteristic UV absorbance at 285 and 330 nm. Two types of phenylpropanoid glycosides were detected in the methanol extract of the leaves of C. inerme. The first type, represented by verbascoside, isoverbascoside, magnoloside A or D (peaks 14 and 15), consists of caffeoyl and dihydroxy phenylethanol moieties linked together with two sugars, hexose and deoxy hexose. The deoxyhexose moiety of verbascoside or its isomer isoverbascoside is rhamnose, while that of magnoloside A is mannose whereas magnoloside D contains allose as the deoxy sugar. In MS/MS analysis, this type is characterized by elimination of 162 atomic mass units (amu) corresponding to caffeoyl ion. The second type, represented by markhamioside B, leonoside B, betonyoside D, angoroside C or lamiophlomiside A (peak 20), consists of feruloyl and hydroxy methoxy phenylethanol moities linked together with three sugars, hexose, deoxy hexose and pentose. This type is characterized in MS/MS analysis by elimination of 176 and 132 amu corresponding to feruloyl and pentosyl ions, respectively. The methanol extract of the leaves of C. splendens was found to contain one type of phenylpropanoid glycosides, represented by markhamioside C, betonyoside F, angoroside A or myricoside (peak 13) and consisting of caffeoyl and dihydroxy phenylethanol moieties linked together with three sugars, hexose, deoxy hexose and pentose and characterized in MS/MS analysis by elimination of 162 and 132 amu corresponding to caffeoyl and pentosyl ions, respectively. The MS/MS data and UV absorbance of the detected phenylpropanoid glycosides were compared to reference literature for confirmation of their identities (Cardoso et al., 2005; Li et al., 2005; Savarese et al., 2007; Lee et al., 2015; Xue et al., 2016). MS/MS analysis of molecular ion [M-H]- at m/z 623.1975 assigned as verbascoside or one of its previously mentioned isomers is represented by figure 27.

Figure 27: MS/MS spectrum of peak 13 in the negative ionization mode

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UPLC-Orbitrap HRMS Profiling

Identification of Iridoid Glycosides Iridoid glycosides were identified in the methanol extracts of the leaves of C. inerme and C. splendens. In MS/MS analysis, signals were assigned for iridoid glycosides viz., melittoside (peak 2), inerminoside A1 (peak 5), teucardioside (peak 6), p-coumaroyl melittoside (peak 12), inerminoside B (peak 17) and inerminoside A (peak 18) at [M-H]- of 523.1668, 507.1706, 489.1602, 669.2020, 673.2704 and 675.2857 m/z, respectively. The identities of the detected iridoid glycosides were confirmed through comparison with MS/MS data of reference literature (Calis, et al., 1993; Karioti et al., 2010). MS/MS analysis of molecular ion [M-H]- at m/z 507.1706 assigned as inerminoside A1 is represented by figure 28.

Figure 28: MS/MS spectrum of peak 5 (inerminoside A1) in the negative ionization mode Identification of Flavonoids Both flavonoid aglycones and glycosides were identified in the methanol extracts of the leaves of C. inerme and C. splendens. Most of the identified flavonoids appeared to belong to the flavone class and this was confirmed through their MS data and UV absorbances. Flavones identified in the methanol extract of the leaves of C. inerme were apigenin (peak 24), pictolinarigenin (peak 30) and hydroxy trimethoxy flavone (peak 31) at [M-H]- of 269.0456, 313.0713 and 343.0816 m/z, respectively. Another MS signal at [M-H]- of 299.0558 (peak 27) was assigned to a flavone; diosmetin, hispidulin or 4'-methyl scutellarein. Naringenin (peak 29) was the only identified flavanone in the leaves of C. inerme with MS signal at [M-H]- of 271.0606 m/z and UV absorbance of 292 nm. The flavonoid glycosides identified in the leaves of C. inerme were found to have flavone nucleus linked to hexuronic acid via O-glycosidic linkage. This was revealed in MS analysis from elimination of 176 amu corresponding to O-hexuronyl ion. MS signals at [M-H]- of 475.0874 and 459.0925 m/z were assigned to 4'-methyl scutellarein-7-O- hexuronide (peak 21) and acacetin-7-O-hexuronide (peak 23), respectively. On the other hand, the two flavonoid glycosides identified in the leaves of C. splendens (peaks 11 and 16) were found to be; a flavonol attached to a disaccharide, rutinose, at position 3 (rhamnazin-3-O-rutinoside) at [M-H]- of 637.1763 m/z and a 7- methoxy flavone attached to disaccharide consisting of hexose and deoxy hexose 107

UPLC-Orbitrap HRMS Profiling through O-glycosidic linkage at position 6 (nitensoside A, flavosorbin or semecapetin) with MS signal at [M-H]- of 607.1658 m/z. For confirmation of the identities of the compounds, their MS/MS data and UV absorbances were compared to reference literature (Grayer et al., 2002; Zhang et al., 2014; Li et al., 2015). MS/MS analysis of molecular ion [M-H]- at m/z 313.0713 assigned as pictolinarigenin is represented by figure 29.

Figure 29: MS/MS spectrum of peak 30 (pictolinarigenin) in the negative ionization mode Identification of Diterpenoids One abietane diterpenoid was detected in the methanol extract of the leaves of C. inerme (peak 32) at [M-H]- of 347.1858 m/z, which was identified through its MS data to be trihydroxy abietatrienoic acid. Another diterpenoid, with clerodane nucleus, was detected in both methanol extracts of the leaves of C. inerme and C. splendens. MS signal at [M-H]- of 331.1910 m/z was assigned to clerodermic acid or its stereoisomer, marrubiagenin (peak 34). For confirmation, the MS/MS data of the identified diterpenes was compared to reference literature of their identities (Efdi et al., 2007). MS/MS analysis of molecular ion [M-H]- at m/z 331.1910 assigned as clerodermic acid or its isomer, marrubiagenin is represented by figure 30.

Figure 30: MS/MS spectrum of peak 34 in the negative ionization mode 108

UPLC-Orbitrap HRMS Profiling

Identification of Phenolic Acid Derivatives Derivatives of coumaric and caffeic acids were identified in the methanol extracts of the leaves of C. inerme and C. splendens as relatively minor constituents, except rosmarinic acid which was detected in the methanol extract of the leaves of C. splendens with high abundance. They were identified through their MS/MS data and characteristic UV absorbance. Glycosides of coumaric acid were detected in the leaves of C. inerme at [M-H]- of 325.0924 and 457.1343 m/z corresponding to coumaric acid hexoside (peak 4) and coumaric acid pentosyl hexoside (peak 7), respectively. In MS/MS anlysis, they both showed fragmentation pattern at 163.0404 and 119.0490 m/z, which is characteristic to coumaric acid. The former showed elimination of 162 amu, corresponding to hexosyl ion while the latter showed elimination of 132 and 294 amu, corresponding to pentosyl and pentosyl hexosyl ions, respectively. Derivatives of caffeic acid were detected at [M-H]- of 473.1292, 335.0765 and 359.0766 m/z corresponding to caffeic acid pentosyl hexoside (peak 8), caffeoyl shikimic acid (peak 10) and rosmarinic acid (peak 19), respectively. Caffeic acid pentosyl hexoside was detected in the methanol extracts of the leaves of C. inerme and C. splendens, caffeoyl shikimic acid and rosmarinic acid were detected only in the methanol extract of the leaves of C. splendens. Another caffeic acid derivative was detected in the methanol extract of the leaves of C. splendens (peak 22) at [M-H]- of 343.0814 m/z but its exact structure was not fully identified. All caffeic acid derivatives identified in the methanol extracts of the investigated species showed MS fragmentation pattern characteristic to caffeic acid at 179.0351 and 135.0454 m/z. For confirmation of the identities of the identified compounds, their MS data and UV absorbances were compared to reference literature (Bais et al., 2002; Sanchez-rabaneda et al., 2003; Cech et al., 2006). MS/MS analysis of molecular ion [M-H]- at m/z 359.0766 assigned as rosmarinic acid is represented by figure 31.

Figure 31: MS/MS spectrum of peak 19 (rosmarinic acid) in the negative ionization mode

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UPLC-Orbitrap HRMS Profiling

Identification of Fatty Acid Derivatives Hydroxylated or oxygenated derivatives of saturated and unsaturated fatty acids were identified in the methanol extracts of the leaves of C. inerme and C. splendens as minor constituents. They showed differences in their retention time based on chain length, number of double bonds and number of hydroxyl, or oxo, functions. Five fatty acid derivatives were detected in in the methanol extracts of the leaves of C. inerme and C. splendens. They were tentatively identified as 2-Hydroxy-2- methyl butyric acid hexoside (peak 3), tri hydroxy octadecenoic acid (peak 26), di hydroxy hexadecenoic acid (peak 28), monolinolein tri hexoside (peak 33) and hydroxy octadecadienoic acid (peak 36) at [M-H]- of 279.1080, 329.2300, 287.2226, 837.4135 and 295.2274 m/z. The exact structures of fatty acid derivatives could not be assigned as the position of hydroxl, or oxo, groups and E/Z geometry of double bonds could not be determined.

110

Chapter V Quantitative Estimation of Phenolic Content

Quantitative Estimation of Phenolic Content Chapter V Quantitative Estimation of Phenolic Content Introduction Phenolic compounds are secondary plant metabolites that are involved in plant defense mechanisms against pathogens and in plant-plant communication (Mendonça et al., 2018). Fruits and vegetables are the richest sources of polyphenols including flavonoids, phenolic acids, curcuminoids, coumarins, lignans and stilbenes (Corrêa and Rogero, 2018). The human health benefits attributed to polyphenolics are numerous; they are reported to have antioxidant and free-radical scavenging properties (Callcott et al., 2018; Mendonça et al., 2018), as well as anti-inflammatory effects (Callcott et al., 2018; Corrêa and Rogero, 2018). They are also claimed to exert a protective action against cardiovascular disorders (Tresserra-Rimbau et al., 2014; Mendonça et al., 2018) possibly through reduction of blood pressure and inhibition of platelet aggregation thus delaying development of atherosclerosis (Milenkovic et al., 2013; Corrêa and Rogero, 2018); beside, preventing obesity (Corrêa and Rogero, 2018). Recent studies revealed that phenolic compounds have modulatory effect on gene expression (Milenkovic et al., 2013; Corrêa and Rogero, 2018), and hence can regulate different biological functions including inflammation, apoptosis and lipid metabolism (Milenkovic et al., 2013). Moreover, consumption of a polyphenol- rich diet is said to be associated with reduction in mortality risk, possibly due to its anti-inflammatory effect (Pounis et al., 2018). Besides, numerous experimentally based bioactivities support the therapeutic uses of flavonoids for treatment and/or prevention of various ailments that affect human condition and performance. They are reported to exert antioxidant (Gupta and Gupta, 2012; Ruan et al., 2018), anti-inflammatory (Srisook et al., 2015; Rengasamy et al., 2018; Ruan et al., 2018) bronchodilator (Hazekamp et al., 2001), hepatoprotective (Wan and Jiang, 2018) and anticancer (Souza et al., 2018) activities. Moreover, they are found to play a significant role in symptomatic relief and protection against several autoimmune diseases as multiple sclerosis, rheumatoid arthritis, psoriasis and systemic lupus erythematosus (Rengasamy et al., 2018). Data also revealed that flavonoids have neuroprotective and acetylcholinesterase inhibitory activities (Khan et al., 2018; Ruan et al., 2018) and thus can be recommended for management of Alzheimer's disease. The beneficial healthcare impacts of plant polyphenols motivated the continuous search for new sources. In this respect, the content of these constituents in the leaves of the two Clerodendrum species under investigation was estimated. To fulfil this goal, both the total phenolic and total flavonoid contents (TPC and TFC, respectively) were determined through application of appropriate spectrophotometric techniques. The EEs prepared from the defatted leaves of the 111

Quantitative Estimation of Phenolic Content plants (page 94) and responding to positive tests for phenolic compounds including flavonoids (Geissman, 1962) were subjected to this study. I. Determination of Total Phenolic Content (TPC) A. Principle The total phenolic contents (TPC) of the EEs of the leaves of C. inerme and C. splendens were determined using the Folin-Ciocalteu spectrophotometric method according to a published procedure (Nickavar and Esbati, 2012). TPC was deduced from a pre-established calibration curve using gallic acid as standard. Results were expressed as mg of gallic acid equivalent (mg GAE)/ g of dried extract. B. Establishment of the Standard Calibration Curve A series of different concentrations of gallic acid solution in distilled water (10-150 µg/mL) was prepared. An aliquot (1 mL) of each concentration was mixed with 5 mL of Folin-Ciocalteu reagent dissolved in 50% methanol (1 mg/10 mL) and left for 10 minutes, followed by addition of 4 mL of sodium carbonate solution in distilled water (75 mg/mL). The absorbance of the blue color produced in the resulting mixture was measured after 30 minutes, at 765 nm by means of a UV- Visible spectrophotometer, against a blank experiment prepared concurrently using 1 mL of distilled water instead of the standard solution. For each concentration, triplicate experiments were carried out and the average absorbance determined. The standard calibration curve (Figure 32) was established by plotting the averages of the measured absorbances against the different gallic acid concentrations. Results are recorded in table 23. Table 23: Absorbances of the different concentrations of standard gallic acid Concentration (μg/mL) Absorbance* 10 0.0761 25 0.1401 50 0.2831 75 0.5813 100 0.8491 150 1.2401 *Average of three determinations

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Quantitative Estimation of Phenolic Content

1.4 y = 0.0087x - 0.0679 1.2 R² = 0.9877

0.8

0.6 Absorbance 0.4

0.2

0 0 20 40 60 80 100 120 140 160 Concentration (ug/mL)

Figure 32: Standard calibration curve of gallic acid C. Preparation of Samples The dried EEs of the leaves of C. inerme and C. splendens (1 mg, each) were separately dissolved in 1 mL of distilled water with the aid of sonication. The resulting solutions were individually treated with Folin-Ciocalteu reagent and sodium carbonate, as previously mentioned. The absorbances of the prepared samples were recorded, as for the standard, at 765 nm and TPC (expressed as gallic acid equivalent, GAE µg/mL solution of tested extract) was deduced from the calibration curve equation as follows: Y = 0.0087 X - 0.0679, R2 = 0.9877 Where Y=absorbance, X=corresponding concentration (µg/mL) and R2=correlation coefficient. D. Results and Discussion The measured absorbances of the C. inerme and C. splendens samples were 0.4828 and 0.4124 corresponding to 63.3 and 55.21 μg GAE/mL solution of the respective EE. Results indicated that the total phenolic content, calculated on dry weight basis of the EEs, of the leaves of C. inerme exceeded that of C. splendens leaves (63.30 vs. 55.21 mg GAE/g dry extract). II. Determination of Total Flavonoid Content (TFC) A. Principle The total flavonoid content (TFC) of the EEs of the leaves of C. inerme and C. splendens were determined by measuring the intensity of the yellow color produced

113

Quantitative Estimation of Phenolic Content when flavonoids are complexed with aluminium chloride reagent (Nickavar and Esbati, 2012) and expressed as mg of quercetin equivalent (mg QE)/ g dried extractive. B. Establishment of the Standard Calibration Curve A series of different concentrations (5-50 µg/mL) of quercetin solution in methanol was prepared. A measured volume (2.5 mL) of each concentration was mixed with an equal volume of aluminium chloride reagent dissolved in distilled water (20 mg/mL). The mixture was allowed to stand for 40 minutes and the absorbance measured at 415 nm, using a UV-Visible spectrophotometer, against a blank prepared at the same time using 2.5 mL of pure methanol instead of the quercetin solution. Triple experiments were carried out for each concentration, and the average absorbance determined. The standard calibration curve (Figure 33) was constructed by plotting the averages of the measured absorbances versus the corresponding quercetin concentrations. Results are recorded in table 24. Table 24: Absorbances of the different concentrations of standard quercetin

Concentration (μg/mL) Absorbance* 5 0.0221 10 0.0522 15 0.0726 25 0.1232 35 0.2186 50 0.3517 *Average of three determinations

0.4

0.35 y = 0.0073x - 0.0297 R² = 0.9799 0.3

0.25

0.2

Absorbance 0.15

0.1

0.05

0 0 10 20 30 40 50 60 Concentration (ug/mL)

Figure 33: Standard calibration curve of quercetin 114

Quantitative Estimation of Phenolic Content C. Preparation of Samples Samples (2 mg, each) of the dried EEs of the leaves C. inerme and C. splendens were separately dissolved in 1 mL of methanol with the aid of sonication. The resulting solutions were individually treated with aluminium chloride reagent as mentioned previously. The absorbances of the prepared samples were measured by the use of a UV-Visible spectrophotometer set at 415 nm. The total flavonoid contents (TFC), expressed as quercetin equivalent (QE) were calculated by means of the following equation: Y = 0.0073 X - 0.0297, R2 = 0.9799 Where Y=absorbance, X=corresponding concentration (µg/mL) and R2=correlation coefficient. D. Results and Discussion The absorbances recorded for the reaction mixtures of the EEs of C. inerme and C. splendens were 0.04797 and 0.04476, respectively, corresponding to 5.32 and 5.01 mg QE/g of dry EE. These results revealed that TFC of the leaves of C. inerme was slightly higher than that of C. splendens.

115

Chapter VI In-vitro Evaluation of the Biological Activities

In-vitro Evaluation of the Biological Activities Chapter VI In-vitro Evaluation of the Biological Activities I. Evaluation of the Antioxidant Activity

Introduction

Natural antioxidants are important for maintaining human health through preventing cell damage caused by oxidative stress and free radical scavenging (Rajendran et al., 2014). They are efficient in protection against many diseases including cancer (Rajendran et al., 2014), Inflammatory Bowel Diseases (Patlevič et al., 2016), cardiovascular ailments (Sugamura and Keaney, 2011; Rajendran et al., 2014) and neurological disorders such as Alzheimer's disease (Rajendran et al., 2014), Parkinson's disease and Amyotrophic Lateral Sclerosis (Chang et al., 2018). The antioxidant activity of the EEs of the leaves of C. inerme and C. splendens was assessed in-vitro through application of the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical spectrophotometric assay. The latter is considered as a simple, rapid and convenient method for screening of different types of samples (independent of polarity) for radical scavenging activity (Koleva et al., 2002); besides, DPPH is a stable free radical at room temperature (Molyneux, 2004). These advantages led to the selection of DPPH assay for evaluation of the antioxidant potential of plant extracts and/or isolated compounds to be proposed as candidates for implementation in pharmaceutical preparations. A. Principle of the Assay In the radical state, the methanolic solution of DPPH is purple in colour and absorbs light at 492 nm. When the odd electron on the nitrogen atom is paired by the presence of an antioxidant (e.g, flavonoid), the free radical is reduced to the corresponding hydrazine molecular form (DPPHH), which is yellow in colour and does not absorb at 492 nm (Sreenivasan et al., 2007). This quenching of absorbance is taken as a measure of the free radical scavenging power of the tested sample. The following equation illustrates the mechanism of free radical scavenging.

116

In-vitro Evaluation of the Biological Activities B. Procedure The assay was performed by adopting a modified procedure as described by (Romano et al., 2009). A set of serial dilutions of each EE and standard reference antioxidant (gallic acid) were prepared and subjected to the DPPH assay. Results are recorded in tables 25 and 26 and displayed in figures 34 and 35. Experiments were carried out by using a 96-wells plate, and were performed in triplicates. The reaction mixture, in each case, consisted of 22 µL of the tested sample and 200 µL of 0.004% DPPH in methanol. Blank experiments were similarly prepared using 22 µL pure methanol instead of sample to get rid of any inherent solvent activity. Negative controls, consisting of 22 µL of the tested sample and 200 µL of methanol, were also run in parallel to correct for any non- DPPH absorbance by coloured extracts at the test wavelength (492 nm). The plates were immediately shaken for 30 seconds followed by incubation in the dark for 30 minutes, at room temperature. The absorbance of remaining DPPH was measured in a microplate reader at 492 nm. The percentage of DPPH quenching was calculated using the following equation:

% DPPH quenched = 100 (Ablank - Asample)/Ablank

Where; Ablank and Asample are the averages of triplicate determinations of the absorbance of the blank and sample at 492 nm, respectively. The percentages of DPPH quenching of the different dilutions of each extractive were plotted against the corresponding concentrations as represented in figures 34 and 35. The EC50 value (effective concentration of the sample in methanol required for quenching 50% of DPPH radicals) was calculated for each extract and for gallic acid. The EC50 values of the tested extracts as compared to the reference anti-oxidant gallic acid are recorded in table 27 and represented in figure 36. Table 25: Percentage of DPPH quenching of serial dilutions of the EEs of the leaves of C. inerme and C. splendens

Concentration % DPPH quenching of the EE* (µg/ml) C. inerme C. splendens 125 0.23178 13.29299 250 21.43961 20.0103 500 56.92763 60.4172 1000 71.23358 66.36621 2000 78.85655 75.94643 4000 93.38999 90.47129 *Average of three determinations

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In-vitro Evaluation of the Biological Activities Table 26: Percentage of DPPH quenching of serial dilutions of the reference antioxidant gallic acid

Concentration (µg/ml) % DPPH quenching of gallic acid*

5.5 13.1 11 33.2 22 60.1 44 77.4 88 92.6 *Average of three determinations

CI., Clerodendrum inerme; CS., Clerodendrum splendens; EE., ethanol 70% extract Figure 34: Line graph representing the concentration-dependent DPPH quenching of the EE of the leaves of C. inerme and C. splendens

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In-vitro Evaluation of the Biological Activities

Figure 35: Line graph representing the concentration-dependent DPPH quenching of standard gallic acid Table 27: DPPH quenching potency of the EEs of the leaves of C. inerme and C. splendens, expressed as EC50 (µg/mL), compared to standard gallic acid

Sample/ Standard *EC50 ± SD (µg/mL) C. inerme EE 627.246 ± 37.261 C. splendens EE 583.905 ± 25.315 Gallic acid 18.37 ± 0.59

* EC50=effective concentration of the sample required for quenching of 50% of the DPPH free radical as compared to the solvent control, SD=standard deviation EE., ethanol 70% extract

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In-vitro Evaluation of the Biological Activities

CI., Clerodendrum inerme; CS., Clerodendrum splendens; EE., ethanol 70% extract Figure 36: Bar chart representing the in-vitro anti-oxidant activity of the EEs of the leaves of C. inerme and C. splendens compared to standard gallic acid C. Results and Discussion From the previous results, tables 25-27 and figures 34-36, both EEs of the leaves of C. inerme and C. splendens showed weak antioxidant activity with respective EC50 values of 627.246 ± 37.261 and 583.905 ± 25.315 µg/mL, where the EE of the leaves of C. splendens exhibited a slightly higher antioxidant activity. The results were compared to the reference anti-oxidant, gallic acid whose EC50 value was 18.37 ± 0.59 µg/mL.

