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STUDIES ON CHEMICAL CONSTITUENTS AND BIOLOGICAL ACTIVITIES OF CONOCARPUS LANCIFOLIUS (COMBRETACEAE)

MALIK SAADULLAH 01-PhDC-12

Session 2012-2015

A thesis submitted in Partial fulfillment of the requirements for the degree of Doctorate of Philosophy in Pharmaceutical Chemistry

FACULTY OF PHARMACY

BAHAUDDIN ZAKARIYA UNIVERSITY, MULTAN

FACULTY OF PHARMACY

BAHAUDDIN ZAKARIYA UNIVERSITY, MULTAN

CERTIFICATE

This is to certify that the thesis entitled, “STUDIES ON CHEMICAL

CONSTITUENTS AND BIOLOGICAL ACTIVITIES OF CONOCARPUS

LANCIFOLIUS (COMBRETACEAE)” submitted by Mr. MALIK SAADULLAH has been thoroughly studied. It is found to be prolific in scope and quality as a thesis for the award of degree of Doctor of Philosophy in Pharmacy (Pharmaceutical Chemistry).

(Prof. Dr. Bashir Ahmad Ch.)

Supervisor

Dedicated to my Beloved Ones

ACKNOWLEDGEMENT

I have no words at my command to express my deepest sense of gratitude to Almighty Allah, the compassionate, the merciful, who enabled me to complete my research work. My special praises are for Holy Prophet, Hazarat Muhammad (Peace Be Upon Him) who is, forever, a source of guidance and knowledge for the whole humanity. It is a matter of great pleasure and honour for me to express my heartiest gratitude and appreciation to my respectable and honourable supervisor Dr. Bashir Ahmad Ch. Professor of Pharmaceutical chemistry Faculty of Pharmacy, Bahauddin Zakariya University, Multan, for his valuable and ever inspiring guidance, persistent encouragement and sagacious advice provided to me throughout this research work. With a deep sense of gratitude I communicate my heartful gratifications to Dr. Muhammad Uzair professor of pharmaceutical chemistry Faculty of Pharmacy, Bahauddin Zakariya University, Multan for his encouragement, useful suggestions and inspiring help during the conduct of my research work. I am grateful to Dr. Shazia Anjum Professor, Department of Chemistry, The Islamia University Bahawalpur for her kind guidance and help in my research work. I am indebted of heartiest thanks and deepest regards for lab staff for their help and immense cooperation for providing me chemicals and equipments.

MALIK SAADULLAH

BRIEF CONTENTS

Serial No. Contents Page No.

1 List of Contents І

2 List of Abbreviations V

3 List of Figures vіі

4 List of Tables Іx

5 Aims of the present work 1

6 Abstract 2

7 Introduction 6

8 Literature Review 21

9 Materials and Methods 75

10 Results and Discussions 98

11 References 158

LIST OF CONTENTS

S.No Desription Page

AIM OF THE PRESENT WORK 1 ABSTRACT 2 1 Introduction 6 1.1 Different medicine systems 9 1.1.1 European system of medicines 9 1.1.2 Chinese system of medicine 9 1.1.3 Egyptian system of medicine 9 1.1.4 Ayurvedic System of Medicine 9 1.1.5 Unani System of Medicines 9 1.2 Herbal Medicines in Islam 10 1.3 Importance of Herbal Medicines 10 1.4 Herbal Medicines Today 10 1.5 Introduction to family combretaceae 13 1.6 Botanical aspects of genus Conocarpus 13 1.7 Conocarpus lancifolius 13 1.7.1 Botanical classification of Conocarpus lancifolius 14 1.8 Secondary metabolites 14 1.8.1 Alkaloids 16 1.8.2 Steroids 17 1.8.3 Terpenoids 18 1.8.4 Tannins 18 1.8.5 Glycosides 19

2 Literature review 21 2.1 Previous reports on the phytochemical studies from various species 21 of genera Terminalia, Combretum and Conocarpus. 2.2 Previous reports on the biological studies from various species of 53 genera Terminalia, Combretum and Conocarpus.

3 Materials and Methods 75 3.1 Plant material 75 3.2 Solvents and chemicals 75 3.3 Preparation of reagents and solutions 75 3.4 Equipments/ instruments/ glasswares 77 3.5 Phytochemical methods 78 3.5.1 Extraction 78 3.5.2 Phytochemical screening 78 3.5.3 Techniques for chromatography 81 3.5.3.1 Thin layer chromatography 81 3.5.3.2 Column chromatography 83 3.6 Biological methods 83 3.6.1 Brine shrimp lethality assay 83 3.6.2 Antibacterial assay 84 3.6.3 Antifungal assay 85 3.6.4 Phytotoxic bioassay 85 3.6.5 Leishmanicidal assay 86 3.6.6 Antioxidant assay 86 3.6.7 Acetylcholinesterase assay 86 3.6.8 Butyrylcholinesterase assay 87 3.6.9 α- Chymotrypsin assay 87 3.6.10 Lipoxygenase assay 88 3.6.11 Urease assay 88 3.6.12 Alpha glucosidase assay 88 3.7 Spectroscopic methods 89 3.8 Physical and spectroscopic data of isolated compounds A-G 90 4 Results and discussions 98 4.1 Phytochemical studies of Conocarpus lancifolius 98 4.1.1 Extraction 98 4.1.2 Phytochemical screening for secondary metabolites 98 4.2 Isolation of compounds A-G 99 4.3 Structural elucidation of isolated compounds 102 4.4 Biological studies of Conocarpus lancifolius 140

4.4.1 Acetylcholinesterase inhibition activities 140 4.4.2 Butyrylcholinesterase inhibition activities 141 4.4.3 Lipoxygenase inhibition activities 141 4.4.4 Carbonic anhydrase inhibition activities 142 4.4.5 Xanthine oxidase inhibition activities 142 4.4.6 β-glucuronidase inhibition activities 143 4.4.7 α-Chymotrypsin inhibition activities 144 4.4.8 Urease inhibition activities 144 4.4.9 Alpha glucosidase inhibitory activities 145 4.4.10 Blood glucose level 146 4.4.11 Hypolipidemic effect 146 4.4.12 Effect on liver function tests (SGPT & SGOT values) 148 4.4.13 In vitro screening on isolated tissue preparation 148 4.4.14 Antimicrobial activity 149 4.4.15 Antifungal activity 150 4.4.16 Phytotoxic assay 150 4.4.17 Brine shrimp lethality assay 151 4.4.18 Antileishmanial activity 152 4.4.19 Antioxidant activity 152 4.5 Total Flavonoid Contents 153 4.6 Quantification of phenolic contents by HPLC 153 4.7 Evaluation of biological activities of isolated compounds 154 5 References 158

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LIST OF ABBRIVIATIONS

µl Microlitre µm Micrometer 1, 2, 3, 4, 5, 6,------Symbols for compounds reported from literature 12(S)-HETE 12-hydroxyeicosatetraenoic acid 12-HHTrE 12-hydroxyheptadecatrienoic acid 13C-NMR Carbon13 nuclear magnetic resonance spectroscopy 1H-NMR Proton nuclear magnetic resonance spectroscopy 5(S)-HETE 5-hydroxyeicosatetraenoic acid A, B, ----- Symbols for compounds isolated in this study AA Arachidonic acid ALP Alkaline phosphatase ALT Alanine transaminase AST Aspartate transaminase BHT Butylated hydroxy toluene BP British pharmacopoeia CCL4 Carbon tetrachloride DCM Dichloromethane DEPT Distortionless enhancement by polarization transfer DMSO Dimethyl sulfoxide DNFB Dinitrofluorobenzene DPPH Diphenyl picryl hydrazide E.coli Escherchia coli Edn Edition EIMS Electron impact mass spectrometry EtOH Ethanol HLE Human leukocyte elastase HMBC Heteronuclear multiple bond correlation HPLC High performance liquid chromatography HR- EIMS High resolution electron impact mass spectrometry HSQC Heteronuclear spin quantum correlation

IC50 Inhibitory concentration50 iv

LCT Long chain triglyceride

LD50 Lethal dose50

LTB4 Leukotriene B4 MeOH Methanol MHZ Megahertz MIC Minimum inhibitory concentration. mmol/L Millimole per litre nm Nanometer NOE Nuclear overhauser effect PMNL Polymorphonuclear leukocytes PMSF Phenyl methyl sulfonyl fluoride SGOT Serum glutamate oxaloacetate transaminase SGPT Serum glutamate pyruvate transaminase TLC Thin layer chromatography TPA Tetradecanoylphorbol acetate USP United state pharmacopoeia UV Ultraviolet

LIST OF FIGURES

S. No. Description Page Figure 1.1 Aerial view of the plant Conocarpus lancifolius 14 Figure 1.2 Interrelationships of biosynthetic pathways leading to 15 secondary constituents in plants. Figure 2.1 Chemical structures of flavonoids (1-51) reported from 23 various species of genera Terminalia, Combretum and Conocarpus. Figure 2.2 Chemical structures of terpens (52-119) reported from various 30 species of genera Terminalia, Combretum and Conocarpus. Figure 2.3 Chemical structures of tannins (120-138) reported from 40 various species of genera Terminalia, Combretum and Conocarpus Figure 2.4 Chemical structures of glycosides (139-164) reported from 46 various species of genera Terminalia, Combretum and Conocarpus. Figure 2.5 Chemical structures of mislinious compounds (165-178) 51 reported from various species of genera Terminalia, Combretum and Conocarpus Figure 4.1 Isolation of compounds A-D from the dichloromethane 100 extract of Conocarpus lancifolius. Figure 4.2 Isolation of compounds E-G from the methanol extract of 101 Conocarpus lancifolius. Figure 4.3 UV spectrum of compound A 103 Figure 4.4 IR spectrum of compound A 103 Figure 4.5 1H-NMR spectrum of compound A 104 Figure 4.6 13C-NMR (BB) spectrum of compound A 104 Figure 4.7 13C NMR (dept) spectrum of compound A 105 Figure 4.8 EI spectrum of compound A 105 Figure 4.9 FAB+ spectrum of compound A 106 Figure 4.10 FAB- spectrum of compound A 106 Figure 4.11 UV spectrum of compound B 108

Figure 4.12 IR spectrum of compound B 108 Figure 4.13 1H-NMR spectrum of compound B 109 Figure 4.14 13C-NMR (BB) spectrum of compound B 109 Figure 4.15 13C NMR (dept) spectrum of compound B 110 Figure 4.16 EI spectrum of compound B 110 Figure 4.17 FAB+ spectrum of compound B 111 Figure 4.18 COSY spectrum of compound B 111 Figure 4.19 HSQC spectrum of compound B 112 Figure 4.20 UV spectrum of compound C 114 Figure 4.21 IR spectrum of compound C 114 Figure 4.22 1H-NMR spectrum of compound C 115 Figure 4.23 13C-NMR (BB) spectrum of compound C 115 Figure 4.24 13C NMR (dept) spectrum of compound C 116 Figure 4.25 EI spectrum of compound C 116 Figure 4.26 FAB+ spectrum of compound C 117 Figure 4.27 FAB- spectrum of compound C 117 Figure 4.28 COSY spectrum of compound C 118 Figure 4.29 HSQC spectrum of compound C 118 Figure 4.30 UV spectrum of compound D 120 Figure 4.31 IR spectrum of compound D 120 Figure 4.32 1H-NMR spectrum of compound D 121 Figure 4.33 13C-NMR (BB) spectrum of compound D 121 Figure 4.34 13C NMR (dept) spectrum of compound D 122 Figure 4.35 EI spectrum of compound D 122 Figure 4.36 FAB- spectrum of compound D 123 Figure 4.37 COSY spectrum of compound D 123 Figure 4.38 HSQC spectrum of compound D 124 Figure 4.39 UV spectrum of compound E 126 Figure 4.40 IR spectrum of compound E 126 Figure 4.41 1H-NMR spectrum of compound E 127 Figure 4.42 13C-NMR (BB) spectrum of compound E 127 Figure 4.43 13C NMR (dept) spectrum of compound E 128 Figure 4.44 EI spectrum of compound E 128

vii

Figure 4.45 UV spectrum of compound F 130 Figure 4.46 IR spectrum of compound F 130 Figure 4.47 1H-NMR spectrum of compound F 131 Figure 4.48 13C-NMR (BB) spectrum of compound F 131 Figure 4.49 13C NMR (dept) spectrum of compound F 132 Figure 4.50 EI spectrum of compound F 132 Figure 4.51 UV spectrum of compound G 134 Figure 4.52 IR spectrum of compound G 134 Figure 4.53 1H-NMR spectrum of compound G 135 Figure 4.54 13C-NMR (BB) spectrum of compound G 135 Figure 4.55 13C NMR (dept) spectrum of compound G 136 Figure 4.56 EI spectrum of compound G 136 Figure 4.57 FAB+ spectrum of compound G 137 Figure 4.58 COSY spectrum of compound G 137 Figure 4.59 HSQC spectrum of compound G 138 Figure 4.60 Structures of isolated compounds A-G from Conocarpus 139 lancifolius Figure 4.61 Total Flavonoids contents of Conocarpus lancifolius. 153 Figure 4.62 Calibration curve for quantification of phenolic contents of 154 Conocarpus lancifolius

viii

LIST OF TABLES S. No. Description Page Table 1.1 Herbal medicine in Islam 7 Table 1.2 Major plant drugs for which no synthetic one is currently available 11 Table 2.1 Flavonoids reported from various species of genera Terminalia, 21 Combretum and Conocarpus. Table 2.2 Terpenes reported from various species of genera Terminalia, 28 Combretum and Conocarpus Table 2.3 Tannins reported from various species of genera Terminalia, 38 Combretum and Conocarpus. Table 2.4 Glycoside reported from various species of genera Terminalia, 44 Combretum and Conocarpus. Table 2.5 Mislinious compound reported from various species of genera 50 Terminalia, Combretum and Conocarpus. Table 3.1 Solvent systems used for TLC analysis of dichloromethane extracts. 82 Table 3.2 Solvent systems used for TLC analysis of methanol extracts. 82 Table 4.1 Results of extraction of Conocarpus lancifolius. 98 Table 4.2 Results of phytochemical screening of Conocarpus lancifolius. 98 Table 4.3 Results of acetylcholinesterase inhibition activity of Conocarpus 140 lancifolius. Table 4.4 Results of butyryl cholinesterase inhibition activity of Conocarpus 141 lancifolius. Table 4.5 Results of lipoxygenase inhibition activity of Conocarpus lancifolius 141 Table 4.6 Results of carbonic anhydrase activity of Conocarpus lancifolius. 142 Table 4.7 Results of xanthine oxidase inhibition activity of Conocarpus 143 lancifolius. Table 4.8 Results of β-glucuronidase activity extracts of Conocarpus lancifolius. 143 Table 4.9 Results of α-Chymotrypsin inhibition activities of extracts of Conocarpus 144 lancifolius Table 4.10 Results of Urease activity of extracts of Conocarpus lancifolius. 144

Table 4.11 Results of α- glucosidase inhibitory activities of extracts of Conocarpus 145 lancifolius. Table 4.12 Effect of Conocarpus lancifolius extract on Blood Glucose level in 146 Alloxan induced diabetic rabbits. Table 4.13 Effect of Conocarpus lancifolius extract on Total Cholesterol level in 146 Alloxan induced diabetic rabbits. Table 4.14 Effect of Conocarpus lancifolius extract on Triglyceride (mg/dl) level in 147 Alloxan induced diabetic rabbits. Table 4.15 Effect of Conocarpus lancifolius extract on HDL and LDL(mg/dl) 147 Table 4.16 Effect of Conocarpus lancifolius extract on Serum SGPT and SGOT 148 (IU/L) in Alloxan induced diabetic rabbits. Table 4.17 Results of antibacterial activities of different extracts of Conocarpus 149 lancifolius at four different concentrations (D1, D2, D3 and D4) Table 4.18 Results of antifungal activities of dichloromethane and methanol extracts 150 of Conocarpus lancifolius. Table 4.19 Results of antifungal activities of dichloromethane and methanol extracts 150 of Conocarpus lancifolius. Table 4.20 Results of In vitro phytotoxic bioassay of dichloromethane and 151 methanol extracts of Conocarpus lancifolius. Table 4.21 Results of brine shrimp lethality bioassay of Conocarpus lancifolius 151 Table 4.22 Results of leishmanicidal activities of methanol extracts of Conocarpus 152 lancifolius Table 4.23 Results of DPPH radical scavenging activities of extracts 152 Conocarpus lancifolius. Table 4.24 Results phenolic contents by HPLC of Conocarpus lancifolius. 154 Table 4.25 Results of DPPH radical scavenging, AChE, BChE inhibition activities of 155 compounds A-G Table 4.26 Results of urease and α- glucosidase inhibition activities of isolated 156 Compounds A-G

AIM OF THE PRESENT WORK

Natural product chemistry serves as a backbone in the drug discovery process. A large number of therapeutically active compounds from plants are now commercially synthesized and available in the market. According to World Health Organization (WHO) more than 80% of the world’s population relies on plants for the treatment of various conditions (Koehn and Carter, 2005) Keeping in view the ethnomedicinal importance plant Conocarpus lancifolius was selected. As literature survey showed the pharmacological and phytochemical importance of genus of selected medicinal plant and the folk history of selected plant give the idea of their potential. A complete understanding of medicinal plants involves a number of disciplines including pharmaceutical chemistry, botany, organic chemistry, enzymology, pharmacology and quality control. The aim of this study is to explore the therapeutic potential of selected medicinal plant Conocarpus lancifolius in terms of biological activities such as antimicrobial, antiinflamatory, cardioprotective, anticancer, anthelmintic, hepatoprotective antileishmanial , phytotoxicity, spasmolytic and bronchodilator assays and enzyme inhibition assays i.e butyrylcholinesterase, alpha-chymotrypsin, urease, lipoxygenase and alpha-glucosidase inhibition assays as well as their phytochemical analysis by using modern isolation techniques and biological assays procedures means that new plant drugs usually find their way into medicine as purified substances rather than in the form of galenical preparation. As a part of research program into the exploitation of natural sources for their biological activities modern techniques for pharmacological and phytochemical studies. The biological activities of extracts and pure compounds will be tested and hope leads to novel bioactive compounds. Plants of the genus Conocarous are reported to contain secondary metabolites such as flavonoids, terpenes, lactones and glycosides. Inspite of the fact that the plant has traditionally been used for diabetes, intermittent fever, gout and asthma, its biological potential has never been explored. Plant extracts were prepared in different solvents by using different extraction techniques. Chromatographic techniques such as thin layer chromatography, open column chromatography have been applied for the isolation of compounds from the plant extracts by using different solvent systems. It needs to explore in detail by adopting new assay techniques such as assays for different enzyme inhibition and other assays of pharmacological importance.