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II. Evaluation of the Cytotoxic Activity Introduction Cancer is expected to be the most important barrier to increasing life expectancy in the world and the leading cause of death in the 21st century (Bray et al., 2018). Lung and breast cancers have the highest incidence worldwide while colorectal cancer comes in the second place. Lung cancer also has the highest mortality rate followed by colorectal and stomach cancers (Parkin et al., 2002; Bray et al., 2018). Among males, lung cancer is the most commonly diagnosed type (14.5%) with the highest mortality rate (22%), whereas the most commonly diagnosed cancer in females is breast cancer (24.2%) with the highest mortality rate (15%) (Bray et al., 2018). The previous data applies also in Egypt (Omar et al., 2003; Bray et al., 2018) except that liver cancer has the highest incidence in males (Omar et al., 2003). Clerodendrum species are reported to exert a cytotoxic effect against various cell lines (Reid et al., 2006; Xu et al., 2013; Sun et al., 2014; Xu et al., 2014; Xu et al., 2016). The ethanolic extract of the leaves of C. inerme exhibited a cytotoxic activity against lung carcinoma (A549) cell line (Kalavathi and Sagayagiri, 2016). In addition, the cytotoxic efficiency of compounds isolated from the leaves of this plant has been proven (Hosny et al., 2004; Xu et al. 2013; Sun et al., 2014). The aerial parts of C. splendens were also reported to have cytotoxic activity against breast, colon and liver carcinoma cell lines (Soliman et al., 2014). The in-vitro cytotoxic activity of the EEs of the leaves of C. inerme and C. splendens, on (H-1299), (HepG-2) and (MCF-7); lung, liver and breast carcinoma cell lines, respectively, was investigated by SRB assay using the method described by (Skehan et al., 1990). These cell lines were selected based on being among the commonest causes of cancers and death both worldwide and in Egypt (Omar et al., 2003; Bray et al., 2018). Safety of the aforementioned extractives was determined by investigating their cytotoxicity on normal fibroblast (BHK) cell lines. A. Principle of the Assay Sulfo-Rhodamine-B (SRB) is a bright pink aminoxanthene dye used for staining of intracellular proteins through binding between its sulphonic and amino groups, under mild acidic conditions, to provide a sensitive index of cellular protein content. This bond is electrostatically and pH dependent, i.e. under mild acidic conditions the bond is formed and under mild basic conditions it can be extracted from cells and solubilized in order to be measured spectrophotometrically at 564 nm. SRB assay depends on incubation of cell lines for 48 hours with a solution of the sample to be investigated in DMSO followed by staining of the surviving cells with SRB stain. The colour intensity of the protein-bound stain is measured in an ELISA microplate reader, at 564 nm, to determine the surviving fraction of the cells. 121

In-vitro Evaluation of the Biological Activities Preparation of cell cultures: Frozen cells were seeded in the supplemented medium and incubated until a confluent growth was achieved. The cells were freshly sub-cultured before each experiment to be in the exponential phase of growth. Collection of cells by trypsinization: The medium was discarded, and the cell monolayer was washed with Phosphate Buffer Saline (PBS). The adherent cells were completely detached from their monolayer by the addition of trypsin. Trypsin was then discarded and the cells separated into single cell suspension and then seeded in supplemented medium. Determination and counting of viable cells: An aliquot (50 μl) of trypan blue solution (0.05%) was added to 50 μl of the single cell suspension. Viable cells (not stained) were counted using a haemocytometer under inverted microscope. The cells were then diluted to give the concentration of single cell suspension required for each experiment. B. Procedure • Cells were arranged in 96-multiwell plate (104 cells/plate) and left for 24 hours to allow their attachment to the plate wall. • Different concentrations (12.5, 25, 50, 100 μg/mL) of each of the tested samples as well as standard doxorubicin were prepared and added to the cell monolayer. Triplicate wells were prepared for each dose. o • The cells were incubated for 48 hours with the sample at 37 C in 5% CO2 atmosphere. • After incubation, the cells were fixed using trichloroacetic acid (TCA, 50%, 50 μl) for 1 hour at 4 °C, washed 5 times with distilled water and stained for 30 min, at room temperature, with 50 μl 0.4% (w/v) Sulfo-Rhodamine-B (SRB) dissolved in 1% acetic acid. • The unbound stain was removed by washing four times with 1% acetic acid and the bound stain was recovered with Tris EDTA buffer (pH 10.5) by shaking for 5 min at 1600 rpm. • The colour intensity was measured in an ELISA microplate reader at 564 nm. The surviving fraction was then calculated from the following equation: Surviving fraction = (CI) of treated cells / (CI) of control cells Where; (CI) is the color intensity measured by the ELISA microplate reader at 564 nm. • Averages of triplicate measurements was calculated for each concentration and graphs were plotted between the surviving fractions of the cells and sample concentrations, then IC50 (The concentration that inhibits the growth 50% of the cells) was determined for each sample on each cell line.

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In-vitro Evaluation of the Biological Activities Results are recorded in table (28) and presented by figure 37. Table 28: In-vitro cytotoxic activity of the EE of the leaves of C. inerme and C. splendens on different cell lines compared to standard doxorubicin

*IC50 (µg/mL) Sample/Standard H-1299 HepG-2 MCF-7 BHK C. inerme EE 23 25 25.5 37 C. splendens EE 9.91 33.6 45.5 36

Doxorubicin 4.28 5.87 2.97 > 50

* IC50=effective concentration of the sample required to inhibit the growth of 50 % of the cells, calculated as average of triplicate measurements. EE, ethanol (70%) extract

CI., Clerodendrum inerme; CS., Clerodendrum splendens; EE, ethanol (70%) extract; Dox, doxorubicin Figure 37: Bar chart representing the in-vitro cytotoxic activity of the EEs of the leaves of C. inerme and C. splendens compared to standard doxorubicin C. Results and Discussion According to the United states National Cancer Institute plant screening program, any crude extract is considered to possess an in-vitro cytotoxic activity if its IC50 123

In-vitro Evaluation of the Biological Activities value is less than 20 μg/mL (Boik, 2001). Thus, from table (28) and figure 37, the EE of the leaves of C. inerme exhibited in-vitro cytotoxic activity against the three carcinoma cell lines while that of C. splendens exihibited strong in-vitro cytotoxic activity against (H-1299) lung carcinoma cell line and weak in-vitro cytotoxic activity against (MCF-7) breast and (HepG-2) liver carcinoma cell lines. The EE of the leaves of C. inerme exhibited higher cytotoxic activity than that of C splendens with IC50 values of 25.5 and 25 μg/mL for C. inerme and 45.5 and 36 μg/mL for C. splendens on (MCF-7) breast and (HepG-2) liver carcinoma cell lines, respectively; whereas, the EE of the leaves of C. splendens exhibited higher cytotoxic activity on (H-1299) lung carcinoma cell line than that of C. inerme with IC50 values of 9.91 vs. 23 μg/mL. EEs of both C. inerme and C. splendens leaves exhibited nearly the same cytotoxic activity on normal fibroblasts (BHK cell line) with IC50 values of 37 and 36 μg/mL respectively meaning that they have nearly the same safety profile.

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III. Evaluation of the Antibacterial Activity against Antibiotic Resistant Bacterial Strains Introduction Antibiotic resistance is a major problem that is spreading and increasing while the development of new antibiotics is ceasing. As a result, some bacterial infections that were once easily treated became untreatable. Many pathogenic bacteria have developed antibiotic resistance over years including; Neisseria gonorrhea, Methicillin and Vancomycin Resistant Staphylococcus aureus (MRSA and VRSA), Vancomycin Resistant Enterococci, Penicillin Resistant Streptococcus pneumonia and multi drug resistant Mycobacterium tuberculosis (TB). These bacteria are the main cause of hospital-acquired infections that lead to death in most cases. Antibiotic resistance can be minimized through increasing awareness about the proper use of antibiotics and development of new antibacterial agents effective against these bacterial strains (Yelin and Kishony, 2018). The EEs of C. inerme and C. splendens were investigated for antibacterial activity against Methicillin Resistant Staphylococcus aureus (MRSA) and Mycobacterium tuberculosis (TB) using the methods reported by (Bonacorsi et al., 2009) and (Franzblau et al., 1998), respectively.

A. Evaluation of the Antibacterial Activity against Methicillin Resistant Staphylococcus aureus (MRSA) 1. Principle of the Assay Measurement of the antibacterial activity is based on measuring the colour intensity of the purple coloured 3- (4,5-dimethyl-thiazol-2-yl)- 2,5- diphenyl-tetrazolium bromide (MTT) at 620 nm using ELISA microplate reader. The change in colour intensity indicates that the bacteria is biologically active. Vancomycin was used as a reference drug. The percentage inhibition of bacterial growth by different concentrations of each sample was calculated (average of triplicate measurements). A dose-response curve was drawn, and the concentrations required to inhibit 90% of bacterial growth (MIC 90) and the lowest concentrations where no change of MTT colour was detected (MIC) were determined. 2. Procedure • An inoculum of MRSA was prepared, and its concentration was adjusted to 106 CFU/mL. • Serial dilutions of the samples to be investigated, EEs of the leaves of C. inerme and C. splendens, and the standard drug (vancomycin) were prepared in dimethyl sulfoxide (DMSO) at concentrations of 0.24 – 125 µg/mL. • The assay was performed in a 96-well microplate. In each well, 40 µL of the growth medium (Brain Heart Infusion, BHI, plus 10% Fetal Bovine Serum, 125

In-vitro Evaluation of the Biological Activities FBS, 10 µL of the inoculum and 50 µL of the sample dilution were added. Vancomycin and DMSO were used as positive and negative controls. o • The plates were incubated at 37 C for 3 days at 5% O2, 10% CO2 and 85% N2 atmosphere. • To each well, 40 µL of MTT, at a concentration of 0.5 mg/mL freshly prepared in water, were added and incubated for 30 minutes. The colour intensity was measured for each well at 620 nm and the percentage inhibition in bacterial growth was calculated using the following formula:

% Inhibition = 100 (Acontrol - Asample)/Acontrol

Where; Acontrol and Asample are the averages of triplicate determinations of the absorbance of both DMSO control and sample at 620 nm, respectively. • MIC 90 and MIC were determined for each sample and for standard vancomycin from dose-response curve as the concentrations required to inhibit 90% of mycobacterial growth and the lowest concentration where no change of MTT colour was detected, respectively. Results are recorded in table 29 and represented by figure 38. Table 29: In-vitro antibacterial activity of the EEs of the leaves of C. inerme and C. splendens against MRSA compared to standard vancomycin

Sample/Standard *MIC 90 µg/mL *MIC µg/mL

C. splendens EE 113.1 <125 C. inerme EE 12.7 31.25 Vancomycin 0.6 1.95 *MIC 90=the concentration required to inhibit 90% of bacterial growth; MIC=the lowest concentration where no change of MTT colour was detected EE, ethanol (70%) extract

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CI., Clerodendrum inerme; CS., Clerodendrum splendens; EE, ethanol (70%) extract; MIC 90, the concentration required to inhibit 90% of bacterial growth; MIC, the lowest concentration where no change of MTT colour was detected Figure 38: Bar chart representing the in-vitro antibacterial activity of the EEs of the leaves of C. inerme and C. splendens against MRSA compared to standard vancomycin 3. Results and Discussion From the previous results (table 29 and figure 38), the EE of C. inerme showed moderate antibacterial activity against MRSA, with MIC 90 value of 12.7 µg/mL and MIC value of 31.25 µg/mL, compared to vancomycin (MIC 90 and MIC values of 0.6 and 1.95 µg/mL, respectively). Meanwhile, the EE of C. splendens showed very weak antibacterial activity against MRSA with MIC 90 value of 113.1 µg/mL. Concentrations up to 125 µg/mL could not inhibit the growth of all of the incubated bacteria and thus the MIC was higher than 125 µg/mL.

B. Evaluation of the Antimycobacterial Activity 1. Principle of the Assay The EEs of the leaves of C. inerme and C. splendens were evaluated for antimycobacterial activity against Mycobacterium tuberculosis using the Microplate Alamar Blue Assay (MABA). The latter is based on measuring the change in colour intensity of the blue colour of Alamar Blue solution by the activity of Mycobacterium tuberculosis. The colour intensity is measured at 590 nm using ELISA microplate reader. The percentage inhibition of mycobacterial

127

In-vitro Evaluation of the Biological Activities growth by different concentrations of each sample was calculated as the average of triplicate measurements. The concentrations required to inhibit 90% of mycobacterial growth (MIC 90) and the lowest concentration that prevented colour change of Alamar Blue solution (MIC) were determined from the dose-response curve. Isoniazide was used as a reference drug. 2. Procedure • The assay was performed in a black, clear-bottomed, 96-well microplate to minimize background effect. • The outer perimeter wells were filled with sterile water to prevent dehydration in experimental wells. • A serial dilution (0.06 –125 µg/mL) of each of the tested extracts and the reference drug, isoniazide, dissolved in DMSO, was prepared in the microplate then 0.1 mL of Mycobacterium tuberculosis inoculum (105 CFU/mL) was added to the wells. Wells containing bacteria only were used as negative control. • Plates were incubated at 37 oC for 4 days, then 20 µL of Alamar Blue solution and 12.5 µL of 20% Tween 80 were added to the entire plate. • The plates were re-incubated at 37 oC for 24 hours then the colour intensity was measured at 590 nm using ELISA microplate reader. • The percentage inhibition of mycobacterial growth was calculated from the following formula; % inhibition = 1 - (mean of test well/mean of B well) ×100 Where mean of test well and mean of B well are the averages of triplicate determinations of the absorbance of both sample and negative control at 590 nm, respectively. • MIC 90 and MIC were determined for each sample and for standard isoniazide from the dose-response curve as the concentrations required to inhibit 90% of mycobacterial growth and the lowest concentration that prevented colour change of Alamar Blue solution indicating no mycobacterial activity, respectively. Results are recorded in table 30 and represented by figure 39. Table 30: In-vitro antimycobacterial activity of the EEs of the leaves of C. inerme and C. splendens compared to standard isoniazide

Sample/Standard MIC 90 µg/mL MIC µg/mL

C. splendens EE 30.8 62.5 C. inerme EE 7.2 15.63 Isoniazide 0.04 0.24

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*MIC 90=the concentration required to inhibit 90% of bacterial growth; MIC=the lowest concentration that prevented colour change of Alamar Blue solution EE, ethanol (70%) extract

CI., Clerodendrum inerme; CS., Clerodendrum splendens; EE, ethanol (70%) extract; MIC 90, the concentration required to inhibit 90% of bacterial growth; MIC, the lowest concentration where no change of MTT colour was detected Figure 39: Bar chart representing in-vitro antimycobacterial activity of the EEs of the leaves of C. inerme and C. splendens compared to standard isoniazide 3. Results and Discussion From the previous results (table 30 and figure 39), it could be concluded that the EE of the leaves of C. inerme exerts moderate antimycobacterial activity against Mycobacterium tuberculosis, with MIC 90 and MIC values of 7.2 µg/mL and 15.63 µg/mL, respectively. Meanwhile, the EE of C. splendens showed very weak antimycobacterial activity against Mycobacterium tuberculosis with MIC 90 and MIC values of 30.8 and 62.5 µg/mL, respectively. The MIC 90 and MIC values of the reference drug, isoniazide were 0.04 and 0.24 µg/mL, respectively.

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Part II Phytochemical and Biological Investigation of Clerodendrum inerme L. Gaertn. Leaves

Chapter I Optimization of Phenolic Extraction from the Leaves of Clerodendrum inerme L. Gaertn.

Optimization of Phenolic Extraction from the Leaves of Clerodendrum inerme Introduction As evidenced in Part I, the small-scale extraction and fractionation of the leaves of C. inerme and C. splendens resulted in higher yields of n-hexane and ethanol extracts (EE), as well as of those of the dichloromethane and ethyl acetate/n- butanol (2:1) fractions obtained from C. inerme leaves relative to those acquired from C. splendens.

TLC examination of the aforementioned extracts and fractions displayed several spots in the chromatograms of the leaves of C. inerme that exceeded in number those detected in their analogues derived from C. splendens. Moreover, comparative UPLC-Orbitrap HRMS metabolic analysis of the leaves of both species showed a chromatographic profile wealthier in components in the C. inerme sample as compared to C. splendens. Besides, quantitative estimation of the total phenolic and flavonoid contents (TPC and TFC) of the examined leaves revealed their presence in higher amounts in those of C. inerme.

From the biological standpoint as well, in-vitro evaluation of the cytotoxic and antibacterial activities of the EEs of the tested plant leaves showed a broader cytotoxic action and a stronger antibacterial efficiency (against both TB and MRSA) for the EE of C. inerme leaves.

Consequently, the leaves of C. inerme were selected for the following more intensive phytochemical and biological investigation aiming to evaluate the possibility of further implementation in herbal formulations.

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Optimization of Phenolic Extraction from the Leaves of Clerodendrum inerme Chapter I Optimization of Phenolic Extraction from the Leaves of Clerodendrum inerme L. Gaertn. Fractionation techniques are continuously developed for separation of complex mixtures of plant metabolites to improve productivity in a more rapid, less expensive and automated manner. Conventional liquid/liquid fractionation (LLF), based on separation of components between two immiscible aqueous and organic solvents, is commonly applied. Despite being successful, this method has certain disadvantages as increasing the risk of hydrolysis of certain constituents due to long contact with water and trapping of highly polar ones in the aqueous layer. In this chapter, a liquid/solid fractionation (LSF) technique aided by sonication was developed to isolate phenolics from the dried ethanol extract (EE) of the defatted leaves of C. inerme in a higher yield. The method efficiency was compared to that of conventional liquid/liquid fractionation in terms of yields of extractives, TPC and TFC of the resulting fractions. I. Extraction and Fractionation of the Leaves of C. inerme The air-dried leaves of C. inerme (1 Kg) were defatted with n-hexane (5 × 1000 mL), and the n-hexane extract evaporated to dryness, under vacuum at 55 oC, to yield 12.2 g of residue. The mark was then extracted with 70% ethanol (20 × 1000 mL), and the solvent similarly removed to give 328 g of dried EE residue. Two different procedures were then applied for EE fractionation. The first involved suspension of an aliquot (10 g) of EE in water, followed by successive liquid/liquid fractionation (LLF) in a separating funnel using dichloromethane (DCM) followed by ethyl acetate/n-butanol mixture (2:1). In the second process (liquid/solid fractionation, LSF), an equal amount of EE (10 g) was spread as a thin layer on the inside wall of a cellulosic extraction thimble and directly extracted, by the aid of sonication, with dichloromethane, ethyl acetate/n-butanol mixture (2:1) and methanol in succession, as represented in figure 40. Weights and percentage yields of extractives of the different fractions obtained by the two methods are comparatively recorded in table 31.

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Step 1 Step 2 Step 3 Step 4

Figure 40:The proposed liquid-solid fractionation technique Step 1: Spreading of the extract as a thin layer lining the wall of the thimble Step 2: Addition of the extracting solvent Step 3: Sonication to aid extraction Step 4: Collection of the extracted fraction Table 31: Weights and percentage yields of extractives of the different fractions Step 3: Sonication to aid extraction % Yield FractionationStep 4: Collection Technique of the extractedFractio fractionn Weight (g) (w/w) DCM 0.9 9 Liquid/liquid EtOAc/BuOH 2:1 1.2 12 Remaining water ~7.8 78 DCM 1.2 12 Liquid/solid EtOAc/BuOH 2:1 0.6 6 MeOH 5 50 DCM, dichloromethane; EtOAc, ethyl acetate; BuOH, n-butanol; MeOH, Methanol II. Quantitative Estimation of the Total Phenolic and Total Flavonoid Contents of the Fractions The total phenolic contents (TPC) of the fractions resulting from the conventional liquid/liquid and the proposed liquid/solid techniques were determined by the procedure formerly applied for TPC evaluation in EEs (pages 112, 113). Results were expressed as mg gallic acid equivalent (mg GAE)/ g dried fraction. The total flavonoid contents (TFC) of the different fractions were as well spectrophotometrically quantified as earlier described for EEs (pages 113-115), except that 1 mg of each dried fraction was dissolved in 1 mL of methanol instead of 2 mg in case of EE. TFC of each fraction was expressed as mg quercetin equivalent (mg QE)/ g dried extractive. Results of determination of TPC and TFC were recorded in table 32

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Optimization of Phenolic Extraction from the Leaves of Clerodendrum inerme Table 32: Total phenolic and total flavonoid contents (TPC and TFC) of the different fractions Fractionation TPC (mg GAE/g TFC (mg QE/g Fraction Technique dried fraction) dried fraction) DCM 41.69 158.52 Liquid/liquid EtOAc/BuOH 2:1 106.71 9.45 DCM 35.67 157.92 Liquid/solid EtOAc/BuOH 2:1 61.78 14.16 MeOH 81.31 12.9 DCM, dichloromethane; EtOAc, ethyl acetate; BuOH, n-butanol; MeOH, Methanol Results and Discussion Data displayed in table 31 revealed that the total yield of organic extractives obtained via application of the proposed liquid/solid technique (6.8 g) exceeded by far that acquired by the conventional liquid/liquid fractionation technique (2.1 g). The TPC of each of the ethyl acetate/n-butanol (2:1) and dichloromethane fractions resulting from conventional liquid/liquid fractionation, as recorded in table 32, were found higher than those of their analogues provided on application of the proposed liquid/solid procedure. Yet, the latter yielded an extra methanol fraction which raised the total TPC obtained by this technique over the corresponding amount resulting from conventional liquid/liquid fractionation. Concerning TFC (Table 32), those of the two dichloromethane fractions were found nearly the same, while that of the ethyl acetate/n-butanol (2:1) fraction obtained via the proposed liquid/solid technique was much higher than its analogue resulting from conventional liquid/liquid fractionation. Moreover, the presence of a final methanol fraction in the proposed technique increased its combined TFC to a much higher extent over that resulting from those of the conventional method. Conclusion These results support the suitability of the proposed liquid/solid fractionation (LSF) procedure in enhancing the yield of phenolic extractives and its expected economic impact in large scale isolation of these constituents. Consequently, this technique was adopted for further fractionation of the EE of the leaves of C. inerme rather than the conventional one.