1 ABSTRACT The research work was carried out for the phytochemical and biological studies of Conocarpus lancifolius (Combretaceae). Collected plant material was subjected for preliminary phytochemical screening and revealed the presence of cardiac glycosides, saponins, flavonoids, and tannins while alkaloids were absent. Dichloromethane and methanol extracts were prepared and screened for antibacterial activity, antifungal activity, brine-shrimp toxicity, phytotoxicity, antioxidant activity, α-chymotrypsin inhibitory activity, urease inhibitory activity, α-glucosidase inhibitory activity and butyrylcholinesterase inhibitory activity. Dichloromethane extract of Conocarpus lancifolius exhibited moderate antioxidant activity with a percentage inhibition of 42.06±0.64 mg/ml, while the percentage inhibition of methanol extract by DPPH method was 92.1± 0.1 with 396.205 IC50 value, percentage inhibition by nitric oxide scavenging activity was 93.35± 0.61 and percentage inhibition by ferric reducing antioxidant power method was 93.2± 1.02. Dichloromethane extract of Conocarpus lancifolius showed significant α- chymotrypsin inhibition activity with percentage inhibition of 92.20 ± 1.82 and 91.17

± 0.82 µg/ml IC50 value. Both the extracts of Conocarpus lancifolius exhibited significant lipoxygenase inhibition activity with percentage inhibition of 65.97 ± 0.55 and 63.89 ± 0.02 with IC50 value 158.61 ± 0.08 and 186.31±0.04 respectively. Methanol extract of Conocarpus lancifolius showed significant acetylcholinesterase inhibition activity with percentage inhibition of 87.48 ± 0.09 with 71.21 ± 0.11 IC50 value. Dichloromethane and methanol extracts of Conocarpus lancifolius have promising butyrylcholinesterase inhibition activity with percentage inhibition of

89.52±0.39 at 0.5 mg/ml and 89.06±0.89 with IC50 values 55.25±0.07 and 52.31± 0.11 respectively. Methanol extract of Conocarpus lancifolius was found toxic with

LD50 value of 115.76 (0.0048 - 13.76) µg/ml against Artemia salina. Methanol extract of Conocarpus lancifolius exhibited a significant antiurease activity having percentage inhibition value 81.1±1.82 with 49.1± 1.31 IC50 value. Dichloromethane extract of Conocarpus lancifolius showed carbonic anhydrase inhibition activity with

56.7 2.44 IC50 values using acetazolamide as standard. The aqueous ethanol extracts of Conocarpus lancifolius relaxed the spontaneous contractions in isolated rabbit jejunum preparations in dose dependant manner at 0.01- 3 mg/ml tissue bath concentrations. However aqueous ethanol extract of Conocarpus lancifolius relaxed K+ (80 mM) induced contractions in isolated rabbit jejunum

2 preparation and also relaxed carbachol (1μM) induced contraction in isolated rabbit tracheal preparations in dose dependant manner (0.1-3 mg/ml). It is to be reported that the observed response is likely to be mediated through either anti muscuranic activity or/ and blockade of voltage dependant Ca+2 channels. The aqueous ethanol extract of roots of Conocarpus lancifolius have shown a significant relaxant effect in isolated rabbit jejunum preparations with an EC50 value of 0.2075 mg/ml (95% CI: 0.0478- 0.9007). Methanol extract of Conocarpus lancifolius showed a significant α-glucosidase inhibitory activity with an inhibition (%) value of 79.56 and 5.59±0.06µg/ml IC50 value at even very low concentration of 0.0039 mg/ml. Acarbose which exhibit 92.23

% inhibition and 38.25 µg/ml IC50 value was used as standard. On the basis of in vitro α-glucosidase inhibitory activity in vivo study was also carried out on rabbits. Methanol extract of Conocarpus lancifolius significantly decreases the blood glucose level in alloxan induced diabetic rabbits. The pharmaceutical industry is facing serious challenges for drug discovery process. The available drugs for various diseases are extremely expensive, riskier, and critically inefficient. Keeping in view these facts isolation of compounds from dichloromethane and methanol extracts was carried out and seven compounds Quercetin 3-O-glucuronide (A), S-4-isopropyl-8-methoxy-6-thia-bicyclo[3.2.1]oct-1- en-2-yl benzothioate (B), 2,3,4,8,9,10,11,13-octahydro-1H-phenanthro[3,2-c]oxocin- 3-yl benzoatem (C), 1,3,4,5,6,8-hexahydroxy-3,4,5,5a-tetrahydro-1H-benzo[g] isochromene-5-carboxylic acid (D),1-amino-1,5,7,8-tetramethoxy-1H- cyclopenta[a]naphthalene-2-carboxamide (E), 2,3,8 tri-O-methyl ellagic acid (F), 3- O-methyl ellagic acid 4-O- β -D glucopyranoside (G) were isolated. The chemical structures of isolated compounds (A-G) were established with the help of spectroscopic techniques such as ultraviolet-visible, infrared, proton nuclear magnetic resonance (1H-NMR), 13C NMR (BB, DEPT-135, 90), two dimensional correlation techniques (COSY, HSQC) and mass spectrometry. All compounds were isolated first time from genus conocarpus. The compounds S-4- isopropyl-8-methoxy-6-thia-bicyclo[3.2.1]oct-1-en-2-yl benzothioate (B), 2,3,4,8,9,10,11,13-octahydro-1H-phenanthro[3,2-c]oxocin-3-yl benzoate (C) and 1-amino-1,5,7,8-tetramethoxy-1H-cyclopenta[a]naphthalene-2-carboxamide (E) not only isolated for the first time from genus conocarpus but have noval structures. The S-4-isopropyl-8-methoxy-6-thia-bicyclo[3.2.1]oct-1-en-2-yl benzothioate compound

3 (B) was subjected to antimicrobial activity test against the clinical microbes to determine its biological activities. The zone of inhibition was observed to be between 20 to 24 mm with Bacillus Subtilis having the largest zone of inhibition of 24 ml. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) was determined. It was found that the MFC was higher than that of standard drug Fluconazole. All compounds were screened for antioxidant, in vitro α- glucosidase inhibitory activity, acetylchohline esterase inhibitory activity, and butyl cholinesterase and urease inhibitory activity. F and G compound showed antioxidant activity which is significant when calculated by DPPH method having percentage inhibition of 83.68±0.18 and 68.43±0.17 at 0.5 mM. A, B, C, D and E compounds represented a moderate antioxidant activity by DPPH method having percentage inhibition 42.14±0.13, 16.23±0.15, 16.55±0.13, 17.15±0.16 and 13.36±0.17. Acetylchohline esterase inhibitory activity of compound C and D has significant values 71.62±0.16 and 65.91±0.17. Compounds A-G has exhibited a little percentage inhibition activity of butyl cholinesterase. C, F and G compounds have significant α- glucosidase inhibition with percentage inhibition of 92.83±0.18, 72.26±0.18 and 92.58±0.2. B compound showed a little percentage inhibition of 64.25±0.24. A, D and E compounds have little activity with percent inhibitory values of 17.74±0.17, 42.24±0.16 and 32.64±0.14. Acarbose was taken as standard drug. G compound has shown significant urease inhibitory activity with percentage inhibition 66.54±0.26. A, B, C, D, E and F compounds have little percentage inhibition of 45.91±0.15, 48.85±0.16, 39.75±0.14, 35.96±0.15, 38.64±0.12 and 40.79±0.14m respectively.

CHAPTER 1

5 1. INTRODUCTION

Unexplored source of drug repository in medicinal plants, have marvellous therapeutic potential and helpful in achieving new challenges of novel drug lead targets with unique mechanism of action. Plants are associated with human life since the human race started. Survival of man could have been impossible if the earth had no plant on it (Nagar 2000). Experience of thousands of years has produced the popular knowledge of plants which is used by human. The most recent study by the World Health Organization (WHO) approximated that for the primary health care needs, four fifth of the total population still confidence on the plant medicine (Farnsworth et al., 1985) and mostly of these plant extracts or their bioactive components. Still today, a huge public is taking the interest to use the herbal remedies. Importance of plant derived medicine for mitigation and treatment purposes is of wonderfull value. Advancement in research increased the knowledge of human being especially in the field of plants having medicinal value. In future scientific pharmacognostic analytical techniques depend upon the methods for authentication, recognition, quality assurance and standardization of herbal medicines (Sultana et al. 2011). Knowledge of drugs which was started in early stages by the Muslims is called Islamic medicinal history (40-247 AH or 661-861 AD). The significance of medicinal plants that are used for different diseases is described in different Surahs of the Holy Quran. The Holy Prophet (P.B.U.H) also exampled the use of hebal medicines in the treatment of common ailments. The Holy Prophet (P.B.U.H) recommended Hordeum vulgare for constipation and different heart problems (Marwat et al. 2012). The knowledge of Quran and Holy Prophet (P.B.U.H) describes the importance of herbs and plants for there curative potential. Surah Baqrah, Al-Bukhari, Ibne maja, Kitabut Tib sahih, and Al-muslim, play important role in fever, stomach ailments ache, constipation, baldness, piles and menstruation. Garlic Allium sativa, has significant in asthma, paralysis, hysteria, Parkinson, tuberculosis and headache, narrated in Surah Baqra (verse no. 61). Holy Quran fourteen citations describe the medicinal significae of date (Phoenix dactylifera) in the field of cardiac, nephro, hepatic, labour pain, eczema, sexual weakness, and diarrhoea and skin diseases.

6 Muscle weakness, measles, baldness and tuberculosis can be cured by using olives (Olea europea) (Fakhri et al., 2003: Kahrizi et al., 2012).

Table 1.1 Herbal medicine in Islam (Ahmad et al., 2009) Botanical name Part used Medicinal uses Intestinal, liver, stomach , heart Sweet flag (Acorus calamus) Roots diseases, asthma and eczema diseases Throat, stomach , infection and to Onion (Allium cepa) Bulb increase production of sperms Tuberculosis, Digestive problems, Garlic (Allium sativum) Bulb asthma, dog bite, wound healer and intestinal pain Skin diseases, heart diseases, Date palm (Phoenix swelling of kidney, antidote, piles, Fruit dactylifera) intestinal pain and for physical strength. Clears blood strengthen body Olive (Olea europaea) Fruit , oil muscles, maleness, piles, tuberculosis and slow down aging. Bark, Release intestinal pain, remove kidney Figs (Ficus carica) leaves and urinary bladder stone,.

The traditional knowledge of herbal medicines, by passing through the process of modification for centuries, is used as guide, which make the way for the chemists towards development of different classes of compounds. Almost 25% of total prescribed drugs have ingridients either directly obtained from plants or semi synthetic plant derived substances. As resultant natural products are of great valuable heritage and must be preserved for the future generations (Vitalini et al. 2009). Justification of plants related medicines is present in history (today and pas) (Wilson, 1992). discovery of plant medicine provide us new chemical entities (NCEs) leading to significant lead compounds used in different pharmacological targets including Alzheimer’s disease (a genetic mental disorder), cancer, AIDS (Acquired immune deficiency syndrome) and malaria etc (Balunas and Kinghorn, 2005). Medicinal plants, due to their economy, medicinal significance,synthesis of advace medicine , providing raw materil for synthetic and semi synthetic purposes and role in primary healthcare of most population are measured as an valuable natural treasure. It is estimated that plant related durgs contributes about 20% in the world (Khan et al. 2013). It is observed thtat even the great intrest in molecular modeling,

7 combinatorial chemistry, and advace techniques for synthetic chemistry by pharmaceutical units, the natural products (medicinal plants) are significant source of novel compounds (therapeutically active). Best selling medicines in (2000 and 2002) about one quarter was based on either directly natural products or derivatives of natural products (Balunas et al. 2005). World Health Organization estimated that about 25% of prescribed medicines and precursors of different synthetic substances are of plant based. The medicinal plants used for treatment of different diseases are more safe, economical and reasonable than those of advace synthetic medicines. It is estimated that about 80% population of developing states, 90% population of African countries and most of population of Pakistan depends medicinal plants native to them for there health issues. Herbal system of medicines evolution mainly due to proficient cure action, ease of access, and low cost (Ullah et al. 2013).

About 500 medicinal plants are highlighted in ancient literature and about 800 medicinal plants have been listed in indigenous medicinal system. Due to low toxicity and less side effects of herbal medicine, they are popular medicines. Herbal medicines are used as major curing system since ancient times. Due to its cultural beliefs and biomedical advantages today these systems contributes a lager in the treatment of human health (Verma and Singh 2008).

The earliest article on Ayurveda Charaka Samhita (600 B.C) total listing of 341 plants and plant products used in health management. Sushruta Samhita also diescribes the medicine related to plants. The author also extended the list of Ayurvedic plant medicines to six hundred different species of plants. The most popular plant Rauwolfia was also listed in ancient Hindu scripture and in Charaka. Mentioned plants are useful as antidote for snake bite as well as insect stings and his effect in treatment of insanity. The significance of this medicine has been approved recently in western medicinal therapy.

8 1.1: Different medicine systems 1.1.1: European system of medicines European medicine system is called as “Galenic medicine”. This system of medicine had origin from Hippocrates and Aristotle (460-377 BC) and (384-322 BC). De Materia Medica is the well known book written by Physician Dioscorides (Weiher, et al. 1999).

1.1.2: Chinese system of medicine About year 5000 back the traditional medicinal system of Chinese is mostly liked and Sung Dynasty Emperor Shen Nong arranged and recorded in written form in 2800 BC. Encyclopedia was published in 1977 of Chinese Materia Medica as refence book of china herbal prescription (Magner, 1992).

1.1.3: Egyptian system of medicine Egyptian system of medicine starts almost 2900 BC dates back. Ebers Papyruis was the first data written was originated approximately 700 medicines from 1500 BC. This record is mostly originated form plants and animals (Weiher, et al. 1999).

1.1.4: Ayurvedic System of Medicine Sanskrit derived ayurved means in essence the Science of life. It is considered as indigenous ancient medicine system about 1500- 800BC Vedic ages. Micinal data form this sytem is recorded in Astanga and Charaka Sushruta (Bhattacharjee, 2001).

1.1.5: Unani System of Medicine The Muslims bring the medicinal system (Unani) to Arab when they were rular. People of Greek people made self drug for different diseases collect the material from Indian medicinal system. When Empire is declined from Greek Roman, the system of medicine from Greek was taken by Muslims as Unani system of medicine. Al Hawi, Ibn Sena’s and Al Qanoon are the best books written by Muslims which describes the medicine of that time. introduced Ayurveda was taken by Abbasid Caliphate and translation of significant Ayurvedic therapies like Charaka Samhita, Sushruta Samhit in Arabic (Bhattacharjee, 2001).

1.2: Herbal Medicines in Islam On observation it is clear that the teaching of Islam focus on the use of medicinal plants to treat ailments. The use of medicines from herbal origin in Islam starts from Hazrat Adam (Alaihi Salaam) and completed by Hazrat Muhammad (Sallallahu Alaihi Wasallam). Good information of plants used for medicine is provided in Holy Quran. The most popular example is honey (the extract of varities of plant) described in Holy Quran. Few examples of medicinal plants are given in Table 1.1. The plants not only in use in Islamic world but also in all countries of world for different aspects for example medicine, food stuff and industrial materials (Ahmad, et al. 2009).

1.3: Importance of Herbal Medicines For medicinal uses about 70,000 plant (towering trees to lichens) and there species are listed. The Plants provide us the material for the purification, isolation and leading to synthesis of drugs. Ayurveda describes about 2,000 plants hve pharmacological properties. Approximate 500 herbs are used in conventional and traditional medicine (Prajapati and Purohit, 2003). A lot of efforts and investments were made in whole world to investigate the pharmacological and therapeutical potential and discovery of more effectie plants for medicines. As resultant some new medicinal plants were screened successfully. Pharmacopoeial demand and products related activities are increasing day by day due to increasing demand of these herbal medicines in India. Now adays these plants for medicines are introduces in farmer. Local cultivation of plant medicine is done (Prajapati and Purohit, 2003).

1.6: Herbal Medicines Today Ancient experience of medicine and traditional health care system has provided us the guidance to advance allopathic sytem of medicine. Efforts are being made to discover and market the new and effective remedies from plant origin. The studies of traditional systems will not only highlightes that evelopment of field but also give idea of our conditions to develop a diversity of cultural practices.

10 Approximate 120 therapeutically active agents of developed structures from 90 flowering plants species which very minute portion. Ayurveda used medicinal plants may provide us a lot of therapeutically active compounds with noval structures have more potency and less toxicity (Joy, 1998). Few potent medicinal plant drugs are listed in Table 1.2.

Table 1.2: Major plant drugs for which no synthetic one is currently available. (Joy, 1998) Drug Structure Use

Vinblastine (Catharanthus roseus) Anticancer

Anticancer, Ajmalicine (Catharanthus roseus) hypotensive

Antimalarial, Quinine (Cinchona sp.) amoebic dysentery

Pilocarpine (Pilocarpus jaborandi) Antiglaucoma

Topical Cocaine (Erythroxylum coca) anesthetic

Morphine (Papaver somniferum) Painkiller

Codeine (Papaver somniferum) Anti cough

Spasmolytic, Atropine (Atropa belladonna) cold

11 Rescinnamine (Rauwolfia Tranquilizer serpentine)

Reserpine (Rauwolfia serpentine) Tranquilizer

Artemisinin (Artemisia annua) Antimalarial

Breast and Taxol (Taxus baccata, ovary cancer, T. brevifolia) antitumor

Pristimerin (Celastrus paniculata) Antimalarial

Antibacterial, Plumbagin (Plumbago indica) antifungal

Antifungal, Allicin (Allium sativum) amoebiasis

Emetine (Cephaelis ipecacuanha) Amoebiasis

Glycyrrhizin (Glycyrrhiza glabra) Antiulcer

12 1.7: Introduction to Family Combretaceae A family of 20 genera and 500 species, widespread in tropical and subtropical regions of the world. Trees, shrubs or lianas. Indumentum of simple hairs often present. Leaves exstipulate, simple, entire, opposite, spirally arranged or alternate. Domatia and glands often present. Flowers bisexual or male, 4-5-merous, actinomorphic, in axillary spikes or racemes, epigynous. Hypanthium usually in 2 distinct parts, the lower surrounding and adnate to the inferior ovary with the upper part projecting to form a short or long tube terminating in the 4-5(8) calyx lobes. Petals 4-5. Stamens usually twice as many as petals (when the latter are present), inserted inside calyx, in 2 series. Ovary unilocular, usually with 2-6 pendulous ovules. Fruit a drupaceous or dry pseudocarp, indehiscent, often winged and ridged (Linsingen et al., 2009).

1.8: Botanical aspects of genus Conocarpus

Conocarpus the button mangrove genus a genus of 2 species distributed in neotropics and tropical western Africa, Saudi Arabia, Somalia, Asia, and Eastern Africa. Leaves alternate, with raised basal glands, petiolate or sessile. Flowers small, densely clustered in globose heads arranged in terminal panicles or racemes, bracts ovate; hypanthium campanulate, the lower part laterally flattened, ± winged; calyx 5-lobed; petals absent; intrastaminal disk 5-lobed, fleshy, stamens 5(10). Fruit scale-like, coriaceous, obcordate, laterally compressed and 2-winged. (Hameed et al., 2012).

1.9: Conocarpus lancifolius Conocarpus lancifolius an ornamental tree is native to coastal and riverine areas of East Africa (Baroon et al. 2012). It is a fast growing tree and grows well in semi-arid conditions and sandy soils. The mature leaves are glossy in appearance with relatively fewer trichomes on both surfaces. Usually trichomes are of two type’s secretory and non secretory trichomes. Secretory trichoms are capitates and short stalked and non secretory have pointed tip and bulbous base. A pair of extrafloral nectaries is present on leaves on both sides. 3-4 pairs of petols are present at distal end near apex of leaf. For secretion of polysaccharides, epicuticlar waxes and polyphenols two cavities or secretory ducts are present on mature leaf (Redha et al., 2011).

13 1.9.1: Botanical Classification of Conocarpus lancifolius

Kingdom: Plantae Subkingdom: Tracheobionta Superdivision: Spermatophyta Class: Equistopsida Subclass: Magnoliide Superorder: Rosanae Order: Myrtales Family: Combretaceae Genus: Conocarpus Species: lancifolius

Figure 1.1: Aerial view of the plant Conocarpus lancifolius

1.10: Secondary metabolites The metabolites not involved in development and normal process of growth are called secondary metabolites. These are organic molecules. Secondary metabolites are diverse class of natural occurring products that are synthesized by plants, animals, fungi, algae and bacteria. Classification is done on biosynthetic bases e.g. alkaloids terpenes and phenolics. They are largly produced during a transition from active growth to stationary phase (Roze et al. 2011).

14 Primary metabolic Primary metabolic Corresponding Pathway of products Secondary Metabolites Carbon

CO2 CO2

Pentose Pathway Glycosides Sugars Carbohydrates Gums and Mucilages Streptomycin

Glycolysis Erythrose PO4

Phosphopenol Shikimic acid Phenols Pyruvate Tannins Aromatic Lignin Amino acids

Pyruvate proteins Alkaloids Aliphatic Peptides Amino acids Penicillin

Acety -CoA Malonyl-CoA Fatty acids Fats and Waxes

Tetracyclines

Anthraquinones Tricarboxylic Acid cycle Isoprene Terpenes

CO2 Squalene Steroids

Figure 1.2: Interrelationships of biosynthetic pathways leading to secondary constituents in plants.

15 1.10.1: Alkaloids Alkaloids are secondary metabolites having one or more heterocyclic nitrogen items. The alkaloids name was derived from the alkaline means to indicate any nitrogenious base. Humans used alkaloids in the different forms from plants extract, stimulants, narcotics, and as medicines. From three thousand families of plants about ten thousand alkaloids of different structures are isolated. That is small portion of plants still reported (Raffauf 1996). Major classes of alkaloids are as follows

 Pyridine/Piperdine: Nicotine (nicotiana tobbacum) is major alkaloid having CNS stimulants and adrenergic proerties (Watson et al. 1983). Piperine is obtain from piper nigrum having stimulant, hepatoprotective properties (Koul and Kapil 1993).  Tropane: Hyoscamine and Hyoscine derived from Atropa Belladonna are the major alkaloids. They are used as anti-cholinergic, reduces salivary secretions, mydriatic effect, and tremors (Evans 1990, Roddick 1991).Cocaine (Erythroxylone coca) having anesthetic, narcotic and CNS stimulant properties. (Tyler et al. 1988).  Quinoline: Quinine and quinidine derived from Cinchona spp. are bthe major alkaloids. Quinine has anti-malarial , muscle relaxant and acute infectious disorders properties. Quinidine has atrial flutter activity, fibrillation and cardiac arrhythmias properties (Harborne and Baxter 1993).  Isoquinoline: Berberine (Berberis spp, Morphine (Papaver somniferum), Boldine (Chelidonium majus), Chelidonine (Pneumus Boldo), and Emetine (Cephaelis ipecacuanha) are the major alkaloids. Berberines have Antimicrobial, antiprotozoal, and cholagogue, wherea properties are Sedative, spasmolytic, choleretic, analgesic and emetic (Speisky et al. 1991).  Quino-lizdine: They are alsocalled as “lupin” alkaloids. Sparteine (Sarothamnus scoparius) are the major alkaloids having Oxytocic, diuretic and cardiotonic properties (Schmeller et al. 1994).  Pyrrolizidine: Sececionine (Senecio jacobeae) and symphetine(Symphytum spp) are major alkaloids. These alkaloids ahave hepatotoxin properties (Harborne and Baxter 1993).

16  Indole: Strychnine, Reserpine, Yohimbine and Ergotamine are major alkaloids Reserpines obtain from Rauwolfia serpentina having Sedative action and antihypertensive properties . Major properties of indol alkaloids are CNS stimulant, vasoconstrictor, hypertensive and toxin as well (Samuelsson 1992).  Imidazole: Pilocarpine obtain from Pilocarpus jaborandi is alkaloid having miotic activity and cholinergic proerties.  Alkaloidal amines: Mescaline, Ephedrine and Colchicine are major classes. Ephedrine obtain from Ephedra sinica having bronchodilator, stimulant activity and sympathetic properties (Blumenthal and King 1995).  Purine alkaloids: Theophylline, Caffeine and Guaranineare are major alkaloids . Caffeine (Coffea arabica) having CNS and sympathetic stimulant (Samuelsson 1992).  Steroidal alkaloids: Solanine and Veratrine are major classes. Solanine is derived from Solanum spp. having anti-inflammatory poperties . Veratrines derived from Veratrum album. having anti-hypertensives and cardiac depressants properties (Bradley et al. 1978 and Robinson 1981).

1.10.2: Steroids The word steroid originated from Greek stereos = solid. The compounds having perhydrocyclopentano phenanthrene nucleus a tetracyclic carbon skeletonare called steroids. They contains a varity of naturally exiting molecules such as bile acids, hormones, sterols, glycosides (cardiac), sapogenins and few alkaloids.

17 1.10.3: Terpenoids

The derivative of isoprene unit (five-carbon and containing two unsaturated bond) are called terpenoids or terpenes. All terpenoids contains isopentane units. The classification is as.

 Monoterpenes (C10H16): Chemical compounds having two isoprene units. They may be linear or acyclic having rings. Paeoniflorin (Paeonia Lactiflora) is the example of monoterpenes. It has sedative, anti-inflammatory, antipyretic and antispasmodic properties (Huang 1993).

 Sesquiterpenes (C15H24): Chemical compounds having three isoprene units. Gossypol (Gossypium herbaceum) having antifertility properties is the example of sesquiterpenes (Hamburger and Hostettmann 1991).

 Diterpenes (C20H32): They are tetracyclic compounds which are lactones. Marrubiin (Marrubium vulgare) having stomachic and tonic properties is the example of diterpenes.

 Triterpenes (C30H48): Organic compounds having C30 precursor are called triterpenes. Configuration is smilar to steroids. Dioscin (Dioscorea villosa) having oestrogenic activity is the example of triterpenes.

 Tetraterpenes (C40H64): Organic compounds having C40 precursor are called tetraterpenes. Lycopene is the example (Eitenmiller and Landen 1999).

1.10.4: Tannins Water soluble oligomers consist of rich in phenolic groups, having precipitating and binding water-soluble proteins. They are classified in two classes. They may be condensed tannins or may be hydrolysable in nature. Biosynthetically condensed tannins are made from flavanols condensation. The esters of a glucose (sugar) having one or more gallic acid (trihydroxy benzene carboxylic acids) are called hydrolyzable tannins.

18 1.10.5: Glycosides

The organic compounds having a sugar portion attached with a bond to one or more non sugar portions. They may be phenolic, alcoholic or sulfur containg compounds. Sugar portion may be fructose, glucose or may be glucuronic acid. (Pengelly, 2004)

They are classified as

 Alcoholic glycoside: Salicin isolated from the genus Salix. By metabolism of salicin salicylic is formed in the body, similar to structure of asprine and have anti inflammatory and antipyretic proerties.  Anthraquinone glycosides: The compounds having aglycones with two or more phenols linked with a quinone ring. Rhubarb, senna, , aloes and cascara sagrada are rich source of anthraquinone glycosides.  Coumarine glycosides: Psoralin and corylifolin (Psoralea corylifolia) having aglycone coumarin. The dilation of coronary arteries by apterin is coumarine glycoside.  Cyanogenic glycoside: The glycoside having nitrogen element in hydrocyanic acid form . Sambunigrin (sambucus nigra) is example of cyanogenic glycoside.  Flavonoid glycosides: The glycosides having aglycone units flavonoids are called flavonoid glycosides . The examples are Querctrin, Hesperidin and Rutin.  Phenolic glycosides: The glycosides having aglycone unit must be simple structure of phenolic e.g. Arbutin.  Saponin glycosides: The C27 rings skeleton presented as nortriterpenes are called saponin glycosides. Liquorice obtaine from Glycyrrhiza glabra is an exampel.  Steroidal glycosides: The glycosides having aglycone unit must be steroidal nucleus structure. The examples are Scillaand , Strophanthus and glycosides of Digitalis.  Thioglycosides: The glycosides having sulfur atom are called thioglycosides. Examples are sinigrin and sinalbin obtained from black and white mustard .

CHAPTER 2

20 2. LITRATURE REVIEW

2.1: Previous phytochemical reports of combretaceae family and genus Conocarpus. 2.1.1: Combretaceae family and genus Conocarpus. Combretaceae is an important family consisting of flowering plants. There are 20 genera included in this family out of which genus Terminalia and Combretum has been most extensively studied for its phytoconstituents and pharmacological activities and commonly available in Asia (Linsingen et al., 2009). Genus Conocarpus consists of only two species namely, C. erectus and C. lancifolius (Abdul Hameed et al., 2012). The genra Terminalia, Combretum and Conocarpus have different secondary metabolites for example flavonoids, phenolics, sterols, tannins, glycosides and terpenes. The isolated compounds from these genra are listed in tables 2.1, 2.2, 2.3, 2.4 and 2.5. The chemical structures of isolated compounds are given in figures 2.1, 2.2, 2.3, 2.4 and 2.5.

Table 2.1: Flavonoids reported from different species of Terminalia, Combretum and Conocarpus genera.

Part Constituents Plant name Reference used Catechin (1), apigenin (2), quercetin (3), quercetin-3-O-glucoside (4), kaemferol-3-O-glucoside (5), rutin (6), Whole Hameed et al. C. erectus quercetin-3-O-glucoside-6-O-gallic plant 2012 acid (7)

(+)-catechin , 5,7,3,5-tetrahydroxy- Banskota et 3,4’-dimethoxyflavone (8) combretol al.2000 (9) (10) pachypodol (11) quercetin C. Whole Adnyana et 3,4-dimethyl ether (12) myricetin quadrangulare plant al.2000 3,3,4-trimethyl ether (13) Toume et al.2011 (14) pentahydroxy 3, 4, 5, 5, 7- flavan (15) isovitexin (16) orientin (17) homoorientin (18) myricetincin-3-O- glucoside (19) galloylvoytexin (20) 2- Whole Welch et C. micranthum O-galloylorientin (21) 2-O- plant al.2010 galloylhomoorientin (22)

21 apigenin (24) genkwanin(25) 5-hydroxy-7,4-dimethoxyflavone (26) C. Whole Martini et Rhamnocitrin(27) kaempferol (28) erythrophyllum plant al.2004 quercetin-5,3-dimethylether(29) rhamnazin (30) dillenetin (31) isorhamnetin (32) quercetin (33) 3-O- C. Whole Araujo et methyl quercetin(34) lanceolatum plant al.2013 (35) 5,3,4-trihydroxy-3,7-dimethoxyflavone (36) Whole Fcundo et al. 5,3’-dihydroxy-3,7,4’- C. leprosum plant 2008 trimethoxyflavone (37)

Pinocembrin (38) 2,4,4- trihydroychalcone(39) eriodictyol Whole Aderogba et C. apiculatum (40)cardamonin (41) plant al. 2012

Arjunore (42) luteolin (43) kaemferol apigenin , quercetin ,catachin, gallo Whole Sharma et al. catachin , epicatachin, rutin Arjunolone T. arjuna plant 1982 (44)baicalein (45)

Whole Eldeen et, al Anolignan B(46) T. sericea plant 2006 8-methyl-5,7,2,4′-tetra-O-methyl- flavanone (47) 5,7,2′-tri-O-methyl- Srivastava et flavanone 4-O-rhamnopyranosyl.- T. alata Root al.1999 (1→4)-β-ᴅ -glucopyranside (48)

7,3-dihydroxy-4′-O-methyl-flavan (49) 7,4 -dihydroxy-3-O-methyl-flavan(50) 7-hydroxy 3-4 methylenedioxy flavan Garcez et (51) T. argentea Bark al.2003

Figure 2.1: Chemical structures of flavonoids (1-51) reported from different species of Terminalia, Combretum and Conocarpus genera.

Figure 2.1: Chemical structures of flavonoids (1-51) continued……..

Figure 2.1: Chemical structures of flavonoids (1-51) continued……

27 Table 2.2: Terpenes reported from different species of Terminalia, Combretum and Conocarpus genera.

Consituents Plant name Part used Reference T. brachystemma Leaves Liu et al. 2009 Ursolic acid(52) T. catappa Leaves Manna et al.2007 T. chebula Leaves Singh 1990 2α-hydroxyursolic acid (53) T. mollis Stem bark Liu et al. 2009 2,3,23-trihydroxy-urs-12-en-28-oic T. catappa Leaves Manna et al.2007 acid (54) T. brassii Wood Asiatic acid (55) Collins et al.1992 T. complanata Wood T. arjuna Root bark Paarakh et al. 2010 Oleanolic acid (56) T. superb Stem bark Wansi et al 2007 Methyl oleonate (57) T.arjuna Fruits Nagar A et al.1979 T. arjuna Fruits Paarakh et al.2010 Arjunic acid (58) T. macroptera Bark Liu et al.2009 T. brassii Wood Manna et al.2007 Arjunolic acid (59) T. complanata Wood Collins et al.1992 23-O-galloyl-arjunolic acid(60) T. macroptera Stem bark Conrad et al.1998 T. arjuna Bark Liu et al.2009 Arjungenin (61) T. bellerica Stem bark Nandy et al.1989 T. macroptera Bark Conrad et al.1998 Bombardelli et T. sericea Roots Sericic acid (62) al.1974 T. macroptera Stem bark Conrad J et al.1998 2α-hydroxymicromeric acid(63) T, chebula Leaves Liu et al.2009 Terminic acid Anjaneyulu et (3β,13β-dihydroxylup-20-en-28- T. arjuna Root bark al.1983 oic acid)(64) T. arjuna Fruits Nagar et al.1979 T. glaucescens Stem bark Atta et al.2002 Friedelin(65) T. mollis Stem bark Liu et al.2009 T. alata Roots Srivastav et al.2001 T. chebula Bark Kundu et al.1993 T. superba Bark Wansi et al.2007 T. bellerica Fruits Row et al.1970 β-sitosterol(66) T. glaucescens Bark Atta et al.2002 T. phanerophlebia Leaves Nair et al.2012 T. arjuna Fruits Paarakh et al.2010

β-sitosterone(67) T. phanerophlebia Leaves Nair et al.2012

Stigma-4-ene-3,6-dione(68) T. phanerophlebia Leaves Nair et al.2012

Terminalin A(69) T. glaucescens Stem bark Atta et al.2002 combretene A (70) combretene B C.molle Leaves (71) Bahar et al.2004

28 Galloylolean-12-en-28-oate (72) Kemyoufo et al. 2008 combregenin(73) arjungenin(74) arjunglucoside 1 C.molle Leaves

(75) combreglucoside (76) Jassang et al. 1996 sericoside (77)

(78), 3 -O-glucoside mollic acid (79), 3 -O-arabinoside mollic acid C. molle Leaves Pegel et al. 1985 (80), 3 -O-xyloside mollic acid (81) quadrangularic acid F(82) quadrangularic acid J(83) 24-epiquadrangularic acid G(84) quadrangularic acid M(85) C. quadrangulare quadrangularic acid H(86) Leaves Banskota et al.2000 quadrangularic cid K(87) quadrangularic acid L(88) 7-hydroxy -23-deoxojessic acid(89) methyl quadrangularate A(90) methyl quadrangularate B(91) methyl quadrangularate C(92) methyl quadrangularate O(93) methyl quadrangularate N(94) quadragularol B(95) quadragularic acid (96) C. 23-deoxojesic acid(97) Leaves Banksota et al.2000 quadrangulare 1-O-acetyl-23-deoxojessic acid(98) 4 ,14 -dimethyl-5 -ergosta-9 ,19- cyclo-24-en-3 -hydroxy-4 - carboxylic acid(99) methyl 23-deoxojessoate(100)

D-glucopyranosyl 2 ,3 ,6 – trihydroxy olean-28-oate(101) C. laxum Roots Bisoli et al. 2008 Bellericoside (102) Amyrin(103) C. micranthum Bark Welch et al.2010 Lupeol(104) Hydroxyoleian-3-oicacid12-ene (105) Dihydroxyolean-12-eny-29- C. imberbe Leaves Angeh et al. 2007 one(106) Trihydiroxy-oleain 29-oic acid(107) Dihydroxyolean-12-ene-23 – rhamnopyranoside(108) C. padoides Leaves Angeh et al.2007 Dihydroxyolean-12-ene-30-oic acid(109)

29 Trihydroxy(3 ,6 ,16 ) lup-20- ene(110) C. leprosum Wood Facundo et al.2010 4 ,carboxy 3 ,16 –dihydroxy- cycloart-24-ene (111) Acetylimberbic acid-29-methyl ester(112) C. oliviforme Bark Wu et al.2010 Rhamnopyranosyl 1,3 - diacetylimberbic acid(113) Acetoxy-25 hydroxy 20,24-epoxy- -3-one(114) C. nigricans Bark Simon et al.2003 11 ,25-dihydroxy-3-one20,24- epoxy-(115) Dihydroxy 12-ène-28-oic acid(116) 6–hydroxymaslenic C. zeyheri Roots Runyoro et al.2013 acid(117) terminolic acid(118) corosolic acid(119) C. vendee Roots Aroke 2012

Figure 2.2: Chemical structures of terpens (52-119) reported from different species of Terminalia, Combretum and Conocarpus genera.