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Chapter II Investigation of the Different Extractives

Investigation of the Different Extractives Chapter II Investigation of the Different Extractives I. Investigation of the n-Hexane Extract

A. Isolation of the constituents The n-hexane extract of the leaves of C. inerme was examined by TLC prior isolation of its major components through vacuum liquid chromatography (VLC) and conventional column chromatography. Results of this investigation, as recorded in table 17 (page 95), revealed that the extract comprises at least 8 constituents (as indicated by the number of spots), 2 of which appeared as major (based on intensity of colour response to p-anisaldehyde/H2SO4 spray reagent). A weighed amount (7 g) of the n-hexane extract was then subjected to fractionation by VLC (silica gel H, 60 g, 11 cm L × 4 cm D). Elution was performed starting with n-hexane: ethyl acetate (95:5) then the polarity was gradually increased by 5% stepwise addition of ethyl acetate till 25% ethyl acetate in n-hexane followed by raising solvent polarity by 25% increments of ethyl acetate up to 100% ethyl acetate. Fractions (200 ml, each) were collected and monitored by TLC (precoated silica gel plates, solvent systems S1 - S5, p-anisaldehyde/H2SO4 spray reagent). Fractions with similar chromatographic pattern were pooled, evaporated under vacuum at 55 oC, weighed and saved in a desiccator. Collective fractions I (5% ethyl acetate in n- hexane) and II (10% ethyl acetate in n-hexane) were subjected to re- chromatography as summarized in the isolation scheme in figure 41.

Collective fraction I: (1.3 g) showed 4 major spots (S2, p-anisaldehyde/H2SO4), and was re-subjected to VLC on a silica gel H column (60 g, 11 cm L × 4 cm D). Elution was started using n-hexane and the polarity of the eluent further increased by stepwise addition of 0.5% ethyl acetate up to 5% ethyl acetate in n-hexane. Fractions, 50 ml, each, were collected and monitored by TLC (S1). Fractions (1-3) eluted with 0.5-1.5% ethyl acetate in n-hexane were pooled and evaporated under vacuum at 55 oC. The resulting orange residue was weighed (200 mg) and saved in a refrigerator. An aliquot (100 mg) of this residue) was dissolved in the least amount of acetone and set aside for 5 min, whereupon a yellowish white precipitate was obtained and separated by filtration. The orange filtrate was then collected and evaporated under o vacuum at 40 C to yield 35 mg of an orange solid (compound H1).

Collective fraction II: (0.8 g) showed one major spot (Rf value 0.29, S5, blue with p-anisaldehyde/H2SO4 spray reagent). This fraction was subjected to column chromatography on a silica gel 60 column (30 g, 19.5 cm L × 2.5 cm D). Elution 134

Investigation of the Different Extractives was started with n-hexane followed by increasing polarity of the solvent system by gradual 2% ethyl acetate increment per fraction up to 20% ethyl acetate in n- hexane.

Fractions, (20 ml, each), were collected and monitored by TLC (S5). Fractions eluted with 10 and 12% ethyl acetate in n-hexane were pooled and evaporated under vacuum at 55 oC whereupon white needle crystals precipitated. The needle crystals were purified by washing with n-hexane, left in desiccator and weighed to give 30 mg of Compound H2.

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n-Hexane extract of the leaves of C. inerme (7 g)

VLC, Silica gel H (60 g, 11 cm L X 4 cm D) Gradient elution with n-hexane: EtOAc) Monitoring by TLC (S1-5)

Collective fraction I (1.3 g) Collective fraction II (0.8 g) 5 % EtOAc/ n-hexane 10 % EtOAc/ n-hexane VLC, Silica gel H (60 g, 11 cm L X 4 cm D) CC, Silica gel 60 Elution with n-hexane: EtOAc (99.5: 0.5- 98.5-1.5 v/v) (30 g, 19.5 cm L X 2.5 cm D) Elution with n-hexane: Four major spots in S EtOAc (90:10- 88:12 v/v) 1 (200 mg) One major spot Rf = 0.29 in S5 Addition of acetone Filtration Evaporation under vacuum Evaporation under vacuum White needle crystals (impure) One major spot Compound H1 Rf = 0.29 in S5 Orange solid Purification by washing (35 mg) Compound H2 White needle crystals with n-hexane (30 mg) Rf = 0.87 in S1

Figure 41: Scheme for chromatographic fractionation of the n-hexane extract of the leaves of C. inerme

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B. Identification of the Isolated Compounds

1. Identification of Compound H1

A small amount of compound H1 (about 0.5 mg) was dissolved in acetone and compared to a solution of standard β-carotene in the same solvent through matching their UV-Visible spectra and co-TLC in S1. The spectra were found identical showing three absorption maxima at 427, 450 and 476 nm (Figure 42).

The acetone solution of compound H1 gave an orange spot (Rf value 0.87, S1, violet with p-anisaldehyde/H2SO4 spray reagent) corresponding to that of standard β-carotene. Based on comparison of the UV-Visible spectra and co- chromatography with standard β-carotene, compound H1 was identified as β- carotene. This report is considered the first on isolation of β-carotene from genus Clerodendrum.

β-carotene

Standard β-carotene Isolated β-carotene

Wavelength nm.

Figure 42: UV-Visible spectra of standard and isolated β-carotene

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2. Identification of Compound H2

Compound H2 was identified through its physicochemical and chromatographic data (Table 33) that indicated its steroidal or triterpenoidal nature (high m.p. and positive response to Liebermann-Burchard’s and Salkowski’s tests). This was further confirmed through interpretation of its spectral data as recorded in table 33 and illustrated by figures 43 and 44. The H1 NMR spectrum of this compound, depicted in figure 43, displayed 5 methyl signals at δ 0.716 (3H, singlet), 0.855 (3H, triplet, J= 7.6 Hz), 1.031, 1.042 (6H, overlapped singlet and doublet) and 1.67 ppm (3H, singlet) corresponding to methyl groups located at C-18, C-29, C-19, C-21 and C-26, respectively. A multiplet proton signal at δ 3.544 ppm was assigned for the proton at C-3. An olifenic proton signal at δ 4.716 ppm (2H, Doublet, J= 3.64 Hz) was assigned for the two protons at C-27. A multiplet olifenic proton signal at δ 5.236 ppm (2H, multiplet) was assigned for protons at C-22 and C-23. A trisubstituted olefinic proton signal at δ 5.363 ppm (1H, br doublet, J= 4.92 Hz) was assigned for the proton at C-6. The C13 NMR spectrum of this compound, represented by figure 44, displayed 29 carbon signals, including five methyl signals at δc 12.057 (C-18), 12.140 (C-29). 19.401 (C-19), 20.222 (C-26) and 20.8 ppm (C-21), six olefinic carbons at δc 109.524 (C-27), 121.69 (C-6), 130.041 (C-23), 137.203 (C-22), 140.766 (C-5) and 148.616 ppm (C-25), in addition to a hydroxylated carbon at δc 71.807 ppm (C-3). 1 13 The H and C NMR spectroscopic data of compound H2 were similar to those of 22-dehydroclerosterol (Wang et al., 2012). Based on the interpretation of its spectral data and by comparison with published ones, compound H2 was identified as 22-dehydroclerosterol, which was previously isolated from the aerial parts of C. inerme (Akihisa et al., 1989).

22-dehydroclerosterol

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Investigation of the Different Extractives Table 33: Physicochemical, chromatographic and spectral data of compound H2

White needle crystals, 30 mg, soluble in chloroform, Physicochemical data m.p. 158-160°C, positive tests for sterols and/or triterpenes. Rf value (0.29, S5; blue colour with Chromatographic data p-anisaldehyde/H2SO4 spray reagent) 0.716 (3H, s, Me-18), 0.855 (3H, t, J= 7.6 Hz, Me-29), H1 NMR  ppm 1.031, 1.042 (6H, overlapped s and d, Me-19 and Me- (400 MHz, CDCl3) 21), 1.67 (3H, s, Me-26), 3.544 (1H, m, H-3), 4.716 (2H, (Figure 43) d, J= 3.64 Hz, H-27), 5.236 (2H, m, H-22 and H-23) and

5.363 (1H, br d, J= 4.92 Hz, H-6).

a 37.262 (C-1), 31.669 (C-2), 71.807 (C-3), 39.681 (C-4), 140.766 (C-5), 121.69 (C-6), 31.897 (C-7 and C-8), 50.156 (C-9), 36.516 (C-10), 21.072 (C-11), 40.189 (C-

C13 NMR  ppm 12), 42.312 (C-13), 56.849 (C-14), 24.320 (C-15), Spectral dat Spectral (100 MHz, CDCl3) 28.705 (C-16), 55.885 (C-17), 12.057 (C-18), 19.401 (Figure 44) (C-19), 42.258 (C-20), 20.8 (C-21), 137.203 (C-22), 130.041 (C-23), 51.998 (C-24), 148.616 (C-25), 20.222 (C-26), 109.524 (C-27), 25.711 (C-28) and 12.140 (C- 29).

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1 Figure 43: H NMR spectrum of compound H2

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21 26

- -

C C

6 27

- -

22 -

C C

C -

13 Figure 44: C NMR spectrum of compound H2

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II. Large Scale Fractionation and Investigation of the 70% Ethanol Extractives The dried EE of the defatted leaves of C. inerme (260 g) was transferred to 8 large cellulosic extraction thimbles (13 cm in length and 2 cm in diameter each, about 30-35 g per thimble) and subjected to LSF as previously mentioned in page 131. The individual fractions; dichloromethane, ethyl acetate/n-butanol (2:1) and methanol, were evaporated under vacuum at 55-60 oC. The solvent-free residues were weighed and kept in desiccator till further investigation. Yields of the different fractions relative to the parent extract are recorded in table 34. Table 34: Yields of the different fractions of the EE of the leaves of C. inerme

Fraction Yield (g/260 g dry EE) DCM 12.9 EtOAc/BuOH 2:1 4.18 MeOH 104.17 EE, ethanol (70%) extract; DCM, dichloromethane; EtOAc, ethyl acetate; BuOH, n-butanol; MeOH, Methanol

A. Investigation of the Dichloromethane Fraction The dichloromethane fraction of the EE of the leaves of C. inerme was subjected to TLC investigation prior isolation of its components through column chromatography. 1. TLC screening This was performed on precoated silica gel plates using different solvent systems, S7-S9, and p-anisaldehyde/H2SO4 as visualizing agent. Best resolution was obtained when using S8 for development. Results were recorded in table 35. At least 9 constituents could be detected, among which 4 appeared to be major (based on intensity of response to visualizing agents).

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Table 35: Results of TLC investigation of the dichloromethane fraction

Response to p-anisaldehyde spray reagent Spot No. Rf value Colour Colour intensity 1 0.9 Violet + 2 0.74 Green ± 3 0.6 Violet ±

4 0.52 Blue +

5 0.4 Yellow +

6 0.39 Blue + 7 0.2 Green ± 8 0.13 Green ± 9 0.06 Green ±

Rf= Rf values in S8: Dichloromethane-Methanol 95:5 v/v; (+) = Dark, (±) = faint 2. Isolation of the constituents The dichloromethane extract (11 g) was purified by dissolving in methanol (300 mL), mixing with powdered charcoal (20 g) and filtration. The powdered charcoal was washed twice with methanol (100 mL × 2) and filtered. The filtrates were combined and the solvent removed by vacuum distillation at a temperature not exceeding 55 oC and the purified extract weighed (5.5 g). An aliquot (4 g) of the extract was suspended in dichloromethane: n-hexane (90:10 v/v) and subjected to fractionation by VLC on a silica gel H column (50 g, 11 cm L × 4 cm D) as indicated in figure 45. Elution was started with 10% dichloromethane in n-hexane, followed by increasing the polarity of the eluent via 5% increments of dichloromethane until 50% dichloromethane was reached then increasing the polarity of the eluent through 10% increments of dichloromethane up to 100% dichloromethane. The polarity of the eluent was further increased by stepwise addition of 10% ethyl acetate till 100% ethyl acetate. Fractions (50 mL, each) were collected, monitored by TLC (systems S5-S9, p-anisaldehyde/H2SO4). Fractions of similar chromatographic pattern pooled, evaporated under vacuum, and the residues weighed and saved in a desiccator for further investigation. Upon concentration of the fraction eluted with 90% ethyl acetate in dichloromethane, a white precipitate was formed. The concentrated fraction was then centrifuged at 5000 RPM for 5 min, the precipitate collected and purified by solubilization in ethyl acetate and filtration. The filtrate was evaporated under vacuum to yield 8 mg of a pure white powder, Compound D1 (Rf value 0.48, S9, blue with p-anisaldehyde/H2SO4).

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Dichloromethane fraction of the 70% ethanol extract of the leaves of C. inerme (11 g)

Purification using powdered charcoal

Purified dichloromethane fraction (5.5 g)

4 g; VLC, Silica gel H (50 g, 9 cm L X 4 cm D) Gradient elution with n-hexane-dichloromethane followed by dichloromethane-ethyl acetate

Fr. 13'

(90 % Ethyl acetate/ Dichloromethane)

Concentration under vacuum

White precipitate

One major spot Rf= 0.48 in S9

Centrifugation and purification by dissolving in EtOAC and filtration

Compound D1 White amorphous powder (8 mg) Figure 45: Scheme for chromatographic fractionation of the dichloromethane fraction of the leaves of C. inerme

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3. Identification of the Isolated Compound

Compound D1 was identified through physicochemical, chromatographic and spectral examination (Table 36) that suggested a steroidal or triterpenoidal nature; this was confirmed through interpretation of its 1D and 2D NMR spectral data illustrated by figures 46-51. The H1 NMR spectrum of this compound (Figure 46) displayed 5 methyl signals at δ 0.649 (3H, singlet), 0.883 (3H, triplet, J= 7.44 Hz), 0.926 (3H, singlet), 1.025 (3H, doublet, J= 6.56 Hz) and 1.713 ppm (3H, singlet) corresponding to methyl groups located at C-18, C-29, C-19, C-21 and C-26, respectively. An overlapped multiplet proton signal at δ 3.979 ppm was assigned for the proton at C-3 together with a C-5' sugar proton. Four oxy-proton signals at δ 4.056 (1H, triplet, J= 7.72 Hz), 4.285 (2H, triplet, J= 5.16 Hz), 4.417 (1H, doublet of doublet, J= 4.96, 11.72 Hz) and 4.548 ppm (1H, doublet, J= 10.4 Hz) were assigned for sugar protons at C- 2', C-3' and C-4' and the last two for C-6' protons. Two singlet olefinic proton signal at δ 4.838 and 4.860 ppm were assigned for the two protons at C-27. A doublet proton signal at δ 5.041 ppm (J= 7.68 Hz) was assigned for the anomeric sugar proton. An olefinic proton signal at δ 5.291 ppm (2H, triplet, J= 5.64 Hz) was assigned for protons at C-22 and C-23. A trisubstituted olefinic proton signal at δ 5.332 ppm (1H, br doublet, J= 4.84 Hz) was assigned for the proton at C-6. 13 The C NMR spectrum of D1 (Figure 47) displayed 35 carbon signals, including five methyl signals at δc 11.922 (C-18), 12.167 (C-29), 19.197 (C-19), 20.091 (C- 26) and 20.793 ppm (C-21), seven hydroxylated carbons at δc 62.626, 71.494, 75.126, 77.894, 78.262, 78.391 and 102.356 ppm corresponding to sugar carbons; C-6', C-4', C-2', C-3, C-5', C-3' and C-1', respectively, in addition to six olefinic carbons at δc 109.950 (C-27), 121.679 (C-6), 130.100 (C-23), 137.292 (C-22), 140.745 (C-5) and 148.403 ppm (C-25). The HMQC spectrum, represented by figures 48 and 49, confirmed the proton- carbon correlations between the previously mentioned protons and carbons. The HMBC spectrum of the compound (Figures 50 and 51) revealed a correlation between the proton signals at δ 4.838 and 4.860 ppm and the carbon signals at δc 20.091 and 52.067 ppm (C-26 and C-24) confirming the position of a double bond between C-25 and C-27. The correlation observed between the anomeric proton signal at δ 5.041 ppm and the carbon signal at δc 77.894 (C-3) confirms the 3-O-linkage of the glucose unit. The correlation shown between the proton signal at δ 5.291 ppm and carbon signals at δc 40.252 and 52.067 ppm (C- 20 and C-24) indicates the presence of a double bond between C-22 and C-23. Similarly, the correlation between the proton signal at δ 5.332 ppm and the carbon signals at δc 31.183, 36.719 and 39.124 (C-7, C-10 and C-4, respectively) confirms the position of a double bond between C-5 and C-6.

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1 13 The H and C NMR spectroscopic data of compound D1 were in accordance with those reported for the steroidal glucoside 22-dehydroclerosterol-3-Oβ-D-glucoside (Leitao et al., 1992). Based on interpretation of its spectral data and through comparison with those published, compound D1 could be identified as 22-dehydroclerosterol-3-Oβ-D- glucopyranoside. Although being previously isolated from certain Clerodendrum species namely C. indicum and C. villosum (Somwong et al., 2015), this is the first report on its isolation from C. inerme.

22-dehydroclerosterol-3-Oβ-D-glucopyranoside

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Table 36: Physicochemical, chromatographic and spectral data of compound D1

White amorphous powder, 8 mg, soluble in pyridine, Physicochemical data m.p. 258-260°C, positive tests for sterols and/or triterpenes. Rf value (0.48, S9; blue colour with Chromatographic data p-anisaldehyde/H2SO4) 0.649 (3H, s, Me-18), 0.883 (3H, t, J= 7.44 Hz, Me- 29), 0.926 (3H, s Me-19), 1.025( 3H, d, J= 6.56 Hz Me-21), 1.713 (3H, s, Me-26), 3.979 (2H, m, H-3 and H-5'), 4.056 (1H, t, J= 7.72 Hz, H-2'), 4.285 H1 NMR  ppm (2H, t, J= 5.16 Hz, H3' and H-4'), 4.417 (1H, dd, J= (400 MHz, Pyridine-d ) 5 4.96, 11.72 Hz, H-6'a) and 4.548 (1H, d, J= 10.4 Hz, (Figure 46) H-6'b), 4.838 (1H, s, H-27a), 4.860 (1Hm s, H- 27b), 5.041 (1H, d, J= 7.68 Hz, H-1') 5.291 (2H, t,

J= 5.64, H-22 and H-23) and 5.332 (1H, br d, J= 4.84 Hz, H-6). 37.258 (C-1), 30.089 (C-2), 77.894 (C-3), 39.124 (C-4), 140.745 (C-5), 121.679 (C-6), 31.183 (C-7)

Spectral data Spectral 31.939 (C-8), 50.131 (C-9), 36.719 (C-10), 21.040 (C-11), 39.598 (C-12), 42.153 (C-13), 56.678 (C- C13 NMR  ppm 14), 24.277 (C-15), 29.743 (C-16), 55.789 (C-17), (100 MHz, Pyridine-d5) 11.922 (C-18), 19.197 (C-19), 40.252 (C-20), 20.793 (Figure 47) (C-21), 137.292 (C-22), 130.100 (C-23), 52.067 (C- 24), 148.403 (C-25), 20.091 (C-26), 109.950 (C-27), 25.833 (C-28), 12.167 (C-29) 102.356 (C-1'), 75.126 (C-2') 78.391 (C-3'), 71.494 (C-4'), 78.262 (C-5') and 62.626 (C-6').