Figure 2.2: Chemical structures of terpens (52-119) continued………….

37 Table 2.3: Tannins reported from different species of Terminalia, Combretum and Conocarpus genera

Part Consituents Plant name Reference used

T. chebula Fruits Lee et al, 2007 Chebulic acid(120) T. bellerica Fruits Pfundstein et al,2011

Neo-chebulic acid T. chebula Fruits Lee et a, 2007

T. chebula Fruits GaoH et al, 2007 Chebulanin(121) T. bellerica Fruits Pfundstein et al,2011 T. horrida Fruits Pfundstein et al,2011 T. mollis Leaves Liu et al, 2007 T. bellerica Fruits Pfundstein et al, 2011 Chebulinic acid(122) T. chebula Fruits GaoH et al, 2007

T. horrida Fruits Pfundstein et al, 2011 T. chebula Fruits Methyl neo-chebulanin(123) T. bellerica Fruits Pfundstein et al,2011 T. chebula Fruits Pfundstein et al, 2011 Methyl neochebulinate (124) T. bellerica Fruits

T. horrida Fruits T. chebula Seeds Nair et al, 2010 Chebulagic acid(125) T. bellerica Fruits Yang et al, 2013 T. horrida Fruits Pfundstein et al, 2011 T. catappa Leaves Kinoshita et al, 2007 T. chebula Fruits Methyl neochebulagate(126) T. bellerica Fruits Pfundstein et al, 2011 T. horrida Fruits T. chebula Fruits Galloyl 2,4-O-chebuloyl-β-ᴅ -Gly T. bellerica Fruits Pfundstein et al, 2011 (127) T. horrida Fruits T. chebula Fruits Tellimagrandin(128) T. bellerica Fruits Pfundstein et al, 2011

T. horrida Fruits T. chebula Fruits Bag et al, 2013 Corilagin(129) T. horrida Fruits Pfundstein et al, 2011

T. catappa Leaves Kinoshita et al, 2007 Tercatain(130) T. catappa Leaves Tanaka et al, 1986

Arjunin(131) T. arjuna Leaves Kandil et al, 1998

38 T. horrida Fruits Pfundstein et al,2011 Punicalin(132) T. arjuna Leaves Kandil et al, 1998

T. catappa Leaves Tanaka et al, 1986

T. chebula Leaves Bag et al, 2013 T. horrida Fruits Pfundstein et al,2011 T. oblongata Leaves Filippich et al, 1991 Punicalagin(133) T. brachystmma Leaves Liu et al ,2007 T. macroptera Roots Silva et al, 2012 T. catappa Leaves Chen et al, 2000 T. arjuna Bark Lin et al, 2000 T. myriocarpa Leaves Marzouk et al, 2002

2,3:4,6-bis-O-HHDP-1-O-galloyl- T. arjuna Leaves Kandil et al, 1998 β-ᴅ -Glc(134)

Tergallagin(135) T. catappa Leaves Tanaka et al, 1986

T. chebula Fruits Bag et al, 2013 Terflavin (A)(136) T. catappa Leaves Tanaka et al, 1986

T. macroptera Bark Conrad et al, 2001

Terflavin (B)(137) T. bellerica Fruits Pfundstein et al,2011 T. catappa Leaves Tanaka et al, 1986 T. macroptera Bark Conrad et al, 2001

T. chebula Fruits Bag et al, 2013 Terflavin (C)(138) T. catappa Leaves Tanaka et al, 1986

T. arjuna Bark Lin et al, 2000

Figure 2.3: Chemical structures of tannins (120-138) reported from different species of Terminalia, Combretum and Conocarpus genera.

Figure 2.3: Chemical structures of tannins (120-138) continued ……

43 Table 2.4: Glycosides reported from different species of Terminalia, Combretum and Conocarpus genera.

Consituents Plant name Part used Reference

2α,3β-dihydroxyurs-12,18-dien-28-oic acid-28-O- β-ᴅ -glucopyranoside(139) 2α,3β,23 trihydroxyurs-12,18-dien-28-oic acid-28- O-β-ᴅ -glucopyranoside(140) 2α,3β,23 trihydroxyurs-12,19-dien-28-oic acid-28- O-β-ᴅ -glucopyranoside(141) T. arjuna Bark Wang et al.,2010 Quadranoside VIII(142)

(2α,3β,23-trihydroxyurs-12,19-dien-28-oic acid- 28-O-β-ᴅ -glucopyranoside) Kajiichigoside F1(143) (2α,3β,19α-trihydroxyurs-12-en-28-oic acid-28-β- ᴅ -glucopyranoside)

Arjunetin(144) T. argentea Bark Gracez et al.,2003

Arjunosides(145) (3-O-β-ᴅ -galactoside of arjunic acid) T. arjuna Root Paarakh,2010

2α,3β,19α-trihydroxyolean-12-en-28-oic acid- methylester-3-O-rutinoside(146) T. alata Roots Srisvata et al.,2009

2α,3β,19α-trihydroxyolean-12-en-28-oic acid-3- O-β-ᴅ -galactopyranosyl- (1→3)-β-ᴅ - T. alata Roots Srivata et al.,2001 glucopyranoside(147)

2α,3β,19β,23-tetrahydroxyolean-12-en-28-oic acid 3-O-β-ᴅ -galactopyranosyl-(1→3)-β-ᴅ - T. alata Roots Srisvata et al.,2001 glucopyranoside-28-O-β-ᴅ -glucopyranoside(148)

Arjunolitin(149) T. arjuna Stem bark Tripathi et al.,1992

Tormentic acid-β-ᴅ -glucopyranoside(150) T. argentea Bark Gracez et al.,2003

Chebuloside (I) (151) T. chebula Stem bark Kundu,1993 Chebuloside (II) (152)

23-galloylarjunolic acid-28-O-β-ᴅ - T. macroptera Stem bark Conrad et al.,1998 glucopyranoside(153)

44 Fruits, Nandy et al., 1989 T. bellerica Arjunglucoside (I) (154) Stem Cao et al.,2010 T. tropophylla Bark Conrad et al.,1998 T. macroptera

Arjunglucoside (II) (155) T. arjuna Stem Honda et al.,1976

Terminoside (A) (156)

Termiarjunoside (I) (157) T. arjuna Bark Ali et al., 2006

Termiarjunoside (II) (158)

Bombardeli et T. sericea Roots al.,1974 Sericoside(159) T. tropophylla Roots Cao et al.,2010 T. macroptera Stem Conard et al.,1998 T. ivorensis Bark Ponou et al.,2010

Ivorenoside (160) T. ivorensis Bark Ponou et al.,2010

Bellericoside(161) T. chebula Bark Kundu,1993 T. bellerica Bark Nandy et al.,1989

Bellericaside (A) (162) T. bellerica Stem bark Mahato et al.,1992

Bellericaside (B) (163) T. bellerica Stem bark Mahato et al.,1992

T. catappa leaves Lin et al.,2000 Daucosterol(164) T. arjuna Leaves Rass et al.,2013

T. bellerica Fruits Yang et al.,2013

Figure 2.4: Chemical structures of glycosides (139-164) reported from different species of Terminalia, Combretum and Conocarpus genera.

Figure 2.4: Chemical structures of glycosides (139-164) continued……….

Table 2.5: Mislinious compounds reported from different species of Terminalia, Combretum and Conocarpus genera.

Part Consituents Plant name Reference used Leucocyanidin(165) T. arjuna Bark Tripathi et al, 1992

Pelargonidin(166) T. arjuna Bark Dwivedi ,2007 combretastatin A-1(167) C. cafrum Leaves Pettit et al, 1987 Combretasta C. erythrophyl Leaves Schwikkard et al, 2000 (168) combretastatin A-2(169) C. cafrum Root Pettit et al, 1987 combretastatin B(170) C. woodii Bark Eloff et al, 2005

3,4 -dihydroxy-4,5-dimethoxy C. molle Leaves Pettit et al, 1987 bibenzyl(171)

3,4,5trimethoxy 4 hydroxylbibenzy l(172) C. psidioides Leaves Letcher et al, 1972 3,5-dimethoxy 4,4 -dihydroxy bibenzyl(173) 2,3 methoxy 3-hydroxy bibenzyl(174) 3,4,5-trimethoxy2 -hydroxy bibenzyl(175) 2,3-dihydroxy-4,5- dimethoxybibenzyl(176) C. apiculatum Leaves Malan et al, 1993 2,4-dihydroxy-3,5- dimethoxybibenzyl(177) 3,4,5-trimethoxy3-hydroxy bibenzyl(178)

Figure 2.5: Chemical structures of mislinious compounds (165-178) reported from different species of Terminalia, Combretum and Conocarpus genera.

52 2.2: Previous reports on the biological studies of combretaceae family and genus Conocarpus. Biological Activity Botanical Name Part Used Bioassay Model Reference

Acetylcholinesterase C. hartmannianum EtOH ext. of leaves Microplate inhibition and TLC method using Eldeen et al. 2007 Inhibition Ellman technique, Drug -IC50 is 0.250 mg/mL

C. fruticosum MeOH/MeCl2 (50:50) ext. Lung of rabbit was catalyze the cleavage of the Braga et al. 2000 of stem chromophore-fluorophore labeled In vitro-ACE substract dansyltriglycine into dansylglycine Drugs:concentration 0.33 mg/mL Topoisomerase C. apiculatum EtOAc ext. of rootbark Topoisomerase-I and topoisomerase-II inhibition Wall et al. 1996 Inhibition method using Polyvinylpyrrolidine (1:1). n-butanol, hexane and Nematocidal activity using hatched egg and larval Ademola et al. 2010

CHCl3 ext. of leaves stages of Haemonchuscontortus 50% Lethal

Concentration (LC50) of drugs: 0.856, 0.323, 0.823, 0.757 mg/mL. Antimalarial C. micranthum 95% EtOH ext. of leaves Cell culturing technique (erythrocytes) using Karou et al. 2003 activity Plasmodium falciparum, Drug IC50 is 33.05 μg/mL

53 Biological Activity Botanical Name Part Used Bioassay Model Reference

C. molle Acetone ext. of bark Cell culturing technique (Trophozoites) using Arses et al. 2001

Plasmodium falciparum, Drug IC50 is 8.27 μg/mL C. Psidioides EtOH, Pet ether, EtOAc Microdiluition technique by using Plasmodium Gessler et al. 2010 of root bark falciparum,Drugs: 31.0, 37.0 and 6 μg/mL.

Antischistosomal C. aculeatum H2O ext. of leaves Cercaricidal and Miracidicidal assays on Schistoisoma Eldeen et al. 1990 Activity mansoni, Drug conc. : 1,000 ppm

C. imberbe H2O ext. of leaves S. haematobium worms (In vitro) Drug MIC: 12.5 McGaw et al. 2001 mg/mL.

C. kraussii H2O ext. of leaves S. haematobium worms (In vitro) Drug MIC: 12.5 McGaw et al. 2001 mg/mL.

Antitrypanosomal C. dolichopetalum 70% EtOH ext. of root Trypanosoma bruceior and Trypanosoma congolense Udem et al. 1996 activity bark Infected rats Dose for Drug dose is: 80.0 mg / kg (i.p.)

C. quadrangular MeOH and H2O ext. of T. cruzi Epimastigotes Drug conc. : 100.0 μg/mL Kiuchi et al. 2002 leaves

Biological Activity Botanical Name Part Used Bioassay Model Reference

Larvicidal C. collinum Fresen. Ether ext. of shoot bark A. aegypti Larvae by using drug conc. : 0.0120-0.200 Odda et al. 2008 Dengue fever mg/mL.

Antimicrobial C. apiculatum Heat ext. of leaves Microdilation method (B. subtilis, E. coli,and McGaw et al. 2001 activity Staphylococcus aureus) maximum concentration of drug is 12.5mg/Ml

C. bracteatum H2O ext. of stem Diffusion of agar assay (C. diphtheriae) Drug Olukoya et al. 1993 concentration: 0.32 g/mL with zone of inhibition 5–9 mm diameter.

C. glutinosum MeOH ext. of leaves Agar diffusion assay (Eschrshia coli and S. lutea) Laurens et al. 1985 Drug concentration :10.0 and 15.0 mg /mL.

Biological Activity Botanical Name Part Used Bioassay Model Reference

C. hartmannianu MeCl2, EtOAc and Microdilation method (B. subtilis) Drugs MIC: <0.1, Eldeen et al .2007 EtOH ext. of leaves 0.29 and 0.21 mg/mL. Microdilution technique (K. pneumonia) Drugs MIC: Eldeen et al. 2007 0.21, 0.68 and 0.29 mg/mL.

C. imberbe MeCl2 extract of leaves Serial dilution technique by microplate (S. aureus) Angeh et al. 2007 Drug concentration: 38 μg/mL. C. micranthumi 95% EtOH of twigs Diffusion of agar assay (C. diphtheriae) Drug Le Grand et al. 1989 concentration: 0.50 mg/mL with zone of inhibition 15– 9 mm diameter. EtOH ext. of leaves Serial dilution technique (S. aureus, Streptococcuss Adoum et al. 1997 Salmonella , E. coli,P. vulgaris, P. aeruginosa) Drug MIC: 1.00 mg/mL.

C. molle Acetone and H2O ext. of In vitro- method of Radiometric (M. tuberculosis) Lall et al. 1999 bark Drug MIC : 2.0 mg /mL.

Biological Activity Botanical Name Part Used Bioassay Model Reference

C. paniculatum. EtOH ext. of leaves Method of Agar plate diffusion (S. aureus, Desta et al. 1993 Streptococcuss, Salmonella , E. coli,P. vulgaris, P. aeruginosa) Drug conc. : 0.19 mL /disc (1000 µg/ mL.

H2O ext. of leaves Method of Agar plate diffusion (S. aureus, Desta et al. 1993 Streptococcuss, Salmonella , E. coli,P. vulgaris, P. aeruginosa) Drug conc. :0.19 mL /disc (1000µg/ mL. C. quadrangulare. MeOH ext. of leaves Assay of well-diffusion Agar plate (H. pylori) Ohsugiet al. 1997

95% EtOH ext. of seed Assay of well-diffusion Agar plate. Many organisms Somanab et al. 1980 of gram positive used.

C. racemosum 40% EtOH ext. of leaves Assay of well-diffusion Agar plate (S. aureus) Drugs Olukoya et al. 1993 conc.: 0.32 g /mL having zone of inhibition ≥ 20 mm diameter.

C. raimbaultii EtOH /H2O (1:1) ext. Assay of well-diffusion Agar plate. (S. aureus and E. Mela et al. 1950 coli)

Biological Activity Botanical Name Part Used Bioassay Model Reference

C. erectus MeOH ext. of fruit Assay of well-diffusion Agar plate (S. aureus) Drugs McDonnell et al conc.: 0.30 g/mL with zone of inhibition ≥ 21 mm

diameter. T. arjuna MeOH ext. of leaves Assay of well - diffusi on Agar plate ( H. pylori) Drugs conc.: 0.32 g/mL with zone of inhibition ≥ 18 mm Shinde et al. 2009 diameter.

Antifungal C. aculeatum H2O ext. of leaves Assay of well-diffusion Agar plate (Candida albicans) Almagboul et al. 1988 Activity C. acutifolium Hexane and Microdilution technique (C. albicans) Drugs MIC: Masoko et al. 2007 MeOH ext. of leaves 0.15, 2.4, 2.8 mg/mL.

C. apiculatum Acetone, Hexane and Microdilution technique (M. canis , A. fumigates ) MeOH ext. of leaves. Drugs MIC: 2.5, 0.02 mg/mL.

C. albopuctatum Acetone, Hexane and Microdilution technique (C. albicans) Drugs MIC: Masoko et al. 2007

MeCl2 ext. of leaves. 0.54, 2.2, 0.22or 0.22 mg/mL.

Biological Activity Botanical Name Part Used Bioassay Model Reference

C. albopuctatum Acetone, Hexane, MeCl2 Microdilution technique (C. albicans) Drugs MIC: 1.20, 2.0, 2.4 or 1.20 mg/ml.

Antiparasitic C. quadrangulare 95% Ether and EtOH ext. In vitro- techniques for Earthworms, drugs Somana et al. 1980 activity bark concentration: 0.25.0.5 and 1 mg/ mL.