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19 19

21 Me

- Me Me

& H &

' -

22 &23

' '

6 2

3 H &

- - -

H H

1 Figure 46: H NMR spectrum of compound D1 148

Investigation of the Extractives

13 Figure 47: C NMR spectrum of compound D1 149

Investigation of the Extractives

Figure 48: HMQC spectrum of compound D1 150

Investigation of the Extractives

Figure 49: Magnified part of HMQC spectrum of compound D1 151

Investigation of the Extractives

Figure 50: HMBC spectrum of compound D1 152

Investigation of the Extractives

Figure 51: Magnified part of HMBC spectrum of compound D1

153

Investigation of the Extractives

B. Investigation of the Ethyl Acetate/ n-Butanol (2:1) Fraction The ethyl acetate/ n-butanol (2:1) fraction of the EE of the leaves of C. inerme was monitored by TLC and HPLC then subjected to VLC for isolation of its components. 1. TLC screening

This was performed on precoated silica gel plates using S11 and S12 as solvent systems and NP/PEG as visualizing agent. Best resolution was obtained when using S12 for development. Results are recorded in table 37. At least 4 major constituents could be detected. Table 37: Results of TLC investigation of the ethyl acetate/ n-butanol fraction

Response to NP/PEG spray

reagent Spot Colour in R Fluorescence in No. f Visible Colour in Fluorescence UV 325 nm Light Visible in Light UV 325 nm 1 0.91 - Blue - + 2 0.79 - Blue - + 3 0.62 - Blue Yellow + 4 0.4 - Blue - +

Rf= Rf values in S12: Ethyl acetate-Formic acid- Acetic acid-Water 100:10: 10: 10 v/v (+) = higher intensity of fluorescence 2. HPLC Screening This was performed on an Agilent Technologies 1100 series HPLC system, equipped as mentioned before (page 78). Separation was carried out on a LiChrospher 100 RP-C18 column (250 × 4.6 mm, particle size 5 μm), preceeded by RP-C18 guard column (10 × 4 mm, particle size 5 μm); the mobile phase consisted of: component A, acetonitrile and component B, 0.3% phosphoric acid; the stepwise gradient elution program adopted was: 18-38% A (0-13 min), 38-60% A (13-17 min), 60-85% A (17-19 min), 85-100% A (19-21 min), the system being equilibrated with 100% of A for another 2 min at the end of each run; the flow rate was adjusted at: 1mL/min; the UV detector was set at 230 nm and the injected volume, 20 μL. A weighed amount (2 mg) of the ethyl acetate/ n-butanol (2:1) fraction was dissolved in 1 mL of methanol for preparation of the working solution. Samples were directly injected into the HPLC system and the analyses carried out at room temperature.

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Investigation of the Extractives

The HPLC fingerprint of the ethyl acetate/ n-butanol (2:1) fraction (Figure 52) revealed the presence of several peaks among which two were considered major at Rt values of 17.49 and 19.49 minutes. Those two major peaks were targeted for isolation from the ethyl acetate/ n-butanol (2:1) fraction.

Figure 52: HPLC fingerprint of the ethyl acetate/ n-butanol (2:1) fraction 3. Isolation of the constituents A weighed amount (2 g) of the ethyl acetate/ n-butanol (2:1) fraction was suspended in dichloromethane. The dissolved portion was decanted then evaporated to dryness under vacuum and weighed to give 300 mg. The dried residue was dissolved in methanol: water (25: 75 v/v), subjected to VLC on RP-C18 column (25 g, 7 cm L × 3 cm D) and gradually eluted with methanol: water (25: 75 to 100: 0 v/v). The percentage of methanol in the eluent was increased by 5% stepwise addition of methanol up to 50% methanol in water, followed by 2% stepwise addition of methanol until 56% methanol in water was reached. Elution was then continued with 56% methanol in water (500 mL) followed by washing with 100% methanol. Fractions (100 mL, each) were collected and monitored by HPLC and TLC (systems S9-S12, NP/PEG spray reagent). Fractions with similar chromatographic patterns were pooled and evaporated under reduced pressure. The residues obtained were weighed and saved in a desiccator for further purification and isolation of the individual components. The fifth to seventh sub-fractions, obtained by elution with 52-56% methanol in water showed one major peak in HPLC at Rt 17.49 min and one major spot on TLC (Rf= 0.59 in S9, NP/PEG spray reagent: yellow colour in visible light and quenching of fluorescence in UV 325 nm). These sub-fractions were combined and

155

Investigation of the Extractives evaporated under vacuum at 55 oC to give 15 mg of a yellow powder (Compound E1). Sub-fractions 8 - 11, eluted with 56% methanol in water, showed one major peak at Rt 19.49 min and one spot on TLC (Rf= 0.33 in S10, lemon yellow colour with p- anisaldehyde/ H2SO4 and yellow colour with NP/PEG spray reagents, yellow fluorescence in UV 325 nm that increases in intensity upon spraying with NP/PEG spray reagent). The combined sub-fractions were evaporated under vacuum at 55 o C to give 10 mg of a buff powder (Compound E2). The fractionation scheme of the ethyl acetate/ n-butanol fraction of the EE of the leaves of C. inerme is represented in figure 53.

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Investigation of the Extractives

Ethyl acetate/ n-butanol (2:1) fraction of the 70% ethanol extract of the leaves of C. inerme (2 g) Dissolved in dichloromethane, extract evaporated under vacuum, residue dissolved in 25% MeOH VLC, RP-C18 (25 g, 7 cm L X 3 cm D) Gradient elution with MeOH- H O 2 Monitoring by TLC (S9-12) and HPLC

Collective fraction I Collective fraction II 52-56 % MeOH/ H2O 56 % MeOH/ H2O

One spot (Rf = 0.59 in S9) One spot (Rf= 0.33 in S10) One peak in HPLC Rt= 17.49 One peak in HPLC Rt= 19.49

Evaporation under Evaporation under o vacuum at 55 oC vacuum at 55 C

Compound E1 Compound E2 Yellow amorphous Buff amorphous powder powder (15 mg) (10 mg)

Figure 53: Scheme for chromatographic fractionation of the ethyl acetate/ n-butanol (2:1) fraction of the leaves of C. inerme

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Investigation of the Extractives

4. Identification of the Isolated Compounds

Identification of Compound E1

Compound E1 was identified through its physicochemical, chromatographic and spectral data which are recorded in table 38 and indicated that it has a flavonoidal nature. This was further confirmed through interpretation of its spectral data illustrated by figures 54 and 55. The UV spectral data revealed the presence of 2 major UV absorption bands I and II in methanol, at 333 and 285 nm, respectively which are typical of a flavone nucleus. Addition of CH3ONa resulted in a bathochromic shift of (+∆ 38 nm) in band I indicating free hydroxyl groups in rings A and/or B. The bathochromic shift in band I produced on addition of AlCl3 (+∆ 39 nm) indicated the presence of free hydroxyl group at C-5 and/or ortho dihydroxyl groups. The partial decrease in the bathochromic chromic shift on addition of HCl to AlCl3 (-∆ 12 nm) confirmed the presence of free hydroxyl group at C-5 and ortho dihydroxyl groups. The bathochromic shift in band I on addition of Na acetate (+∆ 23 nm) indicated the presence of free hydroxyl group at C-7. Moreover, the bathochromic shift in band I on addition of Na acetate/boric acid (+∆ 16 nm) indicated the presence of ortho dihydroxyl groups. The H1 NMR spectrum of this compound (Figure 54) showed 5 proton signals: a methoxy proton signal at δ 3.779 ppm (3H, singlet) assigned for 4'-methoxy group, 2 singlet olefinic proton signals at δ 6.514 and 6.526 ppm, each corresponding to 1H, assigned for protons at C-8 and C-3, respectively and finally 2 doublet olefinic proton signals at δ 6.974 and 7.868 ppm, each corresponding to 2Hs with J value of 8.88 Hz that suggested ortho-coupling, assigned for the protons at C-3' and C-5' and those at C-2' and C-6', respectively. The C13 NMR spectrum (Figure 55) displayed 14 carbon signals corresponding to 16 carbons. The carbon signal assignments are recorded in table 38. 1 13 The H and C NMR spectroscopic data of compound E1 were similar to those reported for the methoxylated flavone, 5,6,7-trihydroxy-4'-methoxy flavone or 4'- methylscutellarein (Sabina and Aliya, 2009). Based on interpretation of its spectral data and by comparison with published literature, compound E1 was identified as 4'-methylscutellarein, which was previously isolated from the leaves of C. inerme (Vendantham et al., 1977)

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4'-methylscutellarein

Table 38: Physicochemical, chromatographic and spectral data of compound E1 Yellow amorphous powder, 15 mg, soluble in acetone, DMSO Physicochemical and methanol, m. p. 272-274°C, positive tests for phenols and data flavonoids.

Chromatographic Rf value (0.59, S9; NP/PEG spray reagent: yellow colour in data visible light and quenching of fluorescence in UV 325 nm)

MeOH 285, 333 (flavone) NaOMe 275, 371 (free OH on rings A &/or B) 302, 372 (free OH at C-5 &/or ortho OH AlCl 3 groups) UV max nm 301, 360 (free OH at C-5 & ortho OH AlCl / HCl 3 groups) NaOAc 278, 365 (free OH at C-7) NaOAc / 284, 349 (ortho OH groups) H3BO3 H1 NMR  ppm (400 3.779 (3H, s, 4'-O-Me), 6.514 (1H, s, H-8), 6.526 (1H, s, H- MHz, Acetone-d6) 3), 6.974 (2H, d, J= 8.88 Hz, H-3' and H-5') and 7.868 (2H, d, (Figure 54) J= 8.88 Hz, H-2' and H-6'). 163.759 (C-2), 103.068 (C-3), 182.393 (C-4), 150.579 (C-5), C13 NMR  ppm 93.859 (C-8), 152.809 (C-9), 104.562 (C-10), 123.731 (C-1'), (100 MHz, Acetone- 128.069 (C-2' and C6'), 114.465 (C-3' and C5'), 162.696 (C- d ) (Figure 55) 6 4') and 55.064 (4'-O-Me).

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Investigation of the Extractives

1 Figure 54: H NMR spectrum of compound E1

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Investigation of the Extractives

OCH 4' 3

HO O

OH O

5’

6’

’ C &

1’

3’ C &

OCH

10 C

9 5

4 2

4’

- -

C C

C C C

13 Figure 55: C NMR spectrum of compound E1 161

Investigation of the Extractives

2. Identification of Compound E2

Compound E2 was identified through its physicochemical, as well as 1D and 2D NMR spectral data (Table 39) which suggested a diterpene nature. This was confirmed through interpretation of its H1 and C13 NMR, HSQC and HMBC spectra, depicted in figures 56-61. The H1 NMR spectrum (Figure 56) showed 3 methyl signals at δ 0.847 (3H, singlet), 0.865 (3H, doublet, J= 6.16 Hz) and 1.299 ppm (3H, singlet) assigned for the methyl groups at C-20, C-17 and C-19, respectively. An oxy-proton signal at δ 4.892 ppm (2H, doublet, J= 1.52 Hz) was assigned for the two protons at C-16. The two olefinic proton signals at δ 5.912 (1H, triplet, J=1.4 Hz) and 6.653 ppm (1H, triplet, J= 3.7 Hz) were assigned for the olefinic protons at C-14 and C-3, respectively. The C13 NMR spectrum, represented by figure 57, displayed 19 carbon signals corresponding to 20 carbons including 3 methyl signals at δc 14.869 (C-17), 17.219 (C-20) and 19.669 ppm (C-19), a hydroxylated carbon signal at δc 73.596 ppm (C- 16), 4 olefinic carbon signals at δc 113.599 (C-14), 136.509 (C-3), 142.534 (C-4) and 175.719 ppm (C-13) and a carbonyl signal at 173.708 ppm corresponding to 2 carbonyl carbons at C-15 and C-18. The HSQC spectrum (Figures 58 and 59) confirmed the correlation between the proton signal at δ 0.847 ppm and the carbon signal at δc 17.219 ppm, the proton signal at δ 0.865 ppm and the carbon signal at δc 14.869 ppm, the proton signal at δ 1.299 ppm and the carbon signal at δc 19.669 ppm, the proton signal at δ 4.892 ppm and the carbon signal at δc 73.596 ppm, the proton signal at δ 5.912 ppm and the carbon signal at δc 113.599 ppm and between the proton signal at δ 6.653 ppm and the carbon signal at δc 136.509 ppm. The HMBC spectrum (Figures 60 and 61) displayed a correlation between the proton signal at at δ 4.892 ppm and the carbon signals at δc 113.599, 173.708 and 175.719 ppm and between the proton signal at δ 5.912 ppm and the carbon signals at δc 73.596, 173.708 and 175.719 ppm thus confirming the presence of a dihydrofuran ring. In addition, the correlation between the proton signal at δ 6.653 ppm and the carbon signals at δc 17.179 (C-1), 37.302 (C-5) ppm ensured its position between C-3 and C-4. 1 13 The H and C NMR spectroscopic data of compound E2 were in accordance with those reported for the neo-clerodane diterpene, clerodermic acid (Achari et al., 1990).

Based on spectral analysis and via comparison with published data, compound E2 was identified as clerodermic acid, which was formerly isolated from the leaves and aerial parts of C. inerme (Achari et al., 1990; Pandey et al., 2003).

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Investigation of the Extractives

Clerodermic acid

Table 39: Physicochemical, chromatographic and spectral data of compound E2

Physicochemical Buff amorphous powder, 10 mg, soluble in chloroform and data methanol, m. p. 161-162°C.

Rf value (0.33; S10, lemon yellow colour with p- Chromatographic anisaldehyde/ H2SO4 and yellow colour with NP/PEG, data yellow fluorescence in UV 325 nm that increases in intensity after spraying with NP/PEG). H1NMR  ppm 0.847 (3H, s, Me-20), 0.865 (3H, d, J= 6.16 Hz, Me-17), (400 MHz, 1.299 (3H, s, Me-17), 4.892 (2H, d, J= 1.52 Hz, H-16), 5.912 Methanol-d ) 4 (1H, t, J= 1.4 Hz, H-14) and 6.653 (1H, t, J= 3.7 Hz, H-3). (Figure 56) 17.179 (C-1), 21.619 (C-2), 136.509 (C-3), 142.534 (C-4), 13

ctral data ctral C NMR  ppm 38.529 (C-5), 35.693 (C-6), 26.617 (C-7), 36.099 (C-8),

(100 MHz, 37.312 (C-9), 46.649 (C-10), 35.169 (C-11), 26.987(C-12), Spe Methanol-d4) 175.719 (C-13), 113.599 (C-14), 173.708 (C-15), 73.596 (C- (Figure 57) 16), 14.869 (C-17), 173.708 (C-18), 19.669 (C-19), 17.219 (C-20).

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Investigation of the Extractives

O O 15 14 16 13

3 19

4 -

- Me 19 M

HO 18 O Me

3 14

- -

H H

1 Figure 56: H NMR spectrum of compound E2

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Investigation of the Extractives

O O 15 14 16 13

17 20

18 -

19 20

- -

C C C

O 14

HO 16 -

18 -

15 and C

- C C

13 Figure 57: C NMR spectrum of compound E2

165

Investigation of the Extractives

Figure 58: HSQC spectrum of compound E2 166

Investigation of the Extractives

H-16, C-16

H-14, C-14

H-3, C-3

Figure 59: Magnified part of HSQC spectrum of compound E2 167

Investigation of the Extractives

Figure 60: HMBC spectrum of compound E2

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Investigation of the Extractives

H-3, C-1 H-3, C-5

H-14, C-16

H-16, C-14

H-16, C-15 H-14, C-15 H-14, C-13 H-16, C-13

Figure 61: Magnified part of HMBC spectrum of compound E2 169

Investigation of the Extractives

C. Investigation of the Methanol Fraction The methanol fraction of the EE of the leaves of C. inerme was monitored by TLC and HPLC then subjected to VLC for isolation of its components. 1. TLC screening

This was performed on precoated silica gel plates using S11 and S12 as solvent systems, and NP/PEG as visualizing agent. Best resolution was obtained with S12. Results, as recorded in table 40, revealed the presence of at least 4 constituents, among these one (Rf value 0.64) appeared as major (based on intensity of response to visualizing agent).

Table 40: Results of TLC investigation of the methanol fraction

Response to NP/PEG spray

reagent Spot Colour in R Fluorescence in No. f Visible Colour in Fluorescence UV 325 nm Light Visible in Light UV 325 nm 1 0.909 - Blue - ± 2 0.763 - Blue - ±

3 0.64 - Blue Yellow + 4 0.472 - Yellow - ±

Rf= Rf values in S12: Ethyl acetate-Formic acid- Acetic acid-Water 100:10: 10: 10 v/v (+) = higher intensity of fluorescence; (±) = nearly the same intensity of fluorescence 2. HPLC Screening This was performed via the previously mentioned analytical procedure (see page 154). The HPLC fingerprint of the methanol fraction (Figure 62) revealed the presence of several peaks among these one could be considered as major (Rt value, 6.75 min) and was targeted for isolation.

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Investigation of the Extractives

Figure 62: HPLC fingerprint of the methanol fraction 3. Isolation of the constituents A weighed amount (1 g) of the dried methanol fraction was dissolved in distilled water (100 mL) and subjected to VLC fractionation on RP-C18 column (25 g, 7 cm L × 3 cm D). Elution was started with distilled water and the polarity gradually decreased by 5% increments of methanol up to 35% methanol in water followed by washing with 50% then 100% mehanol. Fractions (100 mL, each) were collected and monitored by HPLC and TLC (precoated silica gel plates, S11 and S12, after spraying with NP/PEG spray reagent). Fractions with similar chromatographic patterns were pooled and concentrated under vacuum at 55 oC. The residues obtained were weighed and saved in a desiccator for further purification and isolation of the individual components.

The fraction eluted with 35% methanol in water revealed a major spot on TLC (Rf= 0.64 in S12, NP/PEG: yellow colour in visible light, blue florescence in UV 230 and 325 nm) and a major peak (Rt = 6.75 min) upon HPLC profiling. The residue obtained upon vacuum evaporation was dissolved in the least amount of methanol and the resulting solution added drop-wise to ethyl acetate whereupon a buff precipitate was obtained, collected, washed with ethyl acetate, left to dry and weighed to yield 45 mg of a buff powder (Compound M1). The scheme for fractionation of the methanol fraction of the leaves of C. inerme is represented in figure 63.

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Methanol fraction of the 70% ethanol extract of the leaves of C. inerme (1 g)

VLC, RP-C18 (7 cm L X 3 cm D, 25 g) Gradient elution with MeOH: Water (0:100 to 35:65)

Fraction eluted with 35% M/W One peak in HPLC R =6.75 min t One spot in TLC Rf=0.64 in S12

Evaporation under reduced pressure at 550 C Dissolution in the least amount of methanol then adding dropwise to ethyl acetate

Yellowish buff precipitate

Filtration and purification by washing with EtOAc

Compound M1 Yellowish buff amorphous powder (45mg)

Figure 63: Scheme for chromatographic fractionation of the methanol fraction of the leaves of C. inerme 172

Investigation of the Extractives

4. Identification of the Isolated Compound

Compound M1 was identified through its physicochemical, chromatographic and H1 and C13 NMR and HMBC spectral data that are recorded in table 41 indicating its phenolic nature. This was further confirmed through interpretation of its spectral data illustrated by figures 64-66. The H1 NMR spectrum (Figure 64) displayed one methyl signal at δ 0.956 ppm (3H, doublet, J= 6.12 Hz) assigned for the methyl group of rhamnose sugar (H-6'''), a proton signal at δ 2.695 ppm (2H, multiplet) assigned for the 2 protons attached to C-7', 4 oxy-proton signals at δ 3.898 ppm (2H, multiplet) assigned for the 2 protons attached to C-8', 4.349 ppm (1H, doublet, J= 7.84 Hz) assigned for the anomeric proton of glucose sugar (H-1''), 4.717 ppm (1H, triplet, J= 9.64 Hz) assigned for H-4'' and 5.034 ppm (1H, singlet) assigned for the anomeric proton of rhamnose sugar (H-1'''), the rest of the sugar protons are overlapped in the area between 3 and 3.7 ppm, 2 olefinic proton signals at δ 6.181 and 7.443 ppm (each 1H, doublet, J= 15.8 Hz) assigned for H-8 and H-7, respectively, 2 olefinic proton signals at δ 6.486 and 6.969 ppm (each 1H, doublet of doublet, J= 1.7 and 8 Hz) assigned for H-5' and H-6, respectively, and 4 olefinic proton signals at δ 6.628 ppm (1H, doublet, J= 0.8 Hz) assigned for H-2', 6.636 ppm (1H, doublet, J= 4.6 Hz) assigned for H-6', 6.755 ppm (1H, doublet, J= 8.12 Hz) assigned for H-5 and 7.031 ppm (1H, doublet, J= 1.6 Hz) assigned for H-2. The C13 NMR spectrum (Figure 65) revealed 27 carbon signals corresponding to 29 carbons including 1 methyl carbon signal at δc 18.614 ppm (C-6'''), 11 hydroxylated carbon signals at δc 61.188 (C-6''), 69.194 (C-5'''), 69.580 (C-4''), 70.719 (C-8'), 70.842 (C-3'''), 70.975 (C-2'''), 72.118 (C-4'''), 74.966 (C-2'' and C- 5''), 79.539 (C-3''), 101.683 (C-1''') and 102.733 (C-1''), in addition to 14 olefinic carbon signals at δc 113.962 (C-5), 115.173 (C-5'), 115.953 (C-2'), 116.278 (C-6'), 116.791 (C-2), 119.991 (C-8), 121.895 (C-6), 125.904 (C-1), 129.550 (C-1'), 144.029 (C-4'), 145.474 (C-3 and C-7), 146.065 (C-3'), 149.061 (C-4) and 166.179 ppm (C-9). 1 13 The H and C NMR spectroscopic data recorded for compound M1 were in agreement with those reported for the phenyl propanoid glycoside, verbascoside (Singh et al., 2010).

The HMBC spectrum of compound M1, represented by figure 66, showed correlation between the proton signal at δ 4.717 (H-4'') and the carbon signal at δc 166.179 (C-9) confirming the O-linkage between C-9 and C-4'', thus the identity of isolated compound was confirmed as verbascoside rather than its isomer isoverbascoside in which C-9 is attached to C-6' position. Therefore, on the basis of its spectral data and by correlation with those published, compound M1 was identified as verbascoside, which was previously isolated from the aerial parts of C. inerme (Kanchanapoom et al., 2001; Nan et al., 2005).

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Table 41: Physicochemical, chromatographic and spectral data of compound M1 Yellowish buff amorphous powder, 45 mg, soluble in Physicochemical data DMSO and methanol, m. p. 143-146°C, positive tests for phenols.