C. bracteosum Acetone, Hexane, MeCl2 Microdilution technique (C. albicans) Drugs MIC: andMeOH ext. of leaves 1.20, 2.0, 2.4 or 1.20 mg/ml. C. caffrum Acetone and MeOH Microdilution technique (C. albicans) Drugs MIC: extract of leaves 1.20, 2.0, 2.4 or 1.20 mg/ml

Biological Activity Botanical Name Part Used Bioassay Model Reference

C. collinum MeOH extract of root Assay of well-diffusion Agar plate C. albicans or A. Abreu et al. 1999 niger) Drug c: 2.0 monc.g/mL

Acetone extract of leaves In vitro-Worms of C. elegansvar. Bristol McGaw et al. 2001 Concentration for drugs: 0.5 and 1 mg/mL

C. collinum Hexane, MeCl2 andMeOH Microdilution assay (C. albicans) MIC for all drugs: Masoko et al. 2007 extract of leaves 0.64 mg/mL

C. glutinosum EtOH/H2O (1:1) extract of Microdilution technique (M. gypseum, Baba-Moussa et al.1999 leaves Tricophytonmentagrophytes or Tricophyton rubrum) Drugs MIC: 0.15, 2.0, 1.20 or 0.15 mg/mL.

C. glutinosum H2O / EtOH (1:1) leaves Microdilution technique (C. albicans) Drugs MIC: Baba-Moussa et al.1999 extract 0.15, 2.0, 1.20 or 0.15 mg/mL. C. hereroense Hexane and MeOH Microdilution technique (C. albicans) Drugs MIC: Masoko et al. 2007 extract leaves 0.15, 2.0, 1.20 or 0.15 mg/mL.

Biological Activity Botanical Name Part Used Bioassay Model Reference

C. hispidum H2O .EtOH (1:1) leaves Microdilution technique (C. albicans) Drugs MIC: Moussa et al. 1999 extract 0.15, 2.0, 1.20 or 0.15 mg/mL.

C. imberbe Hexane, MeCl2 Microdilution technique (C. albicans) Drugs MIC: Masoko et al. 2007 MeOH extract of leaves 0.15, 2.0, 1.20 or 0.15 mg/mL.

C. kraussii Acetone, Hexane, MeCl2 Microdilution technique (C. albicans) Drugs MIC: 0.15, 2.0, 1.20 or 0.15 mg/mL.

C. micranthum MeOH and MeOH-H2O Agar plate with diameters inhibition zones (C. Abreu et al. 1999 ext. of leaves albicans) Concentraton for drugs: 5 mg/mL with inhibition zone of 11 mm C. microphyllum Hexane and MeOH ext. of Microdilution technique (C. albicans) Drugs MIC: Masoko et al. 2007 leaves 0.15, 2.0, 1.20 or 0.15 mg/mL.

Biological Activity Botanical Name Part Used Bioassay Model Reference

C. moggi Acetone and Hexane Microdilution technique (C. albicans) Drugs MIC: extract of leaves 0.15, 2.0, 1.20 or 0.15 mg/mL. C. molle MeOH extract of bark Microdilution technique (C. albicans) Drugs MIC: Khan et al. 2000 0.15, 2.0, 1.20 or 0.15 mg/mL. MeOH extract of root tube dilution macro-broth method (C. albicans) MIC Steen kamp et al. 2007 for drug: 1 mg/mL

C. mossambicense Acetoneand Hexane Microdilution technique (C. albicans) Drugs MIC: Masokoet al.2007 extract of leaves 0.15, 2.0, 1.20 or 0.15 mg/mL. C. nelsonii Hexane and MeOH extract Microdilution technique (C. albicans) Drugs MIC: of leaves 0.15, 2.0, 1.20 or 0.15 mg/mL.

C. nigricans EtOH/H2O (1:1) ext. of Microdilution technique (C. albicans) Drugs MIC: Moussa et al. 1999. dried leaf 0.15, 2.0, 1.20 or 0.15 mg/mL.

Biological Activity Botanical Name Part Used Bioassay Model Reference

C. padoides Hexane and MeOH Microdilution technique (C. albicans) Drugs MIC: Masoko et al. 2007 extract of leaves 0.15, 2.0, 1.20 or 0.15 mg/mL.

C. paniculatum H2O extract of leaves Assay of well-diffusion Agar plate (C. albicans) Desta et al, 2000 Drug conc.: 0.10 mL/disc (1,000 µg/mL) C. petrophilum Hexane and MeOH extract Microdilution technique (C. albicans) Drugs MIC: Masoko et al, 2007. of leaves 0.15, 2.0, 1.20 or 0.15 mg/mL. C. zeyheri MeOH extract of stem Assay of well-diffusion Agar plate (C. albicansor Sawhney et al, 1994 T. mentagrophytes Drugs conc.: 0.02 mg/mL Hexane and MeOH extract Microdilution technique (C. albicans) Drugs MIC: Masoko et al, 2007 of leaves 0.15, 2.0, 1.20 or 0.15 mg/mL. Hypoglycemic C. decandrum 70% EtOH ext. of leaves Diabetic rat (Streptozotocin induced) Drug dose: Pannangpetch et al,2003 Activity 0.75 g/ kg (p.o.)

C. micranthum H2O ext. of leaves Diabetes indueced by alloxan (Type 1 and 11) Chika et al, 2003 Drug dose: 150 and 300 mg/kg (p.o.)

Biological Activity Botanical Name Part Used Bioassay Model Reference

T.arjuna Ethanol ext. Bark Diabetic rat (Streptozotocin induced) Drug dose: Raghwan and kumara 2006 0.75 g/ kg (p.o.)

Antiinflammatoy C. collinum H2O ext. of bark Inflammation indced in mice ear by (TPA) Drug Recio et al. 1995 Activity dose: 0.5 mg/ear

C. dolichopetalum MeOH ext. of root Induction of mice paw edema by carrageenan Drug Asuzu et al. 1998 dose: 100, 200 and 400.0 mg/kg (p.o.)

CHCl3 ext. of root Induction of edema in mice ear by Croton oil Drug dose: 0.25, 0.5 and 1.0 mg/ear.

C. apiculatum. H2O, AcOEt ext. of leaves In vitro-Cyclooxygenase-1 (COX-1) inhibition by McGaw et al. 2001 radioactivity bioassay. C. duarteanum EtOH ext. of leaves Induction of paw edema in mice by Arachidonic Gouveia et al. 2011 acid. Drug dose: 150 and 300 mg /kg (i.p)

Biological Activity Botanical Name Part Used Bioassay Model Reference

C. edwardsii H2O and AcOEt ext. of In vitro Radioactivity bioassay through inhibition of McGaw et al. 2001 leaves Cyclooxygenase-1 (COX-1)

C. erythrophyllum H2O and AcOEt ext. of In vitro Radioactivity bioassay through inhibition of leaves Cyclooxygenase-1 (COX-1)

C. hartmannianum. EtOH ext. of leaves In vitro Radioactivity bioassay through inhibition of Eldeen et al. 2007 Cyclooxygenase-1 (COX-1) Drug conc.: 250 μg/mL

C. hereroense H2O ext. of leaves In vitro Radioactivity bioassay through inhibition of Cyclooxygenase-1 (COX-1)

C. imberbe H2O and AcOEt ext. of In vitro Radioactivity bioassay through inhibition of McGaw et al. 2001 leaves Cyclooxygenase-1 (COX-1)

T. arjuna EtOH ext. of Bark In vitro Radioactivity bioassay through inhibition of Halderet al. 2009 Cyclooxygenase-1 (COX-1) dose of 400mg kg-1

Biological Activity Botanical Name Part Used Bioassay Model Reference

C. micranthum. H2O ext. of leaves Induction of paw edema in mice by Cotton pellet Olajideet al. 2003 granuloma formation Doses for drug: 15 mg/kg (p.o.)

Antinociceptive C. duarteanum. EtOH ext. of leaves Acid induction of writhingand hot-plate nociception Gouveia et al. 2011 Activity tests in mice Drug dose : 150 mg/kg(p.o)

C. leprosumMart. EtOH ext. of flowers inducution of nociception by formalin in mice Drug Pietrovski et al. 2006 dose : 150 mg/kg(p.o) T. arjuna EtOH ext. of Bark Acid induction of writhingand hot-plate nociception Halder et al. 2009 tests in mice Drug dose : 400 mg/kg(p.o)

70% EtOH exttract of Tail immersion test and Induction of painby Lira et al. 2002 bark formalin in mice Drug dose: 25.0 mg /kg (i.p.) Antioxidant C. decandrum 70% EtOH extract of Induction of xylenol orange in rats by ferrous ion Pannangpetch et al.2008 Activity leaves oxidation and thiobarbituric acid reactive substance Drug dose: 0.75 g/kg (p.o.)

Biological Activity Botanical Name Part Used Bioassay Model Reference

T. arjuna EtOH extract of Fruit Induction of xylenol orange in rats by ferrous ion Sun et al. 2008 oxidation and thiobarbituric acid reactive substance Drug dose: 0.75 g/kg (p.o.) conc. 25 microM. C. erectus MeOH extract of Fruit Phosphormolybdenum method and scavenging activity of nitric oxide assays.

C. duarteanum EtOH extract of leaves Hydroxyl radical scavenging and nitric oxide Gouveia et al. 2011 activity.

Anti-tumour C. caffrum CHCl3, CCl4 and CH2Cl2 Cell culture technique (in vitro) glioma cell immature Pettit et al. 1987 Activity Ext. of fruit astrocytoma 224c. Drugs conc.: 1.0, 10,100 μg/mL.

MeCl2 ext. of root bark Inhibition cell growth by Murine P-388 lymphocytic George et al. 1982 leukemia.

Biological Activity Botanical Name Part Used Bioassay Model Reference

C. collinum MeOH, and MeOH-H2O Cell culture technique (in vitro) Squamous carcinoma Abreu et al. 1999 ext. of leaves KB, Melanoma SK MEL28, lung carcinoma A549,

Drugs IC50: 21.0 μg/mL T.arjuna MeOH ext. of Bark Comet assay and micro nucleus activity(MN) Reddy et al. 2008

Antitussive C. glutinosum H2O ext. of leaves Using Guinea pig drug dose: 1.0 mg /kg (p.o.) Ngaba et al. 1980 Activity Antiviral C. glutinosum Leaves decoction Viral hepatitis B antigen HBsAg drug IC50:150.0 – Pousset et al. 1993 Activity 550 ng /mL

C. micranthum MeOH ext. of leaves Culturing of cell (In vitro) : infected cells of African Farrea et al. 1993 monkey with virus Herpes simplex and H. simplex drug conc.: 6.0 μg /mL C. paniculatum MeOH ext.of leaves Culturing of cell (In vitro) : MT-4 infected cells Asres et al. 2001 with human virus immunodeficient (HIV 1) for

drug IC50: 5.1 μg/ mL.

Biological Activity Botanical Name Part Used Bioassay Model Reference

C. quadrangulare 95% EtOH and H2O ext. Culturing of cell (In vitro) HIV 1 integrase inhibition Tewtrakul et al. 2003

of leaves. with virus HIV 1) drugs IC50: 2.1 and 2.7 μg/mL. T.arjuna MeOH ext. of Bark in vitro plaque reduction assay 3.6-+0.9 Cheng et al.2002

C. molle. H2O and MeOH ext. of Reverse transcriptase ( HIV1) RNA-dependent-DNA Bessong et al. 2005

roots polymerase (in vitro) activity. Drugs IC50: 36or 9.6 μg /mL. Immunostimulant C. micranthum. Leaves powder Clearance rate of colloidal carbon in mice Drug dose Di Carlo et al. 1964 activity Suspension : 100 mg/ kg (i.v.)

T.arjuna MeOH ext. of Bark Clearance rate of colloidal carbon in mice Drug dose Halder et al ,2009 : 400 mg/ kg (i.v.)

Cardiovascular C. hypopilinum MeOH ext. of seed Cardiac depressant activity in rabbit Bamgboseet al. 1977 Activity

Biological Activity Botanical Name Part Used Bioassay Model Reference

C. ovalifoliumvar EtOH / H2O (1:1) extract Cardiac depressant activity Cat drug dose: 45.0 mg/kg Dharet al. 1973 of leaves C. sokodense MeOH extract of seeds Cat hypotensive drug dose: 200.0 mg/kg (i.v.) Bamgbose et al. 1977

C. verticillatum MeOH extract of seeds Cat hypotensive drug dose: 200.0 mg/kg (i.v.) Dharet al. 1973

T.arjuna MeOH extract of Bark 63,125 and 250 mg kg-1 for kumar et al. 2009 anti fibritic in rats

CNS activity C. hypopilium MeOH extract of seed Mice CSN depressant drug dose: 0.50 mg /kg (i.p.)

C. nigricans MeOH extract of seed Mice CSN depressant drug dose: 0.25 mg /kg (i.p.) C. paniculatum MeOH extract of seed Mice CSN stimulant drug dose: 0.5 mg/ kg (i.p.)

Biological Activity Botanical Name Part Used Bioassay Model Reference

Toxicity C. erythrophyllum H2O extract of roots Agar plate method by S. typhimurium TA 100 and Sohni et al. 1994 Mutagenicity TA 102 drug conc.: 30.0, 60.0, 80.0, 100 μg /disc.

Cytotoxicity C. apiculatum MeOH extract of leaf Cell culturing method (bladder T24 or breast cancer Fyhrquist et al. 2006 MCF7 ) Drug conc.: 30 µg/mL MeOH extract of root Cell culturing method (bladder T24 or breast cancer MCF7 ) Drug conc.: 30 µg/mL C. collinum MeOH extract of leaves Cell culturing method (bladder T24 or breast cancer MCF7 ) Drug conc.: 30 µg/mL MeOH extract of roots Cell culturing method (bladder T24 or breast cancer MCF7 ) Drug conc.: 30 µg/mL C. comosum MeOH extract of leaves Cell culturing method (human THP1 monocytes) Lamidi et al .2005

Drugs IC50 : >120, >150 μg/mL.

C. cuspidatum MeOH extract of roots Cell culturing method (human THP1 monocytes)

Drugs IC50 : >120, >150 μg/mL.

Biological Activity Botanical Name Part Used Bioassay Model Reference

C. duarteanum 95% EtOH ext. of leaves Cell culturing method (KB cells) Drug conc.: 25 Nascimento et al.1990 µg/mL

C. fragrans MeOH extract of leaves Cell culturing method (bladder T24 or breast cancer Fyhrquist et al. 2006 MCF7 ) Drug conc.: 30 µg/mL

C. fruticosum EtOH extract of leaves Cell culturing method (CA-9KB) Drug ED50: 5.5 Nascimento et al. 1990 μg/mL. C.erectus MeOH extract of Fruit Cell culturing method (human THP1 monocytes) Thakore et al.2011

Drugs IC50 : >120, >150 μg/mL.

Gastric activity T.arjuna EtOH ext. of Bark Cell culturing method (bladder T24 or breast cancer Devi et al. 2008 MCF7 ) Drug conc.: 30 µg/mL

wound healing T.arjuna HCl ext. of Bark Cell culturing method Drug conc.: 30 µg/mL Chaudhri et al. 2006 Activity

Biological Activity Botanical Name Part Used Bioassay Model Reference

Reproductive T.arjuna EtOH ext. of Bark 10 mg kg-1b.wt. for 2 days Manna et al. 2008 Activity Antiatherosclerosis T.arjuna EtOH ext. of Bark 50% ethanol extract 100-50mg kg-1 Shaaila et al.1998 Activity

Hepatoprotective C.erectus MeOH ext. of Methanol extract of all parts of plant Hameed et al. 2012 Activity Fruit,roots, leaf of dose 500 mg/kg in mice.

-1 T.arjuna H2O ext. of Bark Aqueous extract of bark 50 mg kg b.wt. Manna et al, 2007

CHAPTER 3

74 3 MATERIALS AND METHODS

3.1: Plant material Conocarpus lancifolius was collected from prepharies of Lahore (Pakistan) in august 2012. The plant was authenticated as Conocarpus lancifolius by Professor Dr. Altaf Ahmad Dasti, Taxanomist Institute of pure and applied Biology, Bahauddin Zakariya University, Multan, Pakistan. The voucher specimen has been deposited in herbarium [R.R. Stewart 271(7).

3.2: Solvents and chemicals Chemicals used fo extraction and isolation purposes are dichloromethane, chloroform, methanol, ethyl acetate, n-hexane, Vanillin, (20 x 20) TLC plates of alluminium and Merck Silica gel 60 F254.

3.3: Reagents and solutions preparation

3.3.1: H2SO4reagent

Conc. H2SO4 (10%) was mixed with ethanol.

3.3.2: Godin reagent Solution: A Prepared by vanillin mixing with ethanol (1%). Solution: B Prepared by purified water mixing with Perchloric acid and having perchloric acid 70%. Mixing of A and B solutions in same volume (Godin 1954)

3.3.3: Dragendorff’s reagent 2.1 g of bismuth oxynitrate (basic bismuth subnitrate) was suspended in tartaric acid 20 g in 40 ml water. Potassium iodide 40 ml, 40% w/v solution was added. Filtration was done after one hour stirring and this solution was protected from heat (B.P 1993).

3.3.4: Mayer’s reagent 1.26 g of mercuric chloride was mixed with 60 ml distelled water and A solution of mercuric chloride was prepared. Potassium iodide 5g was mixed with 20 ml of distelled water and solution B of potassium iodide was prepared. Both solutions (mercuric chloride and potassium iodide) were mixed and make the final volume upto100 ml. (B.P 1988).

75 3.3.5: Wagner’s reagent Potassium iodide 4g was taken and dissolved in 10 ml 0f water. Putt iodine (2 g) into it than make final volume upto 100 ml by adding distlled water (B.P 2012).

3.3.6: Hager’s reagent 1g of picric acid was dissolve in 100 ml waeter. By aadding picric acid saturated solution ismade and than filter the saturated solution (B.P 2012).

3.3.7: Concentrated H2SO4

It has minimum 95% H2SO4 and maximum 100.5% sulphuric acid (B.P 1993).