Rf value (0.64; S12, yellow colour in visible light when sprayed with NP/PEG, blue fluorescence in UV 230 Chromatographic data and 325 nm that increases in intensity after spraying with NP/PEG). 0.956 (3H, d, J= 6.12, Me-6'''), 2.695 (2H, m, H-7'), 3.898 (2H, m, H-8'), 4.349 (2H, d, J= 7.84 Hz, H-1''), 4.717 (1H, t, J= 9.64 Hz, H-4''), 5.034 (1H, s, H-1'''), H1 NMR  ppm 6.181 (1H, d, J= 15.8 Hzm H-8), 7.443 (1H, d, J= 15.8 (400 MHz, DMSO-d6) Hz, H-7), 6.628 (1H, d, J= 0.8 Hz, H-2'), 6.636 (1H, (Figure 64) d, J= 4.6 Hzm H-6'), 6.486 (1H, dd, J= 1.7 and 8 Hz,

H-5'), 6.969 (1H, dd, J= 1.7 and 8 Hz, H-6), 6.755 (1H, doublet, J= 8.12 Hz, H-5) and 7.031 (1H, d, J= 1.6 Hz, H-2). 125.904 (C-1), 116.791 (C-2), 145.474 (C-3), 149.061 (C-4), 113.962 (C-5), 121.895 (C-6), 145.474 (C-7), Spectral data Spectral 119.991 (C-8), 166.179 (C-9), 129.550 (C-1'), 115.953 C13 NMR  ppm (C-2'), 146.065 (C-3'), 144.029 (C-4'), 115.173 (C-5'), (100 MHz, DMSO-d6) 116.278 (C-6'), 35.457 (C-7'), 70.719 (C-8'), 102.733 (Figure 65) (C-1''), 74.966 (C-2''), 79.539 (C-3''), 69.580 (C-4''), 74.966 (C-5''), 61.188 (C-6''), 101.683 (C-1'''), 70.975 (C-2'''), 70.842 (C-3'''), 72.118 (C-4'''), 69.194 (C-5''') and 18.614 (C-6''').

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7 5

- -

6 5'

- -

H H

6''' -

1'''

4'' 1''

- - H H

1 Figure 64: H NMR spectrum of compound M1 175

Investigation of the Extractives

13 Figure 65: C NMR spectrum of compound M1 176

Investigation of the Extractives

H-4'', C-9

Figure 66: HMBC spectrum of compound M1 177

Chapter III Biological Evaluation of the Different Extractives and Isolated Compounds

Biological Evaluation of the Different Extractives & Isolated Compounds Chapter III Biological Evaluation of the Different Extractives and Isolated Compounds Evaluation of the Antioxidant Activity The antioxidant activity of the different fractions of the EE of the leaves of C. inerme was evaluated using the DPPH spectrophotometric assay by adopting the same procedure as for the parent extract (EE, Part I, page 117). Results are recorded in table 42 and represented by figure 67.

Table 42: DPPH scavenging potency of the different fractions of the EE of the leaves of C. inerme, expressed as EC50, compared to standard gallic acid

Fraction/ standard *EC50 ± SD µg/mL DCM 903.688 ± 18.575 EtOAc/BuOH 2:1 65.649 ± 1.601 MeOH 255.137 ± 12.899 Gallic acid (Reference) 18.85 ± 0.59 DCM, dichloromethane; EtOAc, ethyl acetate; BuOH, n-butanol; MeOH, Methanol * EC50=effective concentration of the sample required to scavenge 50 % of the DPPH free radical as compared to the solvent control, SD=standard deviation

DCM, dichloromethane; EtOAc, ethyl acetate; BuOH, n-butanol; MeOH, Methanol Figure 67: Bar chart representing the in-vitro anti-oxidant activity of the different fractions of the EE of C. inerme compared to standard gallic acid 178

Biological Evaluation of the Different Extractives & Isolated Compounds Results and Discussion From table 42 and figure 67, it could be concluded that the ethyl acetate: n-butanol fraction, with lowest EC50 value (65.649 ± 1.601 µg/mL), exhibited the strongest antioxidant activity among other fractions derived from EE, followed by the methanol fraction (EC50 = 255.137 ± 12.899 µg/mL). Meanwhile, the dichloromethane fraction was the least active as compared to the reference antioxidant, gallic acid. III. Evaluation of the Cytotoxic Activity The in-vitro cytotoxic activity of the n-hexane extract alongside that of the different fractions of the EE of the leaves of C. inerme was investigated via the SRB assay on (H-1299), (HepG-2) and (MCF-7); lung, liver and breast carcinoma cell lines, respectively by adopting the previously described experimental protocol (Part I, page 122). Meanwhile, the cytotoxic potential of the compounds isolated from the active fractions was evaluated on (H-1299) and (HepG-2); lung and liver carcinoma cell lines, respectively. The safety of the aforementioned extract/fractions and compounds was determined by investigating their cytotoxicity towards normal fibroblasts (BHK cell line). The activity and safety of the tested samples were compared to those of the reference anticancer drug, doxorubicin. Results are recorded in tables 43 and 44 and represented by figures 68-71. Table 43: In-vitro cytotoxic activity of the n-hexane extract and different fractions of the EE of the leaves of C. inerme, expressed as IC50, compared to standard doxorubicin

*IC50 (µg/mL) Fraction/Standard H-1299 HepG-2 MCF-7 BHK Hex 36.4 23.6 --- 87 DCM 29.3 31.2 100 72 EtOAc/BuOH 2:1 --- 17 --- 34.5 MeOH 32 32.2 --- 57.4 Doxorubicin 4.28 5.87 2.97 > 50 Hex., n-hexane; DCM, dichloromethane; EtOAc, ethyl acetate; BuOH, n-butanol; MeOH, Methanol * IC50=effective concentration of the sample required to inhibit the growth of 50 % of the cells

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100

70 Hex 60 DCM 50

(µg/mL) EtOAc:BuOH 2:1 50

IC 40 MeOH 30 Dox

0 H-1299 HepG-2 MCF-7 BHK

Hex., n-hexane; DCM, dichloromethane; EtOAc, ethyl acetate; BuOH, n-butanol; MeOH, Methanol; Dox, doxorubicin Figure 68: Bar chart representing the in-vitro cytotoxic activity of the different fractions of the EE compared to standard doxorubicin Table 44: In-vitro cytotoxic activity of the compounds, isolated from of C. inerme extracts, expressed as IC50, compared to reference doxorubicin

*IC50 (µg/mL) ± SD Compound H-1299 HepG-2 BHK β-carotene 9.2 ± 0.7 22.5 ± 2.4 28.9 ± 2.2

22-dehydroclerosterol-3β-O-glucoside 32.4 ± 7.5 22.3 ± 2.4 21 ± 1.2

4'-methyl scutellarein 18 ± 4.8 21.3 ± 1.5 34.7 ± 3.7

Clerodermic acid 18.4 ± 4.8 12.2 ± 2.8 19.2 ± 1

Verbascoside 26.8 ± 3.9 22.7 ± 3.1 34.7 ± 1.1

Doxorubicin 4.28 ± 1.6 5.87 ± 1.4 > 50

* IC50=effective concentration of the sample required to inhibit the growth of 50 % of the cells, calculated as average of six measurements. SD=Standard Deviation.

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CAR, β-carotene; D1, 22-dehydroclerosterol-3-Oβ-D-glucoside; E1, 4'-methyl scutellarein; E2, clerodermic acid; M1, Verbascoside; Dox, reference doxorubicin Figure 69: Bar chart representing the in-vitro cytotoxic activity of the compounds, isolated from C. inerme extracts on lung carcinoma cell line (H1299), compared to standard doxorubicin

CAR, β-carotene; D1, 22-dehydroclerosterol-3-Oβ-D-glucoside; E1, 4'-methyl scutellarein; E2, clerodermic acid; M1, Verbascoside; Dox, reference doxorubicin Figure 70: Bar chart representing the in-vitro cytotoxic activity of the compounds, isolated from C. inerme extracts on liver carcinoma cell line (HepG-2), compared to standard doxorubicin 181

Biological Evaluation of the Different Extractives & Isolated Compounds

CAR, β-carotene; D1, 22-dehydroclerosterol-3-Oβ-D-glucoside; E1, 4'-methyl scutellarein; E2, clerodermic acid; M1, Verbascoside; Dox, reference doxorubicin Figure 71: Bar chart representing the in-vitro cytotoxic activity of the compounds, isolated from C. inerme extracts on normal fibroblasts (BHK), compared to standard doxorubicin Results and Discussion 1. As indicated in tables (43 and 44), and figures (68-71), the EE ethyl acetate/ n- butanol (2:1) fraction showed the highest activity among the tested extractives, on (HepG-2) liver carcinoma cell line (IC50 = 17 μg/mL) followed by the n- hexane extract, and the dichloromethane and methanol EE fractions (IC50s 23.6, 31.2 and 32.2 μg/mL, respectively). 2. Concerning lung carcinoma cell line (H-1299), the dichloromethane fraction was the most effective as cytotoxic (IC50 = 29.3 μg/mL) followed by the methanol fraction and n-hexane extract (IC50s = 32 and 36.4 μg/mL), while the ethyl acetate: n-butanol (2:1) fraction was found inactive. On the other hand, all of these samples did not exert any cytotoxic effect against breast carcinoma cell line (MCF-7) except the dichloromethane fraction that exhibited a very weak activity (IC50 = 100 μg/mL). 3. The n-hexane extract showed the weakest cytotoxic activity on normal fibroblasts (BHK cell line, IC50 = 87 μg/mL) and therefore was considered as the safest among all tested samples, followed by the dichloromethane, methanol and ethyl acetate: n-butanol (2:1) EE fractions (IC50s = 72, 57.4 and 34.5 μg/mL). 4. As for the isolated compounds, β-carotene exhibited the highest cytotoxic activity on (H-1299) lung carcinoma cell line (IC50 = 9.2 ± 0.7 μg/mL) 182

Biological Evaluation of the Different Extractives & Isolated Compounds followed by 4'-methyl scutellarein and clerodermic acid which showed moderate cytotoxicity (IC50s = 18 ± 4.8 and 18.4 ± 4.8 μg/mL). Besides, verbascoside and 22-dehydroclerosterol-3-Oβ-D-glucoside were mildly active against the aforementioned cell line (IC50 = 26.8 ± 3.9 and 32.4 ± 7.5 μg/mL), as compared to the reference drug, doxorubicin (IC50 = 4.28 ± 1.6 μg/mL). 5. The effect of the tested compounds on liver carcinoma cell line (HepG-2) was different. Clerodermic acid was the most efficient (IC50 = 12.2 ± 2.8 μg/mL) while the others showed a moderate cytotoxic activity in the following order: 4'-methyl scutellarein, 22-dehydroclerosterol-3-Oβ-D-glucoside, β-carotene and verbascoside (IC50s = 21.3 ± 1.5, 22.3 ± 2.4, 22.5 ± 2.4 and 22.7 ± 3.1 μg/mL) relative to the reference drug, doxorubicin (IC50 = 5.87 ± 1.4 μg/mL). 6. Upon testing the cytotoxicity of the isolated compounds on normal fibroblasts (BHK cell lines), verbascoside and 4'-methyl scutellarein were found the safest followed by β-carotene (IC50s = 34.7 ± 1.1, 34.7 ± 3.7 and 28.9 ± 2.2 μg/mL). Meanwhile, clerodermic acid was the most toxic towards this cell line (IC50 = 19.2 ± 1 μg/mL), thus hindering its use as a cytotoxic drug prior more intense investigation of its efficiency versus safety, and trials to increase the latter via structure modification. Finally, 22-dehydroclerosterol-3-Oβ-D-glucoside could not be considered as suitable candidate for management of either H-1229 or HepG-2 carcinoma since its cytotoxic activity on normal fibroblasts (BHK cell line, IC50 = 21 ± 1.2 μg/mL) exceeded that exerted on the cited cell lines. Taking in consideration that concentrations up to 50 μg/mL of the reference drug, doxorubicin were unable to inhibit the growth of 50% the normal fibroblasts (BHK cell line, corresponding to IC50 > 50 μg/mL).

183

Chapter IV Development and Validation of an HPLC Procedure for Standardization of the Extractives of the Leaves

HPLC Standardization of the Extractives of the Leaves Chapter IV Development and Validation of an HPLC Procedure for Standardization of the Extractives of the Leaves Nowadays, there is an increased attention in natural products that may counteract the detrimental effects of environmental or chemical toxins and prevent multiple hepatic disorders in humans. The abietane diterpene, clerodermic acid, herein isolated from the ethyl acetate/ n-butanol (2:1) fraction of the 70% ethanol extract (EE) of the defatted leaves of C. inerme has been proven to exert the highest cytotoxic activity on HEPG-2 liver carcinoma cell lines among other tested isolates. This compound might therefore be suggested as lead for production of chemo-preventive hepatic anticancer agent. Besides upon chromatographic screening (TLC and HPLC), clerodermic acid appeared to be one of the major constituents of EE. Consequently, an HPLC procedure for reliable standardization of the leaves of C. inerme was developed based on estimation of its clerodermic acid content. This as part of the quality assurance process to facilitate the acceptance of the plant as raw material in herbal formulations. The proposed method was subjected to validation to ensure reliability before application. The amount of clerodermic acid was estimated in both the parent EE of the leaves and its active dichloromethane and ethyl acetate/ n-butanol (2:1) fractions. I. Preparation of the Working Solutions Solutions of the dried EE of the leaves of C. inerme (5 mg: 2 mL) and its dichloromethane (1 mg: 3 mL) and ethyl acetate/ n-butanol (2:1) (1 mg: 1 mL) fractions were prepared in methanol and used as working solutions. Aliquots (20 μL, each) of the working solutions were directly injected into the HPLC system. II. Optimization of the Operating Conditions Several trials were carried out to adjust the HPLC chromatographic conditions; attempts included the use of various mobile phases with different ratios as eluents. Satisfactory separation was achieved at room temperature, within 23 minutes, by gradient elution. The most efficient eluting system consisted of: component A, acetonitrile and component B, 0.3% phosphoric acid. A stepwise gradient elution program was adopted: 18 - 38% A (0-13 min), 38- 60% A (13-20 min), 60 - 100% A (20-22 min). The system was equilibrated with 100% of A, for another 1 min, at the end of each run. The flow rate was adjusted at 1 ml/min and the UV detector set at 325 nm. Aliquots (20 μl, each) of the working solutions were directly injected into the HPLC system for each determination. All injections were repeated in triplicates. 184

HPLC Standardization of the Extractives of the Leaves These conditions were adjusted as those adopted during HPLC screening and isolation of the constituents of the ethyl acetate/ n-butanol (2:1) and methanol fractions (page 154). The HPLC chromatograms of the parent EE, its analyzed fractions and isolated clerodermic acid are represented in figure 72.

Figure 72: HPLC fingerprints of the EE, its dichloromethane (DCM) and ethyl acetate/ n-butanol (2:1) (EtOAc) fractions

185

HPLC Standardization of the Extractives of the Leaves III. Standardization of the Extractives A standard stock solution of clerodermic acid in methanol (1.5 mg/mL) was diluted to yield a series of seven different concentrations (12, 24, 36, 48, 60, 120 and 300 μg/mL) covering the concentration range required, based on the level expected in the plant sample. An aliquot (20 μL) of each prepared dilution was injected in triplicates and corresponding peak areas recorded. The calibration curve (Figure 73) was then constructed by plotting mean peak areas versus corresponding concentrations. Results are recorded in table 45. Table 45: Average peak areas of the different concentrations of clerodermic acid

Concentration (μg/mL) Peak area* 12 101.5 24 236 36 273.5 48 372.5 60 447.5 120 988 300 2401.5 *Average of three determinations

Figure 73: Standard calibration curve of clerodermic acid 186

HPLC Standardization of the Extractives of the Leaves The content of clerodermic acid in each extractive was then determined from the pre-established standard calibration curve and results recorded in table 46. Table 46: Contents of clerodermic acid in the different extractives of the leaves of C. inerme

Extractive Content of clerodermic acid* (mg %) EE 9.76 DCM 70.7 EtOAc/BuOH 46.6 *Average of three determinations EE, ethanol (70%) extract; DCM, dichloromethane fraction; EtOAc/BuOH, ethyl acetate/ n-butanol (2:1) fraction IV. Validation of the Proposed Standardization Method The proposed method was subjected to elementary validation studies according to the guidelines of the International Conference of Harmonization (ICH, 2005) and United States Pharmacopoeia (USP, 2009). The following validation characteristics were examined: specificity, linearity, precision, accuracy, limit of detection, limit of quantification, and sample stability testing. The isolated clerodermic acid was used as standard. A. Specificity The specificity is the ability to detect the analyte peak in the presence of other components or impurities. Specificity was determined by performing three injections: the first with the sample (working solution), the second with the standard (isolated clerodermic acid) and the third with the solvent (blank). All experiments were performed in triplicates. No interference was observed for the main peak with other peaks detected in the chromatogram. B. Linearity Linearity is the ability (within a given range) to obtain a response or signal with a magnitude (y = peak height or peak area) directly proportional to the amount (C = concentration) of the analyte in the sample (ICH, 2005). Linearity was assessed by linear regression analysis, which was calculated by the least square method. As shown previously in figure 73, page 186, the Correlation Coefficient (R2) for the standard calibration curve was 0.999. This indicates linearity of the peak area of clerodermic acid in the range of 12-300 μg/mL.

Correlation coefficient (R2) = 0.999; Regression equation: y = 8.0025x + 2.7162

187

HPLC Standardization of the Extractives of the Leaves C. Accuracy Accuracy expresses the closeness or agreement between the accepted value, either as a conventional true value or an accepted reference value and the test results (ICH, 2005); this is determined by comparing the results obtained for the analyzed samples with the calculated amounts. Recovery experiments were thus performed to evaluate the accuracy of the method. The bias of the analytical procedure was investigated during the method validation study using the recovery data of the tested extract spiked with the standard. Three individual samples of the ethanol (70%) extract (EE, working solution, 2.5 mg/mL) were spiked with three concentration levels of clerodermic acid (24, 48 and 120 μg/mL). The spiked samples were analyzed in triplicates. Accuracy was determined by calculating the difference between the average value found during analysis and the theoretical amount. Accuracy was expressed as mean percent recovery at the three concentration levels examined. The % RSD was calculated and results, presented in table 47 and figure 74, indicate that the data obtained were within acceptance criteria (98-102%).

Figure 74: HPLC chromatograms of the EE of the leaves of C. inerme, isolated Clerodermic acid & EE spiked with Clerodermic acid 188

HPLC Standardization of the Extractives of the Leaves Table 47: Accuracy study of the proposed standardization method

Clerodermic Added *Theoretical Detected acid in clerodermic clerodermic clerodermic Recovery %RSD extract acid acid acid % (μg/mL) (μg/mL) (μg/mL) (μg/mL) 244 24 134 136 3.8 101.49 244 48 146 145 2.3 99.5 244 120 182 179 1.4 98.4 *Mean of clerodermic acid concentration in the extract + added pure clerodermic acid concentration D. Precision Precision expresses the closeness of agreement (degree of scatter) between series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions (ICH, 2005). Precision of the assay is determined by repeatability (intra-day variability) and intermediate precision (inter-day variability). 1. Repeatability (intra-day variability) Repeatability was estimated by analyzing two different concentrations (36 and 60 μg/mL) of clerodermic acid six times in the same day. Repeatability was expressed in terms of relative standard deviation (% RSD) of the peak area and results are recorded in table 48. Table 48: Repeatability for retention time and peak areas expressed as % RSD

C3 (36 μg/mL) C5 (60 μg/mL) Run Peak Area Peak Area R (min) R (min) t (mAU) t (mAU) 1 22.31 284 21.95 442 2 22.1 281 22 458 3 22.28 276 22.07 462 4 22.27 282 22.11 466 5 22.23 272 22.16 454 6 22.19 284 22.31 465 Mean ± SD 22.23 ± 0.076 279.83 ± 4.833 22.1 ± 0.127 457.83 ± 8.95 % RSD 0.343 1.727 0.577 1.956 SD= standard deviation; % RSD= relative standard deviation C3, C5= Clerodermic acid concentrations (36, 60 μg/mL respectively)

189

HPLC Standardization of the Extractives of the Leaves 2. Intermediate precision (inter-day variability or ruggedness) Intermediate precision was determined by analyzing, in triplicates, two concentrations of clerodermic acid (36 and 60 μg/mL) on three different days. Intermediate precision was expressed in terms of relative standard deviation (%RSD) of peak area, and results recorded in table 49. Table 49: Intermediate precision for peak areas of two concentrations of clerodermic acid over three days

Peak Area* (mAU) Day C3 (36 μg/mL) C5 (60 μg/mL) 1 273 447.5 2 285 444 3 274 450 Mean ± SD 277.33 ± 6.658 447.17 ± 3.014 % RSD 2.401 0.674 *Average of three determinations C3, C5= Clerodermic acid concentrations (36, 60 μg/mL respectively) SD = standard deviation; % RSD = relative standard deviation The relative standard deviation values of the repeatability and intermediate precision were less than 2.00 and 3.00 %, respectively and demonstrate that the HPLC method is precise. E. Suitability range The range of suitability of a given analytical procedure is the interval between the minimum and maximum concentrations of the compound to be determined in which the linearity is observed (ICH, 2005), the characteristics of repeatability limits and the accuracy is maintained at a sufficiently high level. The range of purity of the method has been set at concentration range of 12-300 μg/mL of the standard compound, since the method has been shown to be precise, accurate and linear within this range. F. Limit of Detection (LOD) and Limit of Quantification (LOQ) The limit of detection (LOD) is the lowest concentration of an analyte that the analytical process can reliably differentiate from the background level, at a signal- to-noise (S/N) ratio of 3:1. The limit of quantification (LOQ) is the minimum concentration of the analyte that can be determined at an acceptable precision and accuracy under rated conditions of analysis. It is usually regarded as the amount for which the signal-to-noise (S/N) ratio should not be less than 10:1 (ICH, 2005). LOD and LOQ were estimated based on the standard deviation (SD) of the response and the slope of the linearity curve.