3.3.8: Dilute H2SO4 104 g of sulphuric acid is added to purified water (896 g) stirred than cooled (B.P 1993).

3.3.9: Gelatin solution Gelatin solution 1% has sodium chloride 10%.

3.3.10: Chloroform Trichloromethane having usually ethanol 1-2% v/v or amylene 50 mg/liter (B.P 2012).

3.3.11: Concentrated HCL Concentrated HCL has minimum 35% w/w and maximum 39% w/w hydrochloric acid (B.P 1993).

3.3.12: Dilute HCL HCL 274 gm was taken and is mixed with distilled water (726 gm) (B.P 1993).

3.3.13: Solution of lead subaceate lead acetate 40 g was mixed with water 90. PH was adjusted at 7.5 by sodium hydroxide 10 molar solution. Clear supernatant layer solution was obtained by centrifugation. It has minimume 16.7% w/w and maximum 17.4% w/w lead (B.P 2012).

76 3.3.14: 90% Ethanol 934 ml of 96% ethyl alchol is diluted with distilled water upto 1000 ml (B.P 2012).

3.3.15: Dilute acetic acid 33% acetic acid (182 gm) was mixed 818 gm distilled water (B.P 1993).

3.3.16: Strong ammonia solution Strong ammonia solution has maximum of 30% w/w and minimum 25% w/w having ammonia (B.P 2012).

3.3.17: Dilute ammonia solution 10% w/w of ammonia is called dilute ammonia solution. Preparation was done by the dilution of strong solution of ammonia with disttilled water (B.P 2012).

3.3.18: Ferric chloride solution Ferric chloride 9 g is dissolved in distilled water and makethe final volume upto 100 ml.

3.3.19: Glacial acetic acid

It has minimum 99% C2H4O2 and maximum 100.5% of C2H4O2.

3.4: Glasswares / Equipments / Instruments

Silica gel 60 F254 TLC Plates, Mass spectrometer 600H, Analytical balance, Conical flasks, Pasteur pipettes, Quickfit Pyrex Distillation flask, Graduated cylinders, Power Lab Data Acquisition System, EZ-Fit Enzyme Kinetic Software, Rotavapor R-200 having recirculating chiller B-740, Magnetic stirrer (nuova П) thermolyne, High performance liquid chromatography, Sonicator, digital ultrasonic Capillaries of 1.1 mm for spotting, Reagent bottles, NMR ADVANCE AV-600,

Separating funnel, CAMAG Ultaviolet visioner, DESAGAGmbH tank for TLC, TELB- 300 top load balance, Volumetric flasks and Lambda 25 ultraviolet, spectrophotometer

77 3.5: Phytochemical methods 3.5.1: Extraction For successful extraction, plant material (whole plant) Conocarpus lancifolius was kept for 18 days under shade and dried it. The shade dried material was crushed to fine powder with the help of pestle mortar. Extraction of poder material was done by method of maceration with shaking occasionally for 24 hours. The 900 g of powdered material of whole plant was weight and putt it into wide mouth large container having cap. Extraction is carried by dichloromethane (3×2500 ml) and than with methanol (3×2500 ml) was done successively. The both filtrates were solidified to residue by evaporation by using rotavapour. The dichloromethane and methanol residue were collected from flask and shifted to small separate jars. Sonication was also done by dissolving methanol to recover extract.

3.5.2: Phytochemical screening TO evaluate and identify the chemical constituents present in samples of drug the phytochemical screening is performed. The chemical tests may be particular for specific compound or may be general for a particular compounds. Precipitation or coloration may appear. The color must be compared to standard and if precipitation reactions appears turbidity is only detected by comparison with control having reagent only (Brain and Turner 1975).

3.5.2.1: Screening of cardiac glycosides 10 ml of alcohol (70%) was mixed with 1g powdered drug. Than heated the mixture to boil for 2 min with the help of water bath and than filtered. Than distilled water (double volume) was added to filtrate and strong lead sub acetate solution was added to it. The solution was filtered to remove pigments and chlorophyll. Than it was mixed with same volume of chloroform by vigorous shaking. Chloroformlayer was pippett and evaporate it to dryness. Than it was treated with ferric chloride 3.5% as 3 ml in acetic acid and shift it in other test tube. The of conc. H2SO4 about1.5 ml was putted along the wall of tube that makes layer in bottom. If pale green coloration appears in top layer and brownish coloration appears at interface by standing conforims the cardic glycoside (Brain and Turner 1975).

78 3.5.2.2: Anthraquinone detection The test methods performed for screening anthraquinone are given as; 3.5.2.2.1: Borntragers test: Extraction of powder drug (5g) was done with hot water and than filteration was done while it was hot. It was cooled and than extracted with the help of carbon tetrachloride. The layer of carbon tetrachloride removed and by after washing with water treated with dilute solution of ammonia. The appearance of pinkish or cherry red coloration in ammonia layer shows free anthraqunoons in the extract. 3.5.2.2.2: Modified Borntragers test: Extraction of powder drug (5g) was done with solutions of ferric chloride and hydrochloric acid. Than warmd 12 minutes on water bath and filteration was done while it was hot. It was cooled and than extracted with the help of carbon tetrachloride. The layer of carbon tetrachloride removed and by after washing with water treated with dilute solution of ammonia. The appearance of intense pinkish or cherry red coloration in ammonia layer shows the bound anthraqunoons in the extract. (Brain and Turner 1975).

3.5.2.3: Screening of alkaloids 5 g sample was taken and dilute HCL was added than boiled than filtered and dilute ammonia solution was mixed untill it becomes alkalineExtraction of this solution is done by adding chloroform 5ml. Remove chloroform portion and extract with dilute acetic acid 10ml. Take acetic acid portion and add Dragendorff’s reagent few drops. The appearance of Orange precipitate or turbidity shows that alkaloids are present in sample (Brain and Turner 1975).

3.5.2.4: Detection of saponin glycosides Vigorous shakeing of 5g sample with water and than observed the forth production. 20 minutes persistant forh shows that saponins are present (Brain and Turner 1975).

3.5.2.5: Tannins screening 5g sample was taken and extracted by using methanol. 10% ferric chloride was mixed to the methanol extract. The appearance of or greenish grey or dark blue color shows that tannins are present in the sample.

3.5.2.5.1: Catechin test In aqueous plant extract a match stick was dipped, dried than moistened with conc. Hydrochloric acid and warmed near flame.the appearace of red or pink color shows the catechin in the extract (Brain and Turner 1975).

3.5.2.5.2: Ferric chloride test Solution of ferric chloride 1-2 ml was mixed with same volume of extrct solution. The appearanece of blue black precipitate shows the existace of hydrolysable tannin and if brownish green precipitate appears shows that condensed tannins wewre present.

3.5.2.5.3: Gelatin test A portion of filtrate was mixed with 1% gelatin solution having sodium chloride 10%. Appearance of white precipitates appeared shows that tannins were present.

3.5.2.6: Screening of Flavonoids The methods for evulation of flavonoids are as;

3.5.2.6.1: Solution of lead acetate Few drops of 10% lead acetate solution was mixed with a portion of sample as a result the appearance of yellow precipitates shows the flavonoids presence (Bhandary et al. 2012).

3.5.2.6.2: Test of ferric chloride Fecl3 (few drops) was added to sample the black red coloration appearace shows the flavonoids presence (Bhandary et al. 2012).

3.5.2.6.3: Method of alkaline reagent Sample was reacted to sodium hydroxide the appearance of intense yellowish color which disappeared by the mixing dilute HCl some drops shows flavonoids present in sample (Bhandary et al. 2012).

3.5.2..7: Detection of triterpenoids The chloroform solution of extract, acetic anhydride few drops were mixed, heated to boil, cooled and filtaration was done on cooling. To this filtrate some drops of conc.

H2SO4 was mixed with the wall. The appearance of brownish ring at interface of both layers. Deep red color in the lower and green color of the upper layer shows triterpenoids present in sample (Bhandary et al. 2012). 3.5.3: Techniques for chromatography 3.5.3.1: TLC (Thin layer chromatography) 3.5.3.1.1: Material to be required Solvents (ethyl acetate, chloroform, dichloromethane, isopropyl alcohol, n-hexane and methanol), distilled water, testing samples , precoated TLC plates, spotting capillaries, Tanks for TLC, Ultraviolent illuminator, oven and reagents for spot locating.

3.5.3.1.2: Method

Dichloromethane extract (40 mg) was mixed with 3.0 ml of dichloromethane and similarly 40 mg methanol extract was mixed in 3 ml of methanol. The solutions were separately prepared with the help of vortex mixer (stuart) in sample glass vials. Vortex mixer was rotated at 2500 revolutions per minute. To make the solutions clear sonicatar is used. TLC plates of (20x20 cm) were taken and at distance of 1.5 cm from each side marked and than by cutting smaller plates (10x10 cm) was made . The bottom line was marked at distance of one centimeter. By using capillary tube 5-10 ul of all samples (test) were applied. Apply all samples for testing at same space on bottom line and in the center of cutted plate apply standard sample. Mobile phase about 20 ml was taken in saturate TLC tank. After saturation of tank the plate having samples and standered was putted into it and allowed to develop. When one centimeter distance left from top edge the TLC plate was taken out and marked the solvent front. The plate was examined after drying with the help of UV illuminator at 254 nm and 366 nm. Solid lines and dotted lines was drawn for marking of spots and than plate was sprayed with godin reagent and with 10% sulphuric acid reagent. After spraying plates were heated for 5 minutes at 115°C in oven. Different colors appear after heating and observed. The marking of spots was done as

254nm 366nm Godin reagent /////////

81 3.5.3.1.3: System of solvents for TLC Different systems applied for TLC analysis of dichloromethane and methanol samples are presented in table 3.1 and 3.2.

Table 3.1: Solvent systems used for TLC analysis of dichloromethane extracts.

Solvent System Ratio Chloroform : Methanol 97 : 3 95 : 5 89 : 11

n- hexane : Ehtyl acetate 80 : 20 75 : 25 50 : 50

n- hexane : Methanol 90 : 10 80 : 20

n- hexane : Isopropanol 90 : 10 80 : 20

Dichloromethane : Methanol 80 : 20 70 : 30

Table 3.2: Solvent systems used for TLC analysis of methanol extracts. Solvent system Ratio

60 : 40 : 5 Chloroform : Methanol : Water 70 : 30 : 4 80 : 20 : 2 85 : 15 : 1 100 : 14 : 7 100 : 13.5 : 10 100 : 10 : 5 Ethyl acetate : Methanol : Water 98 : 4 : 2 95 : 10 : 5 95 : 5 : 3

82 3.5.3.2: Column chromatography

3.5.3.2.1: Material Required

Stationary phases (Silica gel 60 (40-63 μm and 63-100 μm), Sephadex LH- 20].

3.5.3.2.2: Columns Q 50/28 CR 60/50, Q20/25 CR 40/50, Q 24/14 CR 40/30, Q 25/32 CR 40/40 and Q 44/40 CR 20/30

3.5.3.2.3: Outline of method Glass column was properly washed, cleaned and dried for the column chromatography. Sample applied onto column was made clear solution by dissolving in sufficient quantity of solvent. Sample is loaded on silica gel by thorough mixing and than dried and prepared it into free flowing form. The length of column was measured. Selected mobile phase was filled in one third prtion of column. At the bottom of column a thin cotton plug was placed iside the column. The slurry was made with ratio of sample: silica gel (1:30). By pouring slurry into column by using the selected mobile phase, the column was packed and than allowed to settle. Wait as column fully settled (silica gel) completely. The mobile phase present in excess in column was drained till silica gel shiny surface appered. Sample was applied in column and mobile phase was passed through column already defined rate. Fractions of known volume were collected and analytical thin layer chromatography was performed. Elution was done by isocratic and stepwise methods.

3.6: Biological techniques 3.6.1: Assay of Brine shrimp lethality Materials Brine shrimp eggs (Artemia salina), perforated small tank having cover and dam, syringes (0.5 ml, 5.0 ml, 10 µl and 100 µl), 2 vials dram (1control + 9 each sample), solvents (DCM, MeOH, DMSO, CHCl3), purified water and test samples. Procedure 3Sea salt about 38 g was dissolved in one liter of water and artificial sea water was made. “Sea water wain placed in tiny l irregularly portioned tank and shrimp egg (Artemia salina) was placed in the big section of tank which was made dark with the help of

83 aluminium foil. Nauplii (shrimp larvae) are attracted through perforations in the dam of illuminated portion. The shrimps remain for 48 hr at (22-29°C) temperature where they hatch and mature. Testing vails were organized for each portion and were analysed primarily at 10, 100, and 1000 µg/ml, replication for each strength were made by having 9 vials total. The sample was weighed as 20 mg and 20 mg/2 ml of organic solvent about 2 ml was added to it. 5 µl, 50 µl and 500 µl of this solution were placed to corresponding vials to 10, 100, and 1000 µg/ml. Evaporation of solvents was done wit the help of nitrogen and then placed under high vacuum for about 30 min. Shrimps(10) and sea water (5 ml) were placed after 2 days to every vial by Pasteur pipette having per dilution 30 shrimps. Illumination of these vails was maintained. After 24 hr surviving number of shrimps was noted and LC50 was determine at confidence interval 95%” (Meyer et al. 1982).

3.6.2: Agar well diffusion technique for antibacterial activity Material Bacillus subtilis, Escherichia coli, , Salmonella typhi, sterile cork borers, nutrient broth, pipettes, solvents (DCM, MeOH, , CHCl3, DMSO), standard (Imipenum), incubator, test samples Procedure Nutrient broth (10 ml) with test organism inoculated for 24 hours at 37°C. Broth culture (0.6 ml) and organism for testing was kept to molten agar 60 ml which was cooled already to 45°C. After through mixing it was poured into a sterild well petri dish (0.2 ml culture for 9 cm petri dish is placed to 20 ml of agar). For every organism a duplicate plate was organized. When settled and hardens the agar, it was ensured the prorper number of holes by cork and ensured distribution of cups holes as centric and in the periphery. Plugs of agar were detached. Fresh cork borer were arranged for each organism. Proper organic solvent was dissolved about 100 µl of testing sample and also in labeled cups of same concentration .these were placed for 2 hr at room temperature. And allow sample diffusion. By placing cups straight incubated for 24 hr at 37°C. The inhibited zone and diameter was recorded in mm (size of cup also noted) (Leven et al. 1979).

84 3.6.3: Agar tube dilution technique for antifungal activity Requirments Candida albicans, Aspergillus flavus, Trichphyton longifusus, Candida glabrata, autoclave, screw test tubes, micropipettes, dextrose agar, antifungal drugs (standard). Miconazole, amphotericin-B and test samples Procedure Sterile DMSO was added in testing sampe and made the stock solution. Sabouraud glucose agar (4%) and distilled water with agar are mixed and dextrose agar (sabouraud) was made. Magnetic stirrer was used for stirring and screw cap tubes were used for dispensing. Media containing test tubes were autoclaved for 15 min at 121°C and kept for cooling to 50°C. The sample of known amount and concentration was taken from stock solution by usig pipett and was mixed into sabouraud agar media (non solidified). Wait to solidification in slanting positionat at room temperature. Than inoculated with culture of fungi which is seven day old and 4 mm in diameter. All these tubes (culture containing) were kept for 7-10 days at28-30°C optimum temperature for growth. 40% to 50% humidity condition was maintained with the help of water in incubator open pan. During incubation cultures were analysed twice a week. Minimum inhibitory concentration (µg/ml) was examined after 7-10 days of incubation (Rehman et al. 2001).

3.6.4: Phytotoxic activity assay Materials Lemna minor, 2 dram vials, syringes (5 ml, 2μl, 10μl, 50μ1) container of glass having glass lid for vials holdig, E-Medium, chamber for growth , fluorescent (continuous), incandescent light and samples for test. Procedure KOH pellets were mixed and pH 5.5–6.0 was maintained and inorganic medium was prepared. Vials per dose (500, 50, 5 ppm, control) 10 vials for testing were made. 15 mg of extract was weighed and mixed with solvent about 15 ml. 10, 100, and 1000 μl solutions were mixed with 5, 40 and 500 ppm vails and evaporate the solvent overnight. 2 ml of E-medium with plant having three fronds rosette with every vial. These were kept in water filled 2 cm glass dish. Vials and dish was kept in chamber of growth at 26°C for seven days using fluorescent and incandescent light. After 3 day 3 and 7day data was examined and counted. Analysis of data was done as ED50

85 percentage by using computer program to recorded FI50 values and confidence interval 65% (McLaughlin et al. 1991).

3.6.5: Leishmanicidal assay Saline phosphate buffered having 0.5% DMSO and 7.4 pH 1.0 mg in 1 ml were mixed in test sample. Macrophage cultures (1.5×106 cells/well) 100 μl infected was added in 96 well microtiter plates. Portions (10 μl) having different amount of the test samples were mixed into the culture. Positive and negative controls were setted by mixing, 10 μl of phosphate buffered saline (PBS) having 7.4 pH with MeOH0.5% / DMSO 0.5%) and pentamidine, amphotericin Band managed finally to of 1.0 mg/ ml concentration respectively. They were allowed to grow in humid chamber at temperature 36–37°C existence of CO2 5% minimum for about 6 days.the organisms in this period multiply for about 1.5–3.0 times. Than they were stained with giemsa and measured the ED50 of all compounds (Rehman et al. 2001).

3.6.6: Antioxidant assay For determination of antioxidant assay DPPH was used. Various series of compounds concentration were mixed in defined solvents to same volume (10 µL) to 90 µL of 100 µM methanol DPPH having 100 µL total volumes in plates 0f 96 well. All the contents were assorted and incubated at temperature of 37ºC for about 30 minutes. By use of microplate reader absorbance at 517 nm was measured”. Quercetin was used as standard control. The procedure was repeated in triplicates. By using enzyme kinetic software IC50 values were calculated. When absorbance decreases it in shows scavenging increases which was calculated by formula; Percentage Inhibition = (Control absorption - Test absorption / Control absorption) × 100 Calculation were performed in triplicate and calculations were presented Mean ± SEM (Koleva et al. 2002).

3.6.7: Acetylcholinesterase activity Acetylcholinesterase inhibitory activity was done by well known procedure (Ellman et al. 1961) with little alterations. 100 µL mixture volume was prepared having 60 µL - bufferin the form of Na2HPO4 and concentration 50 mM with 7.7 pH. (0.5 mM well 1) 10 µL test compound was mixed, and than by adding 10 µL enzyme. All

86 ingredints were added and readed at 405 nm. These were pre incubated at 37ºC for 10 minutes. As reaction starts by mixing 0.5 mM well-1 about 10 µL of substrate, and than by adding DTNB 0.5 mM well-1 about10 µL. As 15 min passed after incubation at temperature 37ºC by the use of 96-well plate reader absorbance at 405 nm was measured. All procedures were performed in triplicate. Positive control was used in the form of 0.5 mM well-1 eserine.

3.6.8: Butyrylcholinesterase assay Butyrylcholinesterase assay was done by well known procedure (Ellman et al. 1961) with little alterations . 100 µL of mixture volume was prepared having 60 µL buffer in -1 the form of Na2HPO4 and concentration 50 mM with 7.7 pH. (0.5 mM well ) 10 µL test compound was mixed, and than by adding 10 µL enzyme. All ingredints were added and readed at 405 nm. These were pre incubated at 37ºC for 10 minutes. As reaction starts by mixing 0.5 mM well-1 10 µL of butyrylthiocholine chloride (substrate), and than by adding DTNB of 10 µl As 15 min passed after incubation at temperature 37ºC by the use of 96-well plate reader absorbance at 405 nm was measured. All procedures were performed in triplicate. Positive control was used in the form of 0.5 mM well-1 eserine.

3.6.9: -Chymotrypsin assay The assay was done by well known procedure (Cannell et al. 1988). 100 µL mixture volume was prepared having buffer 60 µL in the form of Tris-HCl and concentration 50 mM with 7.6 pH. (0.5 mM well-1) 15 µL test compound was mixed, and than by adding 15 µL enzyme (0.005 unit well-1). All ingredints were added and readed at 410 nm. These were pre incubated at 37ºC for 10 minutes. As reaction starts by mixing 10 µL and N-succinyl phenylalanine-p-nitroanilide (substrate), and than by adding of 10 µL (0.5 mM well-1) DTNB. As 30-60 min passed after incubation at temperature 37ºC by the use of 96-well plate reader absorbance at 410 nm was measured. All procedures were performed in triplicate. Positive control was used in the form of 0.5 mM well-1 eserine. All data was collected as that of above section.

87 3.6.10: Lipoxygenase activity Lipoxygenase assay was done by well known method (Evans et al. 1987). Total 200 µL volume of reaction mixture was prepared having 150 µL sodium phosphate buffer and concentration 100 mM with 8.0 pH. (0.5 mM well-1) 10 µL test compound was mixed, and than by adding 15 µL enzyme (600 unit well-1). All ingredints were added and readed at 234 nm. These were pre incubated at tempreture 25ºC for 10 min. As reaction starts by mixing 25 µL substrate solution. As 6 min passed the chage of absobance was noted after incubation at temperature 25ºC by the use of 96-well plate reader.0.5 mM well-1 baicalin is used as control (positive). All procedures were performed in triplicate. All data was collected as that of above section

3.6.11: Urease assay Urease assay was done according to the well known method (Weatherburn 1967) with little modification. Total 85 µL volume of reaction mixture was prepared having 10 µL phosphate buffer and concentration 100 mM with 7.0 pH. (0.5 mM well-1) 10 µL test compound was mixed, and than by adding 25 µL enzyme (0.1347 unit). All ingredints were added and readed at 234 nm. These were pre incubated at tempreture 37ºC for 15 min and by aadding 40 µL(20 mM) urea (stock solution) . As the reaction starts by adding 25 µL substrate solution. As some time passed phenol hypochlorite 115 µL was added to every well. To develop color it kept more 10 min. at 370C 37. The absobance was noted at 625 nm. All procedures were performed in triplicate. All data was collected as that of above section.

3.6.12: α-Glucosidase activity assay The assay was done by well known procedure (Chapdelaine et al. 1978) with little modification. Total 100 µL volume of reaction mixture was prepared having 10 µL phosphate buffer and concentration 50 mM (70 µL ) with 6.8 pH. (0.005 mM well-1) 10 µL test compound was mixed, and than by adding 10 µL enzyme (0.005 unit well- 1). All ingridients were kept at 370C for 10 min. As treaction starts by mixing 10 µL of substrate . AS yellowish color appears that shows p-nitrophenol creation. Reading was done at 400 nm with the help of 96-well microplate reader. Acorbose is used as control (positive). All procedures were performed in triplicate.

88 3.7: Spectroscopic methods Ultraviolet spectras were done by using Lambda-25 spectrophotometer (Perkin Elmer). Infrared spectras were recorded on Infrared instruments (Alpha Bruker).

Magnetic resonance of proton was done by 500 MHz and 600 MHz NMR spectrometers (Bruker) TMS is used as internal standerd. The 13C-NMR spectras were recorded at 100 and 150 MHz by using same instruments as that of 1H-NMR. The 2D techniques (COSY, HSQC) spectras were done by 500 MHz and 600 MHz.Finnigan MAT 311 was used for recording EIMS with data system of MassPec. Finnigan MAT 312 mass spectrometer were used for field ionization, fields desorption and Peak matching. HRMS was done by Jeol JMS (HX 110) MS .

89 3.8:Spectroscopic and physical data of compounds A-G 3.8.1: A Compound (Quercetin 3-O-β-D-glucuronide)

Physical Data; Powder of yellowish color Quantity: 10 mg Melting Point:175-180 oC 25 [α]D : + 40(c = 0.021)

Ultraviolet λmax; 253, 267, 291nm -1 IR (KBr) max :3344, 2995, 1701 and 795 cm Proton NMR; (500 MHz) Signals of proton at δ 3.31-3.95 (5H, m, sugar-H), 5.24 (1H, duplet having J = 6.50 Hz, 1H), 6.22 (1H, s, H-6), 6.41, (1H, siglet H-7), 6.85 (1H, duplet J = 7.5 Hz), 6.43 (1H, dd, J = 2.0, 8.5 Hz, H-6'), 7.60 (1H, d, J = 2.0 Hz, H-2) 13C-NMR; (150 MHz) Signals at δ 71.4 , δ 73.8, δ 75.6, δ 75.6, δ 93.6, δ 98.8, δ 101.4, δ 103.9, 115.2, δ 116.2 (C-5'), 120.6, δ 120.9, δ 133.2, δ 144.9, δ 148.6, 156.3 δ, 156.3, δ 161.3, δ 164.3 (C-7), 168.7 (C-6''), 177.2 .92.4 δ 142.7, δ 118.4, δ 71.8. EI-MS; (rel. int.) m/z 478 (40), 324 (90), 232 (60), 184 (43) HR-EI-MS;

478 (for C21H18O13 , 478.3821)

90 3.8.2: B compound (lancifoliate )

Physical Data; Colourless amorphous powder Quantity; 13 mg Melting point; 160-165 oC

Ultraviolet λmax; 230 and 282nm IR (KBr); 2925.8, 2808, 1710, 1610, 1510 cm-1 Proton NMR; using (600 MHz) Resonance signals appears at δ 7.60 (1H, s, for H-8, δ 7.61 (1H, dd having J= 2.2, J= 1.7 Hz, H-6'), δ 7.62 (1H,d, having J= 0.7 Hz, H-2'), δ 6.95 (1H, doublet, J= 7.4 Hz, H-5) and δ 11.56. 13C-NMR; (150 MHz) Signals at δ 172.8, δ 156.7, δ 154.8 , δ 130.4 , δ 131.7, δ 128.7 , δ 129.2 , δ 122.8 (C-4), δ 121.1 (C-3), δ 110.3 (C-2), δ 109.8 (C-1), δ 151.7 (C-5), δ 150.2 (C-3), δ 103.5 (C-9), δ 89.3 (C-3), δ 62.7, δ 61.1, δ 54.2, δ 52.0 and δ 51.9 Methoxyl carbon. EI-MS; (rel. int.) m/z 334 (95), 184 (74), 373 (70), 165 (55), 335 (22), 69 (45)143 (17) and 85 (14) HR-EI-MS;

334.11 (for C18H22O2S2, 334.11).

91 3.8.3: C compound (lancifotarene)

Physical Data; Yellowish powder Quantity; 08 mg Melting point;155-160 oC

UVλmax; 230, 360 nm IR (KBr); 2925, 2866, 1726, 1521 and 1458 cm-1 Proton NMR using (600 MHz) Proton resonate at δ 7.30 (1 H, H-2), δ 7.24 (1H, dd, J = 3.2, J= 1.4 Hz, H-4), δ 7.29 (1H, d having J = 3.2 Hz, H-2'), δ 7.33 (1H, d having J = 0.6 Hz, H-5'), δ 3.82, δ 3.74, δ 3.92, δ 3.91, δ 4.0 and δ 4.02. 13C-NMR; using(150 MHz) Signals at δ 174.01(C-4), δ 162.72 (C-2), δ 104.2 (C-3), δ 150.06 (C-5), δ 127.7 (C- 6), δ 137.6 (C-7), δ 124.6 (C-8), δ 129.3 (C-9), δ 128.2 (C-10), δ 123.9 (C-1), δ 128.3 (C-2), δ 188.5(C-3), δ 130.2(C-4), δ 130.6 (C-5), δ 131.8 (C-6), δ 77.25, δ 77.9. EI-MS; (rel. int.) m/z 386 (65), 387 (100), 357 (10), 354 (15), 344 (22), 326 (19), 197 (22), 147 (16), 73 (15) HR-EI-MS;

386.19 (for C26H26O3, 386.19)

92 3.8.4: D compound (Lancifolian)

Physical Data; Colourless gummy solid Quantity: 07 mg Melting point.:138-145 oC 25 o [α]D : 62.0 (0.12).

Ultaviolet λmax; 230 , 222 nm. Infrared (KBr) ; 3419.6, 2928, 2125.4, 1652 and 1531cm-1. Proton NMR; using(600 MHz) Proton resonate at δ 5.79 (1H, s for 5 – OH), δ 6.72(1H, d having J = 1.4 Hz for H- 4), δ 6.87(1H, d having J = 1.3 Hz, H-6), δ 6.85(1H, d having J = 1.6 Hz, H-2), δ 7.4 (1H, J = 6.3 Hz, H-5), δ 7.35 (1H,dd, J = 6.3, 2.7 Hz, H-4), δ 4.56 (1H, d having J = 7.5, H-1´) . 13C-NMR; using150 MHz) Carbon at δ 61.7 , δ 70.3 (C-4″), δ 73.8 (C-2″), δ 76.8 (C-3″), δ 77.4(C-5″), δ 95.8 (C- 8), δ 100.5(C-6), δ 100.7, δ 103.7 (C-3), δ 106.3 , δ 114.8 (C-2″), δ 115.5 (C-5′), δ 130.3, δ 122.6 , δ 166.9 (C-7), δ 167.8(C-2) and at δ 168.1. EI-MS; (rel. int.) m/z 328 (72), 252 (185), 224 (20), 212 (15), 196 (20), 146 (19), 73 (14

HR-EI-MS. m/z: C14H16O9 ( for C14H16O9, 328.08)

93 3.8.5: E compound (Lancifolamide)

Physical Data; Colourless amorphous solid Quantity: 11 mg Melting point.:210-220 oC

Ultraviolet λmax; 210 and 230 nm - IR (KBr) max : 3657, 2931, 2867, 1624 cm Proton NMR; using(600 MHz) Proton resonate at 6.63 (1H, s, H-2), δ 5.21(1H, s, H-4), δ 5.20 (1H, d having J = 2.3

Hz, H-6), δ 6.19 (1H, d having J = 2.6 Hz for H-3) and δ 3.86 (3H, for -OCH3). 13C-NMR; using(150 MHz) Signals at δ 162.1 (C-1), δ 148.1 (C-4), δ 138.5 (C-9), δ 131.5 (C-8), 128.5 (C-5), 129.5 (C-3), δ 134.5 (C-4), δ 138.1 (C-7), δ 138.1 (C-6), δ 112.0 (C-2) and δ 110.4. EI-MS; (rel. int.) m/z 344 (100), 177 (83), 162.5 (56), 148.5 (65), 135 (33), 120.5 (45), 94.5 (64), 69 (73),(87.5) HR-EI-MS;

344.14 ( for C18H20 N2O5, 344.14)

94 3.8.6: F compound (2, 3, 8 tri-O-methyl ellagic acid)

Physical Data; amorphous powder Quantity: 07 mg Melting point.: 324oC

Ultraviolet λmax; 205.5 and 243 nm 25 [α]D : + 40 (0.021) -1 Infrared (KBr) max: 1723, 3512, 2934, 1661, 1615 and 15761 cm Proton NMR; using(600 MHz) Proton resonate at δ 6.63 (1H, d, J = 1.4 Hz, H-2), δ 5.20 (1H, dd , J = 6.0, 1.20 Hz, H-4), δ 5.21 ( J = 4.1 Hz, H-5), δ 6.62 (1H,d, J = 7.1 Hz), δ 6.38 (1H,d, J = 12 Hz), δ 4.16 (1H, d, J = 7.0 Hz, H-1'), δ 3.32-3.44 (1H, m, H-2'), δ 3.31-3.44. 13C-NMR; (150 MHz) Carbon at δ 126.57(C-1), δ 128.32 (C-2), δ 128.4 (C-3), δ 147.32 (C-4), δ 125.21 (C- 6), δ 124.21 (C-7), δ 136.82 C-7 and δ 114.12 (C-8), δ 161.74 (C-9) and δ 94.65(C- 1ʹ ) EI-MS; 344 (76), 196 (95), 164 (13), 148 (37), 165 (26), 104 (18) and 77 (47) HR-EI-MS;

344.05 (for C17H12O8, 344.05)

95 3.8.7: G Compound (3-O-methyl ellagic acid 4-O-B-D glucopyranoside)

Physical Data; Yellowish solid Quantity: 10 mg 25 []D : -19.3 (c 0.022)

UVλmax; 213, 222 and 229 nm IR; 3355.9 (O-H), 1645 (C=C), 1728 (C=O) and 2923 cm-1 Proton NMR; using(600 MHz) Signals at δ 1.26, δ 5.16 (dd, J = 4.2, 9.1 Hz, H-1), δ 4.10 (dd, J= 2.3, 12.3 Hz, H-1), δ 4.32 (H-5) (dt , J = 2, 9.1 Hz), δ 4.56 (H-4) (dd, J = 3.1, 5.3 Hz), δ 0.72 (6H, t, J = 4.8 Hz, , 3.4), δ 7.26 ( H-3), δ 6.61 H-2), δ 6.72 (1H, d, J = 4.8 Hz, NH). 13C-NMR; using(150 MHz) Signals at δ 169.0 (C-1ʹ ), δ 128.3 (C-4) δ 128.4 (C-1) δ 130.3 (C-4), δ 131.5 (C-2), δ 77.2 (C-3) δ 68.1, δ 28.7-32.3, δ 21.4 for methylene. EI-MS: 464 (32), 268.5 (15), 226 (23), 194 (11), 168.5 (12)

HR-EI-MS; 464.06 (for C20H16O13, 464.06)

CHAPTER 4

97 4 RESULTS AND DICUSSIONS

4.1: Phytochemical studies of Conocarpus lancifolius 4.1.1: Extraction The whole plant of Conocarpus lancifolius was extracted with the help of dichloromethane and methanol (organic solvents) by successive maceration and filtration. The filtrates were concentrated by using the rotary evaporator. Detailed results are shown in tables 4.1. Table 4.1: Results of extraction of Conocarpus lancifolius. Weight of Organic solvents Part used powdered Extract weight (g) plant (g) Dichloromethane (3×2500 ml) 12.0

Whole 900 plant

Methanol (3×2500 ml) 19.0

4.1.2: Screening of secondary metabolites The fine powderd plant material was taken and weighed. Phytochemical tests were performed according to standred procedures for secondry metabolites as given in chapter three. For alkaloids screening Dragendorff’s, Mayer’s and Wagner’s reagents were applied. For cardiac glycosides keller kiliani test was performed and for anthraquinones modified Borntrager,s test and Borntrager,s test was done . Saponins are detected by froth test and tannins were screened by gelatin, catechin and ferric chloride tests. For triterpenoids test of liebermann burchard was performed. The detailed results of screening of secondry metabolites of C. lancifolius are presented in the tables 4.2.

Table 4.2: Results of phytochemical screening of Conocarpus lancifolius. Part Steroids & Cardiac Anthraquinones Alkaloids Saponins Flavonoids Tannins Used Triterpinoids glycosides Free Bound

Whole - + + + + - + + plant + Present - absent

98 4.2: Purification of A-G compounds Fractionation of 12gm of DCM extract of Conocarpus lancifolius (CLD) was done by column using stationary phase silica gel 60 (63-200 μm) and mobile phase chloroform and methanol. Five fractions were collected (CLD1-5). The fraction CLD- 23(1.94 gm) was than processed by using (3:1) n-hex: ethyl acetate and silica gel 60 (40-63 μm) stationary phase by column chromatography. Seven fractions were collected (CLD3-a to CFLD3-g). The CLD3-c (420 mg) was processed by using mobile phase n-hex: isopropyl alchol (9:1) and stationary phase silica gel 60 (40-63μm). Five fractions were collected (CLD3-c1 to CLD3-c5). The CLD3-c1 fraction was achieved as 10 mg pure compound A . The fraction CLD3-c3 was also obtained as pure compound B (13mg). The fraction CLD-3 (1.94 gm) was processed. n-hex: ethyl acetate (3:1) was used as mobile phase and silica gel 60 (40-63μm) as stationary phase. CLD3-a to CLD3-g seven fractions were collected. The CLD3-d (85mg) fraction was processed by using mobile phase chloroform, methanol (29:1) and stationary phase silica gel 60 (40- 63μm). CLD3-d1 to CLD3-d5 five fractions was collected. CLD3-d1 and CLD3-d3 were achieved as pure C (13mg) and D (10 mg). A-D compounds isolation of Conocarpus lancifolius (dichloromethane extract) is presented in figure 4.1. Conocarpus lancifolius methanol extract (CLM) (10 gm) was fractionated with the help of stationary phase silica gel 60 (40-63 μm) and mobile phase chloroform, methanol and water on column by stepwise elution. CLM 1-4 four fractions were collected. The CLM-2(5.2 gm) fraction was processed further using mobile phase chloroform: methanol: water (80:20:2) and stationary phase silica gel 60 (40-63 μm) on column chromatography. CLM-2a to CLM-2f six fractions was collected. Fraction CLM-2b was found to be pure compound E (11mg). CLM-2c (720mg) fraction was processed further by using mobile phase chloroform: methanol: water (80:20:2) and stationary phase silica gel 60 (40-63 μm). Seven fractions were collected. The CLM-2c1 fraction was achieved as a pure compound F (10mg) and CLM-2c7 fraction also pure compound G (8 mg). The purification of compounds E, F and G is listed in figure 4.2.