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HPLC Standardization of the Extractives of the Leaves

Limit of Detection (LOD) = SD × 3/S Limit of Quantitation (LOQ) = SD × 10/S Where: SD = standard deviation of the y intercepts of regression lines S = slope of the linearity curve obtained by regression analysis The LOD and LOQ of clerodermic acid were found to be 1.12 and 3.39 μg/mL, respectively. G. Robustness The robustness of an analytical procedure is the characteristic of its stability with respect to small variations of the different system parameters (main system factors e.g. flow rate, pH, temperature…, etc.) possible under real conditions (ICH, 2005). Preliminary testing of the robustness of the proposed method was established by making deliberate minor variation in the flow rate employed. Flow rate was changed from 1 mL/min to 0.9 and 1.1 mL/min and %RSD was calculated for one clerodermic acid concentration (36 μg/mL). The chromatographic elution pattern remained unaffected and the low values of RSD established the robustness of the method. Results are presented in table 50 and figure 75. Table 50: Results of robustness study for the proposed standardization method for analysis of clerodermic acid based on minor variation in flow rate

Flow rate Mean Peak Mean R * ± SD % RSD t % RSD (mL/min) Area* ± SD (min)

1 280.67 ± 4.163 1.483 22.29 ± 0.021 0.093 0.9 281.33 ± 2.082 0.739 22.32 ± 0.006 0.026 1.1 275.33 ± 3.055 1.109 22.3 ± 0.017 0.078 *Average of three determinations SD= standard deviation; % RSD= relative standard deviation

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HPLC Standardization of the Extractives of the Leaves

Figure 75: HPLC chromatograms of isolated Clerodermic acid at different flow rates (1, 0.9 & 1.1 mL/min) Criteria for validation of the quantitative RP-HPLC-UV method proposed for standardization of the EE of the leaves of C. inerme, using the isolated clerodermic acid as standard, are collectively recorded in table 51.

192

HPLC Standardization of the Extractives of the Leaves Table 51: Criteria for validation of the HPLC-based analytical procedure proposed for standardization of the extractives of the leaves of C. inerme

No. Parameter Acceptance criteria Results

No interference with other 1 Specificity Passes peaks 2 Linearity R2 = 0.995 to 1 0.999 3 Accuracy Recovery = 98-102 % 98.4 to 101.49 Precision 4 Repeatability RSD < 2% 0.343 to 1.956 Intermediate Precision RSD < 3% 2.401 and 0.674 5 Suitability Range ------12-300 μg/mL 6 LOD ------1.12 μg/mL 7 LOQ ------3.39 μg/mL

8 Robustness RSD < 3% 0.026 to 1.483

Conclusion Analysis was performed on a LiChrospher 100 RP-C18 column by stepwise gradient elution using appropriate acetonitrile: 0.3% phosphoric acid mixtures with the UV detector set at 325 nm. The conditions of the developed RP-HPLC/UV standardization method were optimized using the isolated clerodermic acid as standard. The method was validated and all parameters were found within acceptance limits. The method was found to be simple, precise, linear, accurate, reproducible, selective, robust, stable and time saving and thus reliable for identification and quantitation of clerodermic acid in the ethanol extract (EE) of the leaves of Clerodendrum inerme. The contents of clerodermic acid in the parent EE, 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 relative to the dry weight of the individual extractives), respectively.

193

Chapter V Effect of Stage of Plant Development on the Chemical Composition of the Leaves

Effect of Stage of Plant Development on the Chemical Composition of the Leaves

Chapter V Effect of Stage of Plant Development on the Chemical Composition of the Leaves The effect of stage of plant development on the chemical composition of the ethanol extract of the defatted leaves of C. inerme was assessed. Samples of leaves were collected from pre-, intra- and post-flowering plants, air-dried, powdered, defatted, extracted with ethanol and the resulting extracts analyzed by HPLC. I. Preparation of the 70% Ethanol Extracts Samples (10 g, each) of the air-dried leaves gathered from plants at different stages of development (before, during and after flowering) were separately defatted with n-hexane (4 × 50 mL), and the corresponding marcs extracted with ethanol (70%, 7 × 100 mL) with the aid of sonication. The solvents were evaporated under vacuum at a temperature not exceeding 55o C, the dried residues weighed and saved in a desiccator until use. Yields, expressed as percentages relative to dried leaves, and organoleptic characters of the different extracts are recorded in table 52. Table 52: Percentage yield and organoleptic characters of the extracts of the leaves of C. inerme collected at different stages of plant development

Yield (g/100g dried leaves) Extract Pre- Intra- Post- Color Taste Odor flowering flowering flowering n-Hexane 0.291 0.172 0.324 Y. brown Bitter N.C. Ethanol (70%) 3.273 3.933 3.883 D. brown Bitter N.C. D., dark; Y., yellowish; N.C., Not characteristic II. Preparation of the Working Solutions Aliquots (5 mg, each) of the dried EEs of the three different leaf samples (pre-, intra- and post-flowering) were, separately, dissolved in 2 mL methanol for preparation of the working solutions. Aliquots (20 μL) of each working solution were directly injected into the HPLC system. II. HPLC Monitoring of the Extracts Compositions The operating conditions applied for standardization of the extracts (see page 184) were adopted for monitoring the effect of stage of development on the chemical composition of the EE of the defatted leaves of C. inerme and especially on production of bioactive secondary metabolites. HPLC chromatograms of the EEs of 194

Effect of Stage of Plant Development on the Chemical Composition of the Leaves the different leaf samples are illustrated in figure 76. The contents of the three major constituents detected in the analyzed samples namely; verbascoside, 4'- methylscutellarein and clerodermic acid, designated by peak areas, are recorded in table 53 and represented by figure 77.

Figure 76: HPLC chromatograms of the EEs of the leaves of C. inerme collected at different stages of plant development

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Effect of Stage of Plant Development on the Chemical Composition of the Leaves Table 53: Peak areas of the major constituents of the EEs of the leaves of C. inerme collected at different stages of plant development

Peak area (mAU) Stage of Development Verbascoside 4'-Methylscutellarein Clerodermic acid 1105 760 1112 Pre- 1093 748 1032 flowering 1063 758 1064 Mean 1087 755 1069 2149 2795 1895 Intra- 2066 2690 1860 flowering 2160 2758 1893 Mean 2125 2758 1883 2130 1313 1634 Post- 2061 1390 1657 flowering 2110 1434 1696 Mean 2100 1379 1662

V, verbascoside; 4'-M, 4'-methylscutellarein; CDA, clerodermic acid Figure 77: Effect of stage of development on the major constituents of the EEs of the leaves of C. inerme 196

Effect of Stage of Plant Development on the Chemical Composition of the Leaves Results and Discussion The concentrations of clerodermic acid in the EEs of the leaves of C. inerme, evaluated by the proposed HPLC validated method (standard calibration curve figure 73, page 187), were 5.33, 9.4 and 8.29 mg/100 g of the dry EEs of leaves collected from plant at the pre-, intra- and post-flowering stages, respectively. Results represented in table 53 and figures 76 and 77 indicated that the production of major constituents namely; verbascoside, 4'-methylscutellarein and clerodermic acid is obviously affected by the stage of plant development. The highest contents of these metabolites were recorded in leaf samples collected from plants during flowering and the lowest in those obtained from pre-flowering plants. The concentration of the quantified constituents started to decrease in the post- flowering samples. Consequently, from both economical and pharmacological standpoints, the leaves of C. inerme should preferably be collected at the flowering stage to ensure optimum yields of major bioactive metabolites.

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Summary

Summary Summary Family Lamiaceae (the mint family) is the sixth largest family of flowering plants and the largest family of the order Lamiales. It comprises about 236 genera and 7136 species. The greatest genera are Salvia, Clerodendrum, Scutellaria, Stachys, Plectranthus, Hyptis, Teucrium, Vitex, Thymus and Nepeta. The family members are distributed worldwide, many are aromatic and thus used as culinary herbs. Besides volatile constituents, they are reported to contain several pharmacologically active compounds such as phenolics and iridoids and to exert a wide range of biogical effects including antioxidant, cytotoxic, antimicrobial, antifungal and anti-inflammatory. Species of genus Clerodendrum grow mainly in tropical Africa and southern Asia. The two selected species viz., Clerodendrum inerme L. Gaertn. and Clerodendrum splendens G. Don were successfully acclimatized in Egypt as ornamentals. The objective of this work was thus targeted towards assessing the potentialities of these two locally cultivated plants as a source of natural remedies. To fulfil these goals, and after comprehensively reviewing the literature, a comparative study of the two cited species was planned to include the following: 1. Collection of the plant material and establishment of reliable criteria for taxonomical differentiation between the two selected species based on botanical profiling, phytochemical screening and determination of pharmacopoeial constants. In addition to comparative UPLC-Orbitrap HRMS metabolite profiling of the methanol extracts of the leaves. 2. Extraction and bioactivity-guided fractionation of selected extracts of the leaves based on in-vitro evaluation of antioxidant, cytotoxic and antimicrobial activities followed by qualitative and quantitative analyses of the constituents of the bioactive extractives via appropriate procedures (spectrophotometric and HPLC). 3. Isolation, identification and structure elucidation of the main constituents via chromatographic and spectrometric techniques, and correlation of the recorded bioactivities to the isolated compounds. 4. Finally, development and validation of an HPLC procedure for standardization of the bioactive extracts/fractions to facilitate further implementation in herbal formulations.

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Summary Part I: Comparative Study of Clerodendrum inerme L. Gaertn. and Clerodendrum splendens G. Don Chapter I: Botanical Characters I. Macromorphological Characters The leaves and flowers of C. inerme and C. splendens are morphologically different. Leaves of C. inerme are elliptical or obovate, with emarginate, acute or rounded apices, and light green petiole, while those of C. splendens are ovate or cordate, with acuminate apices, dark purplish petiole and are much larger than those of C. inerme. Flowers of C. inerme have white corolla and green calyx, while those of C. splendens have red corolla and calyx and are shorter and wider than those of C. inerme. II. Micromorphological Characters Microscopically, the powdered leaf of C. inerme is characterized by the presence of tortuous fibers, calcium oxalate clusters and oil globules, and that of C. splendens by the presence of crystal sheath. The labiaceous hairs of upper epidermis and stomata are distinctly larger in size in C. inerme whereas the epidermal cells are larger in C. splendens. The epidermal cells of C. inerme have beaded anticlinal walls and are covered with striated cuticle on the lower surface, in contrast the cuticle covering those of C. splendens is smooth. The epidermal cells of the petiole are larger in size and with larger labiaceous hairs in C. splendens compared to C. inerme. The epidermal cells of the petiole of C. inerme are thick-walled while those of C. splendens have beaded walls and show purple-coloured patches. These data could be considered as useful criteria for discrimination between the two species besides allowing their identification among other related members of the genus. Chapter II: Proximate Analysis and Preliminary Phytochemical Screening I. Proximate Analysis The ash values of the leaves of the two species were found distinctly different. The total and acid insoluble ash values were about 1.5 time higher in C. splendens than in C. inerme, while that of water-soluble ash in the latter was almost double that in C. splendens. The crude fibers and moisture contents of C. splendens exceeded those of C. inerme. These data could be considered as quality control criteria for the leaves of the two species. II. Preliminary Phytochemical Screening Results of preliminary phytochemical screening revealed an almost similar response to the applied tests among the leaves of the two species suggesting that they are closely related in composition. Besides, the following could be deduced: 199

Summary 1. Carbohydrates and/or glycosides, free and/or combined flavonoids, sterols and/or triterpenes, saponins, as well as condensed tannins are identified in both samples. 2. Moreover, since both samples gave negative response when tested for crystalline sublimates, cardiac glycosides and alkaloids and/or nitrogenous bases, these constituents may be considered as absent. Chapter III: Extraction, Fractionation and Examination of the Leaves Extractives I. Defatting and Ethanol (70%) Extraction of the Leaves The air-dried powdered leaves of C. inerme and C. splendens (100 g, each) were, separately, defatted with n-hexane, the defatted marc was then extracted with ethanol (70%) with the aid of sonication. The solvents were evaporated under vacuum, the residues weighed, and percentage yields calculated based on dried plant material (g/100g). II. Fractionation of the Ethanol (70%) Extracts (EE) The EE of the leaves of C. inerme and C. splendens (10 g, each) were separately suspended in distilled water and subjected to liquid-liquid fractionation using dichloromethane and ethyl acetate: n-butanol mixture (2:1) in succession. The solvents were removed by evaporation under vacuum, and the dried residues weighed and kept in desiccator for further investigation. III. TLC Fingerprinting of the Different Extractives The extractives of the leaves of C. inerme and C. splendens were subjected to TLC profiling using different solvent systems and visualizing agents to ascertain which could be considered richer in secondary metabolites. Results revealed that the yields of leaf extractives of C. inerme were higher than their C. splendens analogues. Besides, the TLC chromatographic profiles of the different extractives of the leaves of C. inerme showed greater number of spots with stronger response to visualizing agents than those of C. splendens. In conclusion, the leaves of C. inerme could be suggested as a better source of secondary metabolites. Chapter IV: UPLC-Orbitrap HRMS Profiling Comparative UPLC-Orbitrap HRMS metabolic analysis of the leaves of both species showed a chromatographic profile wealthier in components in the C. inerme sample as compared to that of C. splendens. A total of 36 metabolites were detected in the two species under investigation among these 32 were tentatively identified. Twenty eight chromatographic peaks, belonging to various metabolite classes, were assigned in C. inerme, 25 of which were identified. Whereas, among the 14 chromatographic peaks assigned in the C. splendens sample, 12 were

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Summary identified. The main classes of secondary metabolite detected were phenylpropanoid and iridoid glycosides, flavonoids, diterpenoids as well as phenolic acid and fatty acid derivatives. Chapter V: Quantitative Estimation of Phenolic Content I. Spectrophotometric Determination of the Total Phenolic Content (TPC) The TPC of the EEs of the leaves of the two species under investigation were determined based on measuring the intensity of the colour developed when phenolic compounds were complexed with Folin-Ciocalteu reagent. The total phenolic content estimated as gallic acid equivalent in the leaves of C. inerme exceeded that in the leaves of C. splendens (63.3 vs. 55.21 mg/g plant dry weight). II. Spectrophotometric Determination of the Total Flavonoid Content (TFC) The TFC of the leaves of the two species under investigation were determined in the EEs based on measuring the intensity of the colour produced on complexing flavonoids with aluminium chloride reagent. The amount of total flavonoids expressed as mg quercetin /g plant dry weight was slightly higher in the leaves of C. inerme than in those of C. splendens (5.32 vs. 5.01 mg/g plant dry weight). Chapter VI: In-vitro Evaluation of the Biological Activities I. Evaluation of the Antioxidant Activity The antioxidant activity of the EEs of the leaves of C. inerme and C. splendens was evaluated using the DPPH method. Results revealed that EEs of the leaves of C. inerme and C. splendens showed weak antioxidant activity with respective EC50 values of 627.246 ± 37.261 and 583.905 ± 25.315 µg/mL, with that of C. splendens exhibiting a slightly higher efficiency. The results were compared to the reference anti-oxidant, gallic acid whose EC50 value was 18.37 ± 0.59 µg/mL. II. Evaluation of the Cytotoxic Activity The cytotoxic activity of the EEs of the leaves of C. inerme and C. splendens was evaluated using Sulfo-rhodamine B (SRB) spectrophotometric assay. Results revealed that the EE of the leaves of C. inerme exhibited in-vitro cytotoxic activity against the three tested carcinoma cell lines while that of C. splendens exihibited strong in-vitro cytotoxic activity against (H-1299) lung carcinoma cell line and a weak effect against both (MCF-7) breast and (HepG-2) liver carcinoma cell lines. The EE of the leaves of C. inerme exhibited higher cytotoxic activity than that of C splendens with IC50 values of 25.5 and 25 μg/mL for C. inerme and 45.5 and 36 μg/mL for C. splendens on (MCF-7) breast and (HepG-2) liver carcinoma cell lines respectively, whereas the EE of the leaves of C. splendens exhibited higher cytotoxic activity on (H-1299) lung carcinoma cell line than that of C. inerme with IC50 values of 9.91 and 23 μg/mL respectively. Both EEs of C. 201

Summary inerme and C. splendens leaves exhibited nearly the same cytotoxic activity on normal fibroblasts (BHK cell line) with IC50 values of 37 and 36 μg/mL respectively meaning that they have nearly the same safety profile. III. Evaluation of the Antibacterial Activity against Antibiotic Resistant Bacterial Strains A. Evaluation of the Antibacterial Activity against Methicillin Resistant Staphylococcus aureus (MRSA) The antibacterial activity against Methicillin Resistant Staphylococcus aureus (MRSA) of the EE of the leaves of C. inerme and C. splendens was evaluated using MTT assay. Results revealed that the EE of C. inerme showed moderate antibacterial activity against MRSA, with MIC 90 value of 12.7 µg/mL and MIC value of 31.25 µg/mL, compared to vancomycin which had MIC 90 and MIC values of 0.6 and 1.95 µg/mL, respectively. Meanwhile, the EE of C. splendens showed very weak antibacterial activity against MRSA with MIC 90 value of 113.1 µg/mL. Concentrations up to 125 µg/mL could not inhibit the growth of all of the incubated bacteria and thus the MIC was higher than 125 µg/mL. B. Evaluation of the Antimycobacterial Activity The antimycobacterial of the EE of the leaves of C. inerme and C. splendens was evaluated using Microplate Alamar Blue Assay (MABA). The EE of the leaves of C. inerme showed moderate antimycobacterial activity against Mycobacterium tuberculosis, with MIC 90 and MIC values of 7.2 µg/mL value of 15.63 µg/mL, respectively. Moreover, the EE of C. splendens showed very weak antimycobacterial activity against Mycobacterium tuberculosis with MIC 90 and MIC values of 30.8 and 62.5 µg/mL, respectively. The MIC 90 and MIC values of the reference drug, isoniazide were 0.04 and 0.24 µg/mL, respectively.

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Part II: Phytochemical and Biological Investigation of Clerodendrum inerme L. Gaertn. Leaves As evidenced in Part I, the small-scale extraction and fractionation of the leaves of C. inerme and C. splendens resulted in higher yields of n-hexane and ethanol extracts (EE), as well as of those of the dichloromethane and ethyl acetate/n- butanol (2:1) fractions obtained from C. inerme leaves relative to those acquired from C. splendens. TLC examination of the aforementioned extracts and fractions displayed several spots in the chromatograms of the leaves of C. inerme that exceeded in number those detected in their analogues derived from C. splendens. Moreover, comparative UPLC-Orbitrap HRMS metabolic analysis of the leaves of both species showed a chromatographic profile wealthier in components in the C. inerme sample as compared to C. splendens. Besides, quantitative estimation of the total phenolic and flavonoid contents (TPC and TFC) of the examined leaves revealed their presence in higher amounts in those of C. inerme. From the biological standpoint as well, in-vitro evaluation of the cytotoxic and antibacterial activities of the EEs of the tested plant leaves showed a broader cytotoxic action and a stronger antibacterial efficiency (against both TB and MRSA) for the EE of C. inerme leaves.

Chapter I: Optimization of Phenolic Extraction from the Leaves of Clerodendrum inerme L. Gaertn. In this chapter, a liquid/solid fractionation technique aided by sonication was developed to isolate phenolics from the dried EE of the defatted leaves of C. inerme in a higher yield. The method efficiency was compared to that of conventional liquid/liquid fractionation in terms of yields of extractives, TPC and TFC of the resulting fractions. Results revealed that the total yield of organic extractives obtained by the proposed liquid/solid technique (6.8 g) exceeded by far that acquired by liquid/liquid fractionation technique (2.1 g). Although, the TPC of the ethyl acetate/ n-butanol (2:1) and dichloromethane fractions resulting from the conventional liquid/liquid technique were higher than those of the corresponding fractions resulting from the proposed liquid/solid technique. Yet, the latter provided an extra methanol fraction that efficiently raised the combined TPC of the resulting fractions over that yielded by the conventional liquid/liquid technique.

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Summary On the other hand, comparing the TFC of the different fractions, that of the two dichloromethane fractions were found nearly the same, while the TFC of the ethyl acetate/ n-butanol (2:1) fraction resulting from the proposed liquid/solid technique was much higher than that of its analogue obtained by conventional liquid/liquid technique. In addition, the presence of the methanol fraction in the proposed liquid/solid technique increased the combined TFC of the fractions resulting from this technique to a much higher level than those resulting from the conventional fractionation method. These results support the appropriateness of the proposed liquid/solid technique for extraction of higher yields of phenolic constituents, and therefore it was adopted for large scale fractionation of the EE of the leaves of C. inerme. Chapter II: Investigation of the Different Extractives I. Isolation and identification of the constituents of the n-Hexane Extract The n-hexane extract of the leaves of C. inerme was subjected to fractionation by vacuum liquid chromatography (VLC) on a silica gel H column and gradient elution was performed. For isolation and purification of individual compounds, the obtained sub-fractions were subjected to further chromatographic separation by VLC on a silica gel H column and column chromatography (CC) on silica gel 60 column.

Compounds H1 and H2 were isolated and purified. Identification of the compounds was based on determination of their physico-chemical, chromatographic and spectral data, comparison with available authentic samples and/or with published data.

Compounds isolated from this extract were identified as β-carotene (H1) and 22- dehydroclerosterol (H2). This is the first report of isolation of β-carotene from C. inerme. II. Isolation and identification of the constituents of the Dichloromethane Fraction The dichloromethane fraction of the EE of the leaves of C. inerme was subjected to fractionation by VLC on a silica gel H column and gradient elution was performed. Upon concentration of the sub-fraction eluted with 90% ethyl acetate in dichloromethane, a white solid was precipitated. The concentrated sub-fraction was subjected to centrifugation to isolate the white solid which was then purified to yield compound D1.

Compound D1 was identified as 22-dehydroclerosterol-3-O-β-D- glucopyranoside, based on determination of its physico-chemical, chromatographic and spectral data, and by comparison with available authentic samples and/or with published data. This is the first report of its isolation from C. inerme. 204

Summary III. Isolation and identification of the constituents of the Ethyl Acetate/ n- Butanol (2:1) Fraction The ethyl acetate/ n-butanol (2:1) fraction of the leaves of C. inerme was subjected to fractionation by VLC on a RP-C18 column and gradient elution was performed. The sub-fractions were monitored by TLC and HPLC. Two collective sub-fractions were selected for isolation of the major constituents of this fraction. Compounds E1 and E2 were isolated and purified. Identification of the compounds was carried out through determination of their physico-chemical, chromatographic and spectral data, and by comparison with available authentic samples and/or with published data. Compounds isolated from this fraction were identified as 4’-methylscutellarein (E1) and clerodermic acid (E2). IV. Isolation and identification of the constituents of the Methanol Fraction The methanol fraction of the leaves of C. inerme was subjected to fractionation by VLC on a RP-C18 column and gradient elution was performed. The sub-fractions were monitored with TLC and HPLC. The collective sub-fraction eluted with 35% methanol in water was selected for isolation of its major constituent(s). Compound M1 was isolated and purified. Identification of compound M1 was based on determination of its physicochemical, chromatographic and spectral data, comparison with available authentic samples and/or with published data.

Compound M1 isolated from this fraction was identified as verbascoside. Chapter III: Biological Evaluation of the Different Extractives and Isolated Compounds I. Evaluation of the Antioxidant Activity The antioxidant activity of the different fractions of the EE of the leaves of C. inerme was evaluated using the DPPH spectrophotometric assay. Results revealed that the ethyl acetate: n-butanol fraction, with lowest EC50 value (65.649 ± 1.601 µg/mL), exhibited the strongest antioxidant activity among other fractions derived from EE, followed by the methanol fraction (EC50 = 255.137 ± 12.899 µg/mL). Meanwhile, the dichloromethane fraction was the least active as compared to the reference anti-oxidant, gallic acid. II. Evaluation of the Cytotoxic Activity The in-vitro cytotoxic activity of the n-hexane extract alongside that of the different fractions of the EE of the leaves of C. inerme was investigated via the SRB assay on (H-1299), (HepG-2) and (MCF-7); lung, liver and breast carcinoma cell lines, respectively. Meanwhile, the cytotoxic potential of the compounds

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Summary isolated from the active fractions was evaluated on (H-1299) and (HepG-2); lung and liver carcinoma cell lines, respectively. The safety of the aforementioned extract/fractions and compounds was determined by investigating their cytotoxicity towards normal fibroblasts (BHK cell line). Results revealed that the EE ethyl acetate/ n-butanol (2:1) fraction showed the highest activity among the tested extractives, on (HepG-2) liver carcinoma cell line followed by the n-hexane extract, and the dichloromethane and methanol EE fractions. Concerning lung carcinoma cell line (H-1299), the dichloromethane fraction was the most effective cytotoxic followed by the methanol fraction and n-hexane extract, while the ethyl acetate: n-butanol (2:1) fraction was found inactive. On the other hand, all of these samples did not exert any effect against breast carcinoma cell line (MCF-7) except the dichloromethane fraction that exhibited a very weak activity. The n-hexane extract showed the weakest cytotoxic activity on normal fibroblasts and therefore was considered as the safest among all tested samples, followed by the dichloromethane, methanol and ethyl acetate: n-butanol (2:1) EE fractions. As for the isolated compounds, β-carotene exhibited the highest cytotoxic activity on (H-1299) lung carcinoma cell line followed by 4'-methyl scutellarein and clerodermic acid which showed moderate cytotoxicity. Besides, verbascoside and 22-dehydroclerosterol-3-Oβ-D-glucoside were mildly active against the aforementioned cell line, as compared to the reference drug, doxorubicin. The effect of the tested compounds on liver carcinoma cell line (HepG-2) was different. Clerodermic acid was the most efficient while the others showed a moderate cytotoxic activity in the following order: 4'-methyl scutellarein, 22- dehydroclerosterol-3-Oβ-D-glucoside, β-carotene and verbascoside relative to the reference drug, doxorubicin. Upon testing the cytotoxicity of the isolated compounds on normal fibroblasts (BHK cell lines), verbascoside and 4'-methyl scutellarein were found the safest followed by β-carotene. Meanwhile, clerodermic acid was the most toxic towards this cell line, thus hindering its use as a cytotoxic drug prior more intense investigation of its efficiency versus safety and trials to increase the latter via structure modification. Finally, 22-dehydroclerosterol-3-Oβ-D-glucoside could not be considered as suitable candidate for management of either H-1229 or HepG-2 carcinoma since its cytotoxic activity on normal fibroblasts (BHK cell line) exceeded that exerted on the cited cell lines. Taking in consideration that concentrations up to 50 μg/mL of the reference drug, doxorubicin were unable to inhibit the growth of 50% the normal fibroblasts (BHK cell line, corresponding to IC50 > 50 μg/mL).

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Summary Chapter IV: Development and Validation of an HPLC Procedure for Standardization of the Extractives of the Leaves Analysis was performed on a LiChrospher 100 RP-C18 column by stepwise gradient elution using appropriate acetonitrile: 0.3% phosphoric acid mixtures with the UV detector set at 325 nm. The conditions of the developed RP-HPLC/UV standardization method were optimized using the isolated clerodermic acid as standard. The method was validated and all parameters were found within acceptance limits. The method was found to be simple, precise, linear, accurate, reproducible, selective, robust, stable and time saving and thus reliable for identification and quantitation of clerodermic acid in the ethanol extract (EE) of the leaves of Clerodendrum inerme. The contents of clerodermic acid in the parent EE, 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 relative to the dry weight of the individual extractives), respectively. Chapter V: Effect of Stage of Plant Development on the Chemical Composition of the Leaves The effect of stage of plant development on the chemical composition of the ethanol extract of the defatted leaves of C. inerme was assessed. Samples of leaves were collected from pre-, intra- and post-flowering plants, air-dried, powdered, defatted, extracted with ethanol and the resulting extracts analyzed by HPLC. The concentrations of clerodermic acid in the EE of the leaves of C. inerme, evaluated by the proposed HPLC validated method were 5.33, 9.4 and 8.29 mg/100 g of the dry EEs of leaves collected from plant at the pre-, intra- and post-flowering stages, respectively. Results also indicated that the production of the major constituents namely; verbascoside, 4'-methylscutellarein and clerodermic acid is obviously affected by the stage of plant development. The highest contents of these metabolites were recorded in leaf samples collected from plants during flowering and the lowest in those obtained from pre-flowering plants. The concentration of the quantified constituents started to decrease in the post-flowering samples. Consequently, from both economical and pharmacological standpoints, the leaves of C. inerme should preferably be collected at the flowering stage to ensure optimum yields of major bioactive metabolites.

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General Conclusion and Recommendation

General Conclusion and Recommendation General Conclusion and Recommendation General Conclusion 1. Clerodendrum inerme and Clerodendrum splendens can be distinguished from each other by their macro- and micro-morphological characteristics as well as proximate composition. 2. From both chemical and biological standpoints, the leaves of C. inerme appeared more promising than those of C. splendens as raw material for drug development. a. Since they gave higher yields of solvent extractives and revealed a higher number of component metabolites by both TLC and UPLC-Orbitrap HRMS profiling. b. Quantitatively, they were characterized by a higher total phenolic and flavonoid contents (TPC and TFC). c. In addition, their defatted ethanol extract (EE) exerted a relatively broader cytotoxic activity (against lung, liver and breast carcinoma cell lines) and stronger antibacterial action (against the antibiotic-resistant bacterial strains MRSA and TB). 3. UPLC-Orbitrap HRMS profiling of both samples revealed a variety of metabolites including: iridoid and phenylpropanoid glycosides, flavonoids, diterpenoids as well as phenolic acid and fatty acid derivatives. 4. For optimization of phenolic extraction from the leaves of C. inerme, a solid- liquid fractionation procedure (LSF) of its ethanol extract was proposed. Compared to the conventional liquid-liquid fractionation (LLF) method, it was found more efficient in terms of extractives yields, TPC and TFC. 5. Column chromatographic fractionation of the different extractives of the leaves of C. inerme resulted in isolation of 6 compounds (β-carotene, 22- dehydroclerosterol, 22-dehydroclerosterol-3-O-β-D-glucopyranoside, 4’- methyl scutellarein, clerodermic acid and verbascoside), two of these are for the first time reported in the species. 6. Among the isolated compounds, β-carotene exhibited the strongest cytotoxic activity against lung carcinoma cell line (H-1299); while clerodermic acid was the most effective against liver carcinoma cell line (HepG-2); thus suggesting further investigation of their cytotoxic potential. 7. 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.

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General Conclusion and Recommendation 8. Moreover 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 revealing that the leaves should better be collected during the flowering stage. 9. Finally, the standardized ethanol extract of the leaves of C. inerme collected during flowering could be suggested as a promising natural raw material for development of efficient and economic pharmaceuticals. Yet, intensive biological and clinical studies are necessary to support its implementation in drug industry. Recommendation Nowadays, the demand for herbal medicine is continuously increasing due to the serious side effects of synthetic drugs. In this respect, the leaves of Clerodendrum inerme L. Gaertn. might be recommended as a valuable nutritional supplement for prevention and/or control of various ailments including cancer. β-carotene and clerodermic acid isolated from the ethanol extract of the defatted leaves of this plant appeared to be promising as cytotoxic drugs, yet extensive clinical trials must be performed in order to support the evaluated bioactivities and to facilitate implementation of these promising herbals in pharmaceutical formulations.

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Arabic Summary

الملخص العربى الملخص العربى تعتبر العائلة الشفوية أكبر عائلة في رتبة الشفويات، وسادس أكبر عائلة من النباتات ال م زهرة حيث تتضمن 263 جنس و 7136 نوع. تنتشر نباتات العائلة الشفوية في جميع أنحاء العالم وتتميز بكثرة استخدامها في أعمال الطهي الحتواء العديد منها على زيوت عطرية. تحتوي نباتات العائلة الشفوية إلى جانب الزيوت العطرية على الكثير من المواد الفعالة التي لها خصائص عالجية مثل الفينوالت والقزحانيات وقد اثبتت األبحاث العلمية االنشطة البيولوجية لنباتات العائلة وتشمل أنشطة مضادة لألكسدة و للميكروبات و للفطريات و للخاليا السرطانية و االلتهابات. يعد جنس كليرودندرم أحد أكبر األجناس المنتمية للعائلة الشفوية؛ و تنتشر نباتات هذا الجنس فى المناطق االستوائية من قارة أفريقيا و جنوب قارة آسيا و ينتمي لهذا الجنس نباتا الياسمين الزفر وطربوش الملك المختاران للدراسة واللذان تتم زراعتهما فى مصر كنباتات للزينة. يعتبرالغرض من هذه الدراسة تقييم فاعلية نباتى الياسمين الزفر وطربوش الملك المزروعان فى مصر كمصدر لبعض المواد ذات الفاعلية الطبية وذلك بهدف اإلكثار منها واستعمالها فى عالج بعض األمراض. ولتحقيق هذه األهداف، تم عمل مسح مرجعي فى الدوريات والكتب المرجعية على هذين النباتين ثم القيام بدراسة مقارنة بين النوعين وتشتمل على ما يلى: 1- جمع عينات من نوعى النباتين المختارين ووضع معايير مفيدة للتمييز والتصنيف ىبينهما على أساس الدراسة النباتية والمسح الكيميائي وتعيين بعض الثوابت الدستورية باإلضافة إلى إجراء تصنيف مقارن لنواتج األيض الثانوية لخالصات الكحول الميثيلى للنباتين محل الدراسة باستعمال طريقة كروماتوجرافيا السائل فائقة األداء المقترن بمطياف الكتلة. 2- استخالص المواد الفعالة من النبات والقيام بتجزئة المواد المستخلصة بنا ًءا على التقييم البيولوجى للخواص المضادة لألكسدة والخاليا السرطانية والبكتيريا المقاومة للمضادات الحيوية متبوعا بالتحليل الكمى والكيفى للمكونات الموجودة في أجزاء الخالصات المثبت فعاليتها بواسطة الطرق المناسبة كالطرق الطيفية وكروماتوجرافيا السائل عالية األداء. 3- فصل وتنقية وتوصيف المواد الفعالة الرئيسية بالنبات وذلك باستخدام الطرق الكروماتوجرافية والطيفية ومن ثم ربط العالقة بين المواد المفصولة والفعاليات البيولوجية المسجلة. 4- إنشاء طريقة لمعايرة الخالصات الفعالة باستخدام كروماتوجرافيا السائل عالية األداء والتأكد من صحة وفعالية هذه الطريقة لتسهيل استخدام هذه الخالصات كمستحضرات عشبية.

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الملخص العربى الجزء األول: دراسة مقارنة لنباتي الياسمين الزفر و طربوش الملك الفصل األول: التوصيف النباتى أ. المواصفات الشكلية: أثبتت هذه الدراسة وجود العديد من الفوارق الشكلية بين النباتين حيث أن أوراق نبات الياسمين الزفر مدارية أو بيضاوية الشكل وأطرافها العليا حادة أومدورة ولها عنق أخضر فاتح، في حين أن أوراق نبات طربوش الملك بيضاوية أو قلبية الشكل ولها عنق أرجواني داكن وهى أكبرحجما من أوراق الياسمين الزفر، باإلضافة إلى الفوارق الشكلية بين زهور النباتين حيث أن تويج أزهار الياسمين الزفر أبيض اللون بينما الكأس أخضرفى حين أن التويج و الكأس في أزهار طربوش الملك لونهما أحمر وأي ًضا تختلف أزهار النباتين من حيث الحجم، فأزهار طربوش الملك اقل طو ًال و أكثر عر ًضا من أزهار الياسمين الزفر. ب. المواصفات المجهرية: من الناحية المجهرية، تتميز أوراق الياسمين الزفر بوجود ألياف متعرجة، عناقيد أكساالت الكالسيوم وكريات زيتية، بينما تتميز أوراق طربوش الملك بوجود األغمدة البللورية. يختلف النباتان مجهريًا أي ًضا من حيث خصائص البشرة العلوية والسفلية لالوراق حيث أن الثغور والشعيرات الشفوية في البشرة العلوية والسفلية أكبر حج ًم ا في أوراق الياسمين الزفر في حين أن خاليا البشرة تكون أكبر في أوراق طربوش الملك. بينما خاليا بشرة العنق تكون أكبر في الحجم في نبات طربوش الملك مقارنة بالياسمين الزفر. وكذلك خاليا بشرة العنق في نبات الياسمين الزفر سميكة الجدرا ،ن بينما تحتوي خاليا بشرة العنق في نبات طربوش الملك على جدران مخرزة وت ظهر بقعًا أرجوانية اللون. يمكن اعتبار هذه البيانات معايير مفيدة للتمييز بين النوعين بعضهما البعض وأي ًضا للتمييز بين أي منهما وبين النباتات األخرى المنتمية لنفس الجنس. الفصل الثانى: التحليل التقريبى و الوصف الكيميائى األولى أ. التحليل التقريبى تم تعيين بعض الثوابت الدستورية ألوراق الشجر المجففة في الهواء لكل من النوعين محل الدراسة. اشتمل التحليل على تعيين نسبة الرماد الكلي والرماد غيرالقابل للذوبان فى الحمض، والرماد القابل للذوبان في الماء ونسبة الرطوبة واأللياف الخامة. وقد و جد اختالف ملحوظ في نسب هذه الثوابت الدستورية حيث كانت نسب الرماد الكلى و الرماد غير القابل للذوبان في الحمض أعلى بحوالى مرة ونصف في أوراق طربوش الملك مقارنة بأوراق الياسمين الزفرفي حين أن قيمة الرماد الذائب في الماء في األخير كانت تقريبا ضعف قيمته في أوراق الياسمين الزفر. ب. المسح الكيميائي األولى كشفت نتائج الفحص الكيميائي النباتي األولى استجابة مماثلة تقريبا لالختبارات المطبقة بين أوراق النوعين مما يشير إلى ارتباطهما ارتباطا وثيقا في التكوين. إلى جانب ذلك، يمكن استنتاج ما يلي: 1. وجود الموادالكربوهيدراتيه و/أو الجلوكوزايدات والفالفونيدات الحرة و/أو المقترنة واالستيروالت و/أو التربينات الثالثية وكذلك العفصيات في كل العينات التي تم اختبارها. 2. لم يتم العثور على أي من المكونات المتسامية أو الجلوكوزايدات القلبية أو القلويدات و/أو القواعد النيتروجينية أو الصابونينات أو األنثراكينونات.

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الملخص العربى الفصل الثالث: االستخالص و اختبار الخالصات أ. إزالة الدهون و استخالص األوراق باستخدام الكحول اإليثيلى )70%( تم استعمال االوراق المطحونة لكل من نباتى الياسمين الزفر و طربوش الملك )100 جم لكل منهما( باستعمال النقع في الهكسان علي البارد إلزالة الدهون ، وتقطير المذيب تحت ضغط منخفض. بعد تجفيف مسحوق النبات تم نقعها على البارد فى الكحول االيثيلى )%70( ثم تقطير المذيب تحت ضغط منخفض ووزن الخالصات الخالية من المذيب وتحديد نسبها )جم/100 جم(. ب. تجزئة خالصات الكحول اإليثيلى )70%( تم تعليق خالصات الكحول اإليثيلى الجافة للنباتين محل الدراسة )10 جم لكل منهما( في الماء واستخالصها باستعمال ثنائى كلورو الميثايل وخليط من خالت اإليثيل والبيوتانول بنسبة )1:2( ثم تقطير المذيبات تحت ضغط منخفض و وزن الخالصات الخالية من المذيب و االحتفاظ بها فى مجفف لحين إجراء المزيد من الفحوصات. ج. اختبار المستخلصات بواسطة كروماتوجرافيا الطبقة الرقيقة تم اختبار مستخلصات أوراق النباتين محل الدراسة من حيث وجود المكونات المختلفة عن طريق كروماتوجرافيا الطبقة الرقيقة باستخدام أنماط مختلفة من المذيبات وعوامل التصور للتأكد مما يمكن اعتباره أكثر ثرا ًء ا في نواتج األيض الثانوية. أظهرت النتائج ارتفاع نسب مستخلصات أوراق الياسمين الزفر مقارنة بمثيلتها في أوراق طربوش الملك. باإلضافة إلى ذلك، أظهر فحص المستخلصات بواسطة كروماتوجرافيا الطبقة الرقيقة عددًا أكبر من البقع مع استجابة أقوى لعوامل التصور لمستخلصات أوراق الياسمين الزفر من تلك الموجودة في أوراق طربوش الملك. في الختام ، يمكن اقتراح أوراق الياسمين الزفر كمصدر أفضل لنواتج األيض الثانوية. الفصل الرابع: دراسة نواتج األيض الثانوية بإستعمال طريقة كروماتوجرافيا السائل فائقة األداء المقترن بمطياف الكتلة تممم عمممل دراسممة مقارنممة لنممواتج األيممض الثانويممة لخالصممات الكحممول الميثيلممى ألوراق كممل مممن النبمماتين محممل الدراسممة. أظهممر التحليممل األيضممي المتقمدم صمورة كروماتوغرافيمة أكثمر ثمرا ًء ا فممي المكونممات فممي عينممة أوراق الياسمين الزفر مقارنةً بعينة أوراق طربوش الملك. تم اكتشاف ما مجموعه 36 ناتج في النموعين قيمد الدراسمة تم التعرف عليهم جميعا. من بين هذه األنواع تم تعيين 28 في عينة أوراق الياسمين الزفر و تم التعمرف علييمم بينما تم تعيين 14 ناتج في عينة أوراق طربوش الملك و تم التعرف عليمم ايضا جميعا. تنتمي هذه النواتج إلى فئات األيمض المختلفمة وكانمت الفئمات الرئيسمية المتعمرف عليهما همي جليكوسميدات فينيمل بروبانويمد وإيريويمد، والفالفونويدات، والتربينات الثنائية، وكذلك مشتقات حمض الفينول واألحماض الدهنية. الفصل الخامس: التقدير الكمي لمحتوى الفينول أ. التقدير الطيفى للمحتوى الكلى للفينوالت تم تحديد المحتوى الكلى للفينوالت بخالصات الكحول اإليثيلى )%70( ألوراق النباتين محل الدراسة على أساس قياس شدة اللون الناتج من تفاعل المركبات الفينولية مع كاشف فولين سيوكالتو. و قد أظهرت النتائج أن إجمالي محتوى الفينول المقدّر كمكافيء لحمض الجاليك في أوراق الياسمين الزفر قد تجاوز مثيله في أوراق طربوش الملك )63.3 مقابل 55.21 مج / جم من وزن النبات الجاف(. ب. التقدير الطيفى للمحتوى الكلى للفالفونويدات - 3 -

الملخص العربى تم تحديد المحتوى الكلي للفالفونويدات بخالصات الكحول اإليثيلى )%70( ألوراق النباتين محل الدراسة على أساس قياس شدة اللون الناتج من تفاعل المركبات الفينولية مع كاشف ثالثى كلوريد األلومنيوم. و قد أظهرت النتائج أن كمية الفالفونويدات الكلية المقدّرة كمكافئ للكويرسيتين في أوراق الياسمين الزفر أعلى قلي ًال مقارنة بمثيلتها فى أوراق طربوش الملك )5.33 مقابل 5.01 مج / جم من وزن النبات الجاف(. الفصل السادس:تقييم األنشطة البيولوجية داخل المختبر أ. تقييم النشاط المضاد لألكسدة تم تقييم النشاط المضاد لألكسدة لخالصات الكحول اإليثيلى )% (70 ألوراق نباتي الياسمين الزفر وطربوش الملك باستخدام طريقة DPPH. و قد أظهرت النتائج أن خالصات الكحول اإليثيلى )%70( للنباتين قد أبدتا نشا ًطا ضعي ًفا كمضاد لألكسدة بالمقارنة مع حمض الجاليك المستخدم كمعيار لقياس النشاط المضاد لألكسدة. ب. تقييم النشاط السام للخاليا أظهرت نتائج تقييم النشاط السام للخاليا أن خالصة الكحول اإليثيلى )%70( ألوراق نبات الياسمين الزفر قد أبدت نشا ًطا سا ًما للخاليا في المختبر ضد خطوط الخاليا السرطانية الثالثة )الرئة، الكبد و الثدى( في حين أن خالصة الكحول اإليثيلى )%70( ألوراق نبات طربوش الملك قد أظهرت نشا ًطا قويًا ضد خط خاليا سرطان الرئة و لكن لم تبدي أي نشاط سام للخاليا ضد أي من خطوط خاليا سرطان الكبد أو الثدى. و أظهرفحص السمية الخلوية تجاه الخاليا الليفية الطبيعية أن لكلتا الخالصتين نفس النشاط السام للخاليا تقريبا مما يعني أن لديهم خصائص السالمة ذاتها بالتقريب. ج. تقييم النشاط المضاد للبكتيريا ضد بعض السالالت البكتيرية المقاومة للمضادات الحيوية 1. تقييم النشاط المضاد للبكتيريا ضد المكورات العنقودية الذهبية المقاومة للميثيسيلين )MRSA( تم تقييم النشاط المضاد للبكتيريا لخالصات الكحول اإليثيلى )% (70 ألوراق نباتى الياسمين الزفر وطربوش الملك ضد المكورات العنقودية الذهبية المقاومة للميثيسيلين باستخدام مقايسة MTT. أظهرت النتائج أن خالصة الكحول اإليثيلى )%70( ألوراق نبات الياسمين الزفر قد أبدت نشا ًط ا متوس ًطا بينما أبدت خالصة الكحول اإليثيلى )%70( ألوراق نبات طربوش الملك نشا ًطا ضعي ًفا جدًا كمضاد للبكتيريا ضد المكورات العنقودية الذهبية المقاومة للميثيسيلين مقارنة بالدواء المرجعى، فانكومايسين. 2. تقييم النشاط المضاد للبكتيريا المسببة لمرض السل تم تقييم النشاط المضاد للبكتيريا المسببة لمرض السل لخالصات الكحول اإليثيلى )% ( 70 ألوراق نباتى الياسمين الزفر وطربوش الملك باستخدام فحص (MABA). أظهرت خالصة الكحول اإليثيلى )%70( ألوراق نبات الياسمين الزفر نشا ًط ا متوس ًط ا كمضاد للبكتيريا المسببة لمرض السل بينما أظهرت خالصة الكحول اإليثيلى )%70( ألوراق نبات طربوش الملك نشا ًطا ضعي ًفا جدًا كمضاد للبكتيريا المسببة لمرض السل مقارنة بالدواء المرجعي، أيزونيازيد.

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الملخص العربى الجزء الثانى: دراسة فايتوكيميائية و بيولوجية ألوراق نبات الياسمين الزفر كما اتضح في الجزء األول، أن نسب المستخلصات المستخرجة من أوراق نبات الياسمين الزفر كانت أعلى بكثير من مثيالتها فى نبات طربوش الملك. كما أظهر فحص هذه المستخلصات بواسطة كروماتوجرافيا الطبقة الرقيقة وأي ًضا التحليل األيضى المقارن ألوراق كال النوعين بواسطة كروماتوجرافيا السائل فائقة األداء المقرونة بمطياف الكتلة صورة كروماتوغرافية أكثر ثرا ًء ا في أوراق الياسمين الزفر مقارنة بأوراق طربوش الملك. إضافة إلى ذلك، كشف التقدير الكمي للمحتويات الكلية للفينوالت والفالفونويدات لألوراق التي تم فحصها عن وجودها بكميات أعلى في أوراق الياسمين الزفر. من الناحية البيولوجية أي ًضا، أظهر التقييم داخل المختبر للنشاط السام للخاليا والنشاط المضاد للجراثيم ألوراق النباتين محل الدراسة نشا ًطا سا ًما للخاليا ذا نطاق أوسع وأي ًضا كفاءة أقوى كمضاد للجراثيم )ضد كل من السل و MRSA( لخالصة الكحول اإليثيلى )%70( ألوراق الياسمين الزفر . ونتيجة لذلك، تم اختيار أوراق الياسمين الزفر إلجراء مزيد من الدراسات الكيميائية النباتية والبيولوجية التالية المكثفة عليها.

الفصل األول: تحسين استخالص المكونات الفينولية من أوراق نبات الياسمين الزفر في هذا الفصل، تم تطوير تقنية تجزئة سائلة/صلبة بمساعدة الموجات فوق الصوتية لتحسين استخالص المكونات الفينولية من خالصة الكحول اإليثيلى )%70( ألوراق الياسمين الزفر منزوعة الدهون بنسب أعلى. تمت مقارنة كفاءة الطريقة مع تجزئة السائل/السائل التقليدية من حيث نسب المستخلصات الناتجة عن التجزئة و أي ًضا من حيث المحتويات الكلية للفينوالت و الفالفونويدات لكل من المستخلصات الناتجة عن طريقتى التجزئة. كشفت النتائج أن إجمالي العائد من المستخلصات العضوية التي تم الحصول عليها بواسطة تقنية السائل/ الصلبة المقترحة )6.8 جم( تجاوز إلى حد بعيد تلك الناتجة عن تقنية تجزئة السائل/السائل )2.1 جم(. على الرغم من أن المحتوى الكلى للفينوالت فى مستخلصات خالت اإليثيل/ بيوتانول )1:2( و ثنائي كلورو ميثان الناتجة عن تقنية التجزئة التقليدية كانت أعلى من تلك الخاصة بالمستخلصات المماثلة الناتجة عن تقنية التجزئة المقترحة إال أن التقنية المقترحة قدمت مستخل ًص ا إضافيًا من الميثانول أدى إلى رفع المحتوى الكلى للفينوالت للمستخلصات الناتجة عن الطريقة المقترحة مجتمعة بالمقارنة بتلك الناتجة عن التقنية التقليدية. من ناحية أخرى، عند مقارنة المحتوى الكلى للفالفونويدات للمستخلصات المختلفة، و جد أن المحتوى الكلي للفالفونويدات لمستخلص خالت اإليثيل/ بيوتانول )1:2( الناتج عن التقنية المقترحة كان أعلى بكثير من نظيره الناتج عن التقنية التقليدية. باإلضافة إلى ذلك، أدى وجود مستخلص الميثانول في التقنية المقترحة إلى زيادة المحتوى الكلى للفينوالت للمستخلصات الناتجة عن الطريقة المقترحة مجتمعة إلى مستوى أعلى بكثير من تلك الناتجة عن تقنية التجزئة التقليدية. تدعم هذه النتائج مدى مالئمة التقنية السائلة/الصلبة المقترحة الستخالص المكونات الفينولية بعائد أعلى من اوراق نبات الياسمين الزفر.

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الملخص العربى الفصل الثانى: فحص المستخلصات المختلفة أ. فصل المكونات الرئيسية بخالصة الهكسان و التعرف عليها تمت تجزئة خالصة الهكسان ألوراق نبات الياسمين الزفر بواسطة الكروماتوجرافيا السائلة تحت ضغط منخفض )VLC( على عمود من السيليكا H. لعزل وتنقية المركبات الفردية، تمت تجزئة األجزاء الفرعية التي تم الحصول عليها لفصل كروماتوجرافي إضافي بواسطة الكروماتوجرافيا السائلة تحت ضغط منخفض على عمود من هالم السيليكا H متبوعة بكروماتوجرافيا العمود )CC( على هالم السيليكا 60.

تم فصل المركبين H1 و H2 وتنقيتهما. استند تحديد هويتهما على تحديد بياناتهما الفيزيائية والكروماتوغرافية والطيفية، باإلضافة إلى المقارنة مع العينات األصلية المتاحة و/أو مع البيانات المنشورة.

تم تعريف المركبين المفصولين على أنهما بيتا كاروتين )H1( و 22- ديهيدروكليروستيرول )H2(. و يعد هذا التقرير هو األول لفصل البيتا كاروتين من نبات الياسمين الزفر. ب. فصل المكونات الرئيسية بمستخلص ثنائى كلورو الميثان و التعرف عليها تم تجزئة مستخلص ثنائي كلورو الميثان بواسطة الكروماتوجرافيا السائلة تحت ضغط منخفض على عمود من السيليكا H بواسطة مذيبات متدرجة القطبية. عند تركيز الجزء الفرعي المزال مع 90% من خالت اإليثيل في ثنائي كلورو الميثان، ترسبت مادة صلبة بيضاء وتم فصلها من الجزء الفرعى المر َّكز باستخدام الطرد المركزي ثم تنقيتها بعد ذلك وتكون المركب .D1

تم تحديد هوية المركب D1 على أنه 22- ديهيدروكليروستيرول O-3--جلوكوزايد، بنا ًء ا على تحديد البيانات الفيزيائية والكيميائية والكروماتوغرافية والطيفية، وبالمقارنة مع العينات األصلية المتاحة و/أو مع البيانات المنشورة. وهذا هو التقرير األول أي ًضا لفصل هذا المركب من نبات الياسمين الزفر. ج. فصل المكونات الرئيسية بمستخلص خالت االيثيل/ بيوتانول )1:2( و التعرف عليها تمت تجزئة مستخلص خالت اإليثيل/بيوتانول )1:2( بواسطة الكروماتوجرافيا السائلة تحت ضغط منخفض على عمود الطور المعكوس باستخدام ميثانول:ماء بنسب متدرجة. تم رصد األجزاء الفرعية الناتجة بواسطة كروماتوجرافيا الطبقة الرفيعة وكروماتوجرافيا السائل عالية االداء.

تم اختيار اثنين من األجزاء الفرعية الناتجة لفصل المكونات الرئيسية بها. تم فصل وتنقية المركبين E1 و E2 وتحديد هويتهما من خالل تحديد بياناتهما الفيزيائية والكروماتوغرافية والطيفية والمقارنة مع العينات األصلية المتاحة و/ أو مع البيانات المنشورة.

تم التعرف على المركبين المفصولين على أنها 4’-ميثيل سكوتيالرين )E1( وحمض كليروديرميك )E2(.

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الملخص العربى ج. فصل المكونات الرئيسية بمستخلص الميثانول و التعرف عليها تمت تجزئة مستخلص الميثانول بواسطة الكروماتوجرافيا السائلة تحت ضغط منخفض على عمود الطور المعكوس باستخدام ميثانول:ماء بنسب متدرجة. تم رصد األجزاء الفرعية الناتجة بواسطة كروماتوجرافيا الطبقة الرفيعة وكروماتوجرافيا السائل عالية االداء.

تم فصل المركب M1 و تنقيته من الجزء الفرعى المجمع و المزال بواسطة 35% من الميثانول في الماء. استند تحديد هوية المركب M1 إلى تحديد بياناته الفيزيائية والكروماتوغرافية والطيفية والمقارنة مع العينات األصلية المتاحة و/ أو البيانات المنشورة.

تم التعرف على المركب M1 على أنه فيرباسكوزايد. الفصل الثالث: التقييم البيولوجي لمختلف المستخلصات والمركبات المفصولة أ. تقييم النشاط المضاد لألكسدة تم تقييم النشاط المضاد لألكسدة للمستخلصات المختلفة ألوراق نبات الياسمين الزفر باستخدام طريقة DPPH. أظهرت النتائج أن مستخلص خالت اإليثيل/بيوتانول )1:2( قد أظهرأقوى نشاط مضاد لألكسدة بين المستخلصات األخرى المشتقة من خالصة الكحول اإليثيلى )% (70 لألوراق، متبو ًعا بمستخلص الميثانول بينما كان مستخلص ثنائى كلورو الميثان األقل نشا ًطا. وقد تمت مقارنة جميع النتائج بحمض الجاليك.

ب. تقييم النشاط السام للخاليا تم تقييم النشاط السام للخاليا في المختبر لخالصة الهكسان إلى جانب المستخلصات المختلفة المشتقة من خالصة الكحول اإليثيلى )%70( لألوراق ضد خطوط خاليا سرطان الرئة والكبد والثدى. وفي الوقت نفسه، تم تقييم النشاط السام للخاليا للمركبات المفصولة ضد خطوط خاليا سرطان الرئة والكبد. تم تحديد سالمة المستخلصات والمركبات المذكورة أعاله من خالل فحص السمية الخلوية تجاه خط خاليا الليفية الطبيعية. أظهرت النتائج أن مستخلص خالت اإليثيل/بيوتانول )1:2( كان األعلى نشا ًط ا بين المستخلصات المختبرة ضد خط خاليا سرطان الكبد متبو ًعا بخالصة الهكسان ثم مستخلصات الميثانول و ثنائى كلورو الميثان. فيما يتعلق بخط خاليا سرطان الرئة كان مستخلص ثنائى كلورو الميثان األكثر فاعلية يليه مستخلص الميثانول وخالصة الهكسان، بينما وجد أن مستخلص خالت اإليثيل/بيوتانول )1:2( غير نشط. من ناحية أخرى، لم يكن ألى من هذه العينات أي تأثير على خط خاليا سرطان الثدي باستثناء مستخلص ثنائي كلورو الميثان الذي أظهر نشا ًطا ضعي ًفا للغاية. أظهرت خالصة الهكسان أضعف نشاط سام للخاليا على الخط خاليا الليفية الطبيعية وبالتالي تم اعتبارها األكثر أما ًنا بين جميع العينات التي تم اختبارها، يليها مستخلصات ثنائى كلورو الميثان، ميثانول ثم خالت اإليثيل/بيوتانول )1:2(. أما بالنسبة للمركبات المفصولة، فقد أظهر بيتا كاروتين أعلى نشاط سام للخاليا ضد خط خاليا سرطان الرئة بينما كان حمض كليروديرميك األكثر كفاءة ضد خط خاليا سرطان الكبد مقارنة بالدواء المرجعي، دوكسوروبيسين.

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الملخص العربى عند اختبار السمية الخلوية للمركبات المفصولة على الخاليا الليفية الطبيعية، وجد أن فيرباسكوزايد و 4'- ميثيل سكوتالرين هما األكثر أما ًنا يليهما البيتاكاروتين. وفي الوقت نفسه، كان حمض كليروديرميك األكثر سمية تجاه هذه الخاليا. الفصل الرابع: إنشاء طريقة لمعايرة الخالصات الفعالة باستخدام كروماتوجرافيا السائل عالية األداء والتأكد من صحة وفعالية هذه الطريقة تم إجراء التحليل على عمود LiChrospher 100 RP-C18 باستخدام نسب متدرجة بخليط من أسيتونيتريل : حمض الفوسفوريك 0.3% مع كاشف األشعة فوق البنفسجية المحدد في 325 نانومتر. استخدم حمض كليروديرميك المفصول كمعيار. تم التحقق من صحة الطريقة وكانت جميع العوامل ضمن حدود القبول حيث ثبت أن هذه الطريقة بسيطة ودقيقة وخطية وقابلة للتكرار و انتقائية و قوية و مستقرة و موفرة للوقت وبالتالي موثوقة لتحديد تركيز حمض كليروديرميك في خالصة الكحول اإليثيلى )%70( ألوراق الياسمين الزفر. الفصل الخامس: دراسة تأثير مراحل تطور النبات على التركيب الكيميائي لألوراق تم تقييم تأثير مراحل تطور النبات على التركيب الكيميائي لخالصة الكحول اإليثيلى )%70( ألوراق الياسمين الزفر. تم جمع عينات من األوراق من النبات قبل و أثناء و بعد مرحلة اإلزهار، تم تجفيف األوراق بالهواء، وطحنها ثم إزالة الدهون و استخالص األوراق منزوعة الدهون باستخدام الكحول اإليثيلى )70%( وتم تحليل الخالصات المحضرة من عينات األوراق المجمعة فى المراحل المختلفة بواسطة كروماتوجرافيا السائل عالية االداء. أشارت النتائج إلى أن إنتاج المكونات الرئيسية وهي؛ فيرباسكوزايد، 4' ميثيل سكوتالرين وحمض كلوديرميك يتأثر بشدة اختالف مراحل تطورالنبات. سجلت أعلى محتويات هذه المكونات في عينات األوراق التي تم جمعها أثناء مرحلة اإلزهار؛ نتيجة لذلك، من الناحية االقتصادية والصيدالنية على حد سواء، ي فضل أن يتم جمع أوراق نبات الياسمين الزفر في مرحلة اإلزهار لضمان أفضل عائد من المكونات النشطة حيوي ًا.

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المستخلص العربى المستخلص العربى نباتا الياسمين الزفر و طربوش الملك، هما من أعضاء العائلة الشفوية و تمت زراعتهما بنجاح في مصر كنباتات للزينة. تهدف الدراسة الحالية إلى تقييم النباتات المحلية تمهيدا إلدخالما في الصناعات الدوائية، مما يستلزم إجراء تحقيقات مكثفة من ناحية السالمة والفعالية الحيوية للنباتات المذكورة. تم وضع معايير لوصف و/أو التمييز بين النوعين عن طريق التوصيف النباتي، التحليل التقريبي، الفحص الكيميائي النباتي وتحليل كروماتوجرافيا السائل فائقة األداء المقرونة بطيف الكتلة. تمت دراسة األوراق بيولوجيًا وكيميائيًا دراسة مقارنة لتحديد األنسب من الناحية الطبية واالقتصادية. في هذا الصدد، تم تقييم األنشطة البيولوجية للنباتين، و تضمن ذلك تقييم النشاط المضاد لألكسدة والنشاط المضاد للميكروبات وكذلك التأثير المثبط لنمو الخاليا السرطانية الخاصة بسرطان الكبد والرئة والثدى لخالصة الكحول اإليثيلى )%70( ألوراق النباتين المذكورين، وفي الوقت نفسه، تم فحص التركيب الكيميائي لألوراق من خالل التحليالت النوعية والكمية للمكونات الفينولية. في هذا الصدد، تم اختيار أوراق نبات الياسمين الزفر إلجراء المزيد من البحث الكيميائي النباتي والبيولوجي، عالوة على ذلك، تم اقتراح إجراء تقنية تجزئة سائلة/صلبة لخالصة الكحول اإليثيلى )% ( 70 ألوراق الياسمين الزفر لتحسين استخراج المكونات الفينولية ومقارنتها بطريقة تجزئة السائل/السائل التقليدية لتسميل دمج مستخلصات أوراق الياسمين الزفر في المستحضرات العشبية وتم أي ًضا إنشاء طريقة بسيطة وصحيحة وموفرة للوقت لمعايرة المستخلصات الفعالة و لتحديد محتوى حمض كليروديرميك فى هذه المستخلصات باستخدام كروماتوجرافيا السائل عالية األداء. و لزيادة إنتاجية المكونات النشطة حيوياً في مستخلص اإليثانول من أوراق الياسمين الزفر، تم تقييم تأثير مراحل تطور النبات على التركيب الكيميائى لألوراق. أظهرت النتائج أن خالصة الكحول اإليثيلى )%70( ألوراق الياسمين الزفر كانت أكثر فعالية من خالصة الكحول اإليثيلى )%70( ألوراق طربوش الملك. تم فصل ست مركبات من أوراق الياسمين الزفر وهم بيتا كاروتين ، 22-ديهيدروكليروستيرول، و -22 ديهيدروكليروستيرول O-3-- جلوكوزايد ، ’4 -ميثيل سكوتيالرين و فيرباسكوزايد. اثنان من هذه المركبات وهما بيتا كاروتين و 22- ديهيدروكليروستيرول O-3--جلوكوزايد تم فصلهما ألول مرة من هذا النبات فى هذه الدراسة. أخي ًرا، كشفت دراسة تأثير مراحل تطور النبات على التركيب الكيميائي لألوراق أن أعلى محتويات للمكونات الرئيسية تم تسجيلها في عينات األوراق التي تم جمعها أثناء مرحلة اإلزهار، وبالتالي ي فضل جمع أوراق الياسمين الزفر أثناء مرحلة اإلزهار لضمان أفضل عائد من المكونات النشطة حيويًا. الكلمات الدالة: الياسمين الزفر، طربوش الملك، التوصيف النباتى، النشاط الحيوي، التنميط الفينولي، معايرة بكروماتوجرافيا السائل عالية الجودة، التحقق من الصحة، تأثير مراحل تطور النبات.

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Arabic Abstract

دراسة عقاقيرية مقارنة لبعض أنواع كليرودندرم )العائلة الشفوية( المزروعة فى مصر

رسالة مقدمة من

أسماء محمد أحمد خليل

للحصول على درجة الماجستير فى العلوم الصيدلية )عقاقير(

تحت اشراف كل من

أ.د. سهير محمد الزلبانى أ.د. هشام إبراهيم العسكرى أستاذ العقاقير أستاذ العقاقير كلية الصيدلة-جامعة القاهرة كلية الصيدلة-جامعة القاهرة

أ.م.د. عمر محمد صبرى أستاذ العقاقير المساعد كلية الصيدلة-جامعة القاهرة

قسم العقاقير كلية الصيدلة-جامعة القاهرة جمهورية مصر العربية 2019