99 CLD (12gm) CC Silica gel 60(40-63μm) Chloroform: Methanol (39:1→29:1→19:1→9:1→Methanol) ______

CLD-1 CLD-2 CLD-3 CLD-4 CLD-5 1.10gm 4.27gm 1.94gm 680mg 300mg

CC Silica gel 60(40-63μm) N-hexane: Ethyl acetate (3:1→5:2→7:4→Ethyl acetate)

CLD-3a+3b CLD- CLD-3d CLD-3e+3f+3g 555 mg 420 mg 85 mg 300 mg

CC CC Silica gel 60(40-63μm) Silica gel 60(40-63μm) n-hexane: Isopropyl alchol(9:1) Chloroform: Methanol (29:1→19:1→9:1→Methanol)

CLD-3c-1 CLD-3c-2 CLD-3c-3 CLD-3c-4 CLD-3c-5 10 mg* 130 mg 13 mg* 120 mg 33 mg (Compound –A) (Compound –B)

CLD-3d-1 CLD-3d-2 CLD-3d-3 CLD-3d-4 +5 08 mg* 10 mg 07 mg* 27 mg (Compound –C) (Compound –D)

Figure 4.1: Purification of compounds A-D from the dichloromethane extract of Conocarpus lancifolius.

100 CLM (10gm) CC Silica gel 60(40-63μm) C: M: W (85:15:1→80:20:2→ 70:30:4→60:40:5→Methanol) ______

CLM-1 CLM-2 CLM-3 CLM-4 950mg 5.2gm 1.72gm 782mg CC Silica gel 60(40-63μm) C: M: W (85:15:1→80:20:2→ 70:30:4→60:40:5→Methanol)

CLM-2a CLM-2b CLM-2c CLM-2d CLM-2e CLM-2f 220mg 11mg* 720mg 200mg 520mg 800mg (Compound –E) CC Silica gel 60(40-63μm) C: M: W (85:15:1→80:20:2→ 70:30:4→60:40:5→Methanol)

CLM-2c-1 CLM-2c-2+3 CLM-2c-4+5 CLM-2c-6 CLM-2c-7 07mg* 39mg 110mg 82mg 10mg* (Compound –F) (Compound–G)

Figure 4.2: Purification of compounds E-G from methanolic ext. of Conocarpus lancifolius.

101 4.3: Determination of Structures of purified Compounds Compound A

A compound was purified from dichloromethane fraction of Conocarpus lancifolius as yellowish amorphous powder. Figure 4.4 shows the IR spectra of compound A. Carbonyl group was indicated by absorption at1650 cm-1. Other bands in Infrared spectrum A are 3050, 3446 and at 815 cm-1. The melting point of compound A was o 182 C. The UV spectrum (figure 4.4) highlightes λmax at 253, 267 nm as that of flavonoids.

The formula C21H18O13 for molecule was illucidated by using HR-EI-MS indicating ion peak for molecule at m/z 478.07 (for C21H18O13, 478.07). The spectrum Figure 4.5 the proton NMR of A compound indicates sugar moity of 5H having δ 3.32-3.92 .The other proton resonate at 5.24 (1H, dd, J = 6.0 Hz, H- 1'), 6.34 (1H, s, H-6), 6.36, (1H, s, H-8), 6.70 (1H, dd, J = 2.5 Hz, H-5'), 7.48 (1H, d, J = 3.2, 6.4 Hz), 7.62 (1H, d, J = 2.3 Hz.The signal between δ 3.16 - 3.34 proton multiplets shows glucose oxymethine and at δ 4.54 shows anomeric proton. The 13C-NMR (BB and DEPT) spectrum (figure 4.6, 4.7) corrborateb the existace of 21 carbon signals, showing ten methine and eleven quaternary carbon atoms. All mentioned values of spectral and physical data conforims the identification of compound A as Quercetin 3-O-glucuronide. It was previously isolated from the arial part of Nelumb nucifera (Jerome and Mrina, 2012).

102 Figure 4.3: A Compound UV spectrum

Figure 4.4: A Compound IR spectrum

103

Figure 4.5: A Compound 1H NMR spectrum

Figure 4.6: A Compound 13C NMR (BB)

104

Figure 4.7: A Compound 13C NMR (dept)

Figure 4.8: A Compoun EI spectrum

105

Figure 4.9: A Compound FAB+ spectrum

Figure 4.10: A Compound FAB spectrum

106 Compound B

B compound was obtained from dichloromethane fraction of Conocarpus lancifolius as yellowish amorphous powder. Figure 4.12 shows the IR spectra of compound B. Carbonyl group was indicated by absorption at1708 cm-1. The signals for C = C appears at 1610 cm-1. The Sp3 C-H strtching was shown at 2871 cm-1 and for Sp2 C- H was observed 2925.8 cm-1. Ultraviolet spectrum illustrates the absorption at 230 and 282 nm (figure 4.11). The spectrum Figure 4.13 the proton NMR of B compound represents the aromaticity in a molecule having protons at δ 7.24 (1H, dd, J = 2.3 Hz), δ 7.26 (1H, d, with J = 3.1, 2.4, Hz), 6.67 (1H, dd, J = 3.4, 4.6, Hz). The isopropyl proton display there signals at δ 0.92 (6H, d, having J = 2.4 Hz). The broad band and depth (13C- NMR) spectra (figures 4.14, 4.15) of B compound shows eighteen signals for carbon representing three methyl, nine methine, two methylene and four signals for quaternary carbon atoms . The δ 131.0 signals singlet representing downfield region indicates that carbonyl carbon exist in a molecule. The δ 16.0 - 18.4 signals shows the presence of aliphatic carbon. The δ 129.8, 129.9, 131.2 and 131.1 signals shows that aromatic carbon atoms are present. The positions of atoms were conformed by COSY and HSQC (figures 4.18, 4.19). All mentioned values of spectral and physical data conforms the identification of compound B as S-4-isopropyl-8-methoxy-6-thia-bicyclo[3.2.1]oct-1-en-2-yl benzothioate and name was given on species basis as lancifoliate.

107

Figure 4.11: B compound UV spectrum

Figure 4.12: B compound IR spectrum

108

Figure 4.13: B compound 1H NMR spectrum

Figure 4.14: B compound 13C NMR (BB) spectrum

109

Figure 4.15: B compound 13C NMR (dept) spectrum

Figure 4.16: B compound EI spectrum

110

Figure 4.17: B compound FAB + spectrum

Figure 4.18: B compound COSY spectrum

111

Figure 4.19: B compound HSQC spectrum

112 Compound C

C compound was obtained from dichloromethane fraction of Conocarpus lancifolius as amorphous powder. The moleculer formula C26H26O3 was illucidated by using HR-

EI-MS and showed ion peak for molecule at m/z, 386 ( for C26H26O3, 386). Figure 4.21 shows the IR spectra of C compound. It shows absorption at 2925.8 cm-1, at 1726.2 cm-1 for (C = 0) and 1458.1 cm-1 absorption showed the aromaticity in a compound. The Sp2 C-H stretching was observed at 2866 cm-1. The ultraviolet spectrum (figure 4.20) shows that of conjugated system is present in compound at 230 nm and 260 nm.” The spectrum Figure 4.22 the proton NMR of C compound shows nine aromatic proton appeared at δ7.24 (1H, dd having J = 2.3 Hz), 7.29 ( d, having J = 3.0, 3.4 Hz), 7.30 (dd, J = 0.5 Hz), 7.33 (1H, d, J = 0.6 Hz), 7.35 (1H, d, J = 2.7 Hz). The δ4.32 (1H, dt having J = 3.6, 4.2 Hz) signal shows protons for lactone. The alkene proton was observed at δ4.11 (siglet for 1H) in the downfield region. The broad band an depth (13C- NMR) spectra (figures 4.23, 4.24) of C compound revealed the total twenty six carbon signals for 8 methines, 10 methylenes and 8 quaternary carbon signals. The signals at δ 129.3, 128.5, 129.9, 131.1, 131.2, and 133.1 (downfield) is the indication of aromaticity in molecule. The δ 158.0 signals singlet representing downfield region indicates that carbonyl carbon exist in a molecule. The signals at δ 77.25 showed that lactone carbons are present. The positions of atoms were conformied by COSY and HSQC. All mentioned values of spectral and physical data conforms the identification of compound C as 2,3,4,8,9,10,11,13-octahydro-1H-phenanthro[3,2-c]oxocin-3-yl benzoate a novel natural product. The name was given on species basis as lancifotarene.

113

Figure 4.20: C compound UV spectrum

Figure 4.21: C compound IR spectrum

114

Figure 4.22: C compound 1H NMR spectrum

Figure 4.23: C compound 13C NMR (BB) spectrum

115

Figure 4.24: C compound 13C NMR (dept) spectrum

Figure 4.25: C compound EI spectrum

116

Figure 4.26: C compound FAB+ spectrum

Figure 4.27: C compound FAB- spectrum

117

Figure 4.28: C compound COSY spectrum

Figure 4.29: C compound HSQC spectrum

118 Compound D

D compound was obtained from dichloromethane fraction of Conocarpus lancifolius as amorphous powder. Ultraviolet spectrum illustrates the absorption at 230 and 222 nm (figure 4.30). Figure 4.31 shows the IR spectrum of D compound. It shows absorption at 3419.6 cm-1for hydroxyl and at 1513.1 cm-1 for (C = C) . The Sp2 C-H stretching was observed at 2928.3 cm-1. Carbonyl group was indicated by absorption -1 -1 at 2125.4 cm and lactone moity at 1513.1 cm . The formula C14H14O9 for molecule was illucidated by using HR-EI-MS indicating ion peak for molecule at m/z 328 ( for

C14H14O9, 328.08). The spectrum Figure 4.32 the proton NMR of D compound shows signals for alkenes at δ 5.78 (1H, siglet) and at δ 4.71 (1H, dt having J = 4.2, 2.4 Hz) for alcoholic protons. The signals at δ 4.58(1H, d having J = 6.3 Hz) represents lactone moity. The broad band and depth (13C- NMR) spectra (figures 4.33, 4.34) of D compound indicates fourteen carbon signals for six hydroxyl group, seven methane one methylene and five quaternary carbon signals. The δ 189.9 signals singlet representing downfield region indicates that carbonyl carbon exist in a molecule. The signals at δ167.9 showed that lactone carbons are present. The signal at δ 114.9, 115.9, 130.7 -1 cm showsthat molecule has alkene carbon. All mentioned values of spectral and physical data conforims the identification of compound D as 1,3,4,5,6,8-hexahydroxy- 3,4,5,5a-tetrahydro-1H-benzo[g]isochromene-5-carboxylic acid a novel natural product. The name was given on species basis as lancifolian.

119

Figure 4.30: D compound UV spectrum

Figure 4.31: D compound IR spectrum

120

Figure 4.32: D compound 1H NMR spectrum

Figure 4.33: D compound 13C NMR (BB) spectrum

121

Figure 4.34: D compound 13C NMR (dept) spectrum

Figure 4.35: D compound EI spectrum

122

Figure 4.36: D compound FAB- spectrum

Figure 4.37: D compound COSY spectrum

123

Figure 4.38: D compound HSQC spectrum

124 Compound E

E compound was obtained from methanol fraction of Conocarpus lancifolius as amorphous powder. Figure 4.40 shows the IR spectra of compound E. Carbonyl group was indicated by absorption at1723 cm-1. The signals of absorption at 3654 cm- 1 indicates the N-H group in a molecule. The 1634 cm-1 absorption was due to amide group in a molecule. The Sp2 C-H and Sp3 C-H stretching were present at 2867 cm-1 and 2931 cm-1. The spectrum Figure 4.41 the proton NMR of E compound shows signals at δ 6.62 ppm siglets indownfield region indicate that functional group N-H was present. The signals at δ 5.52 (1H, d J = 2.5 Hz), 5.15 (1H, d, J = 3.9, 2.6 Hz) shows the alkene nature of molecule.

The formula C18H20N2O5 for a molecule was obtained by HR-EI-MS having ion peak 13 at m/z 344 ( for C18H20N2O5: 344.14). The broad band and depth ( C- NMR) spectra (figures 4.42, 4.43) of E compound showed eighteen signals of carbon indicating four methoxy, four methine and 10 quaternary carbons. Signals at δ 140.4, 141.7, 152.3 and 152.7 which were downfield indicate the existace of alkene carbon. All mentioned values of spectral and physical data conforms the identification of compound E as 1-amino-1,5,7,8-tetramethoxy-1H-cyclopenta[a]naphthalene-2- carboxamide a novel natural product. The name was given on species basis as Lancifolamide

125

Figure 4.39: E compound UV spectrum

Figure 4.40: E compound IR spectrum

126 Figure 4.41: E compound 1 H NMR spectrum

Figure 4.42: E compound 13C NMR spectrum(BB)

127

Figure 4.43: E compound 13C NMR (dept) spectrum

Figure 4.44: E compound EI spectrum

128

Compound F

F compound was achieved as pale yellowish solid. The ultraviolet spectrum (figure 4.45)shows absorption at 206 and 243 nm. The IR spectrum (figure 4.46) showed absorption bands at 3510, 2844, 1561, 1515 and 1474 cm-1. The HR-EI-MS gives ion peak for amolecule at 344 illucidating formula as C17H12O8, ( for C17H12O8, 344.5). The fragmentation pattern by retro diels alder in EIMS gives the peak at m/z 196 which shows that two methoxyl gruops in ring B one methoxyl and one hydroxyl group in ring A are present.

The spectrum Figure 4.47 the proton NMR of F compound shows three methoxy groups signals in downfield region. Aromatic region protons are appeared at δ 6.36 (1H, singlet, H-8), and other indicates the pattern of a disubstituted (ABX system) representing signals at δ 6.95 (1H, d,having J= 4.2 Hz, H -5) and δ 11.47 that shows hydroxyl group chelation and indicating H-5 position.

The broad band and depth (13C- NMR) spectra (figures 4.48, 4.49) of F compound gives 17 signals displaying 3 methyl, 2 methine and 12 signal for quaternary carbons. All mentioned values of spectral and physical data conforms the identification of compound F as 2, 3, 8 tri-O-methyl ellagic acid. It was previously reported in Irvingia gabonensis bark (George and yimin zaho 2007).

129

Figure 4.45: F compound UV spectrum

Figure 4.46: F compound IR spectrum

130

Figure 4.47: F compound 1H NMR spectrum

Figure 4.48: F compound 13C NMR (BB) spectrum

131

Figure 4.49: F compound 13C NMR (dept) spectrum

Figure 4.50: F compound EI spectrum

132 Compound G

G compound was obtained from methanol fraction of Conocarpus lancifolius as amorphous powder. Ultraviolet spectrum illustrates the absorption at 233, 222 and 229 nm (figure 4.51). Figure 4.52 shows the IR spectrum of G compound. It shows absorption at 3355.9 cm-1 for hydroxyl and at 1645cm-1 for (C = C). The Sp3 C-H stretching was observed at 2923.9 cm-1. Carbonyl group was indicated by absorption at 1728.1 cm-1. The spectrum Figure 4.53 the proton NMR of G compound indicates the existence of aromaticity in amolecule having resonance of protons at δ7.58 (1H, dhaving J = 4.2, Hz), 7.62 (1H, dd, J = 3.1, 1.2, Hz), 7.52 (1H, dd, J = 0.4, 2.6, Hz). The signals at δ7.43 (1H, singlet) shows phenolic proton. The signals at δ 4.21 (d having J = 2.3 Hz) indicates the presence of lactone moity.” “The broad band and depth (13C- NMR) spectra (figures 4.54, 4.55) of G compound indicates twenty carbon signals for two methylene, eight methine, and nine quaternary carbon signals. The δ 169.0 signals singlet representing downfield region indicates that carbonyl carbon exist in a molecule. The signals at δ77.6, 68.0 showed that lactone carbons are present. The signals at δ 128.7, 128.9, 130.8, and 130.9 showed aromatacity in amolecule. All mentioned values of spectral and physical data conforms the identification of compound G as 3-O-methyl ellagic acid 4-O-B-D glucopyranoside. It was previously reported from Anisophyllea dichostyla root bark (khallouki et al, 2006).

133

Figure 4.51: G compound UV spectrum

Figure 4.52: G compound IR spectrum

134

Figure 4.53: G compound 1H NMR spectrum

Figure 4.54: G compound 13C NMR (BB) spectrum

135

Figure 4.55: G compound 13C NMR (dept) spectrum

Figure 4.56: G compound EI spectrum

136

Figure 4.57: G compound FAB+ spectrum

Figure 4.58: G compound HSQC spectrum

137

Figure 4.59: G compound COSY spectrum

138

A B

C D

E F

Figure 4.60: Structures of isolated compounds A-G from Conocarpus lancifolius.

139 4.4: Biological studies of Conocarpus lancifolius

Conocarpus lancifolius extracts dichloromethane and methanol of aerial parts was subjected for antifungal bioassay, cytotoxic bioassay, antibacterial bioassay, phytotoxicity activity and anticancer activity The aerial parts Dichloromethane and methanol extracts of Conocarpus lancifolius were also screened for inhibition of enzyme activities as Antglycation activity, carbonic anhydrase, α -glucosidase and ß glucuronidase, antioxidant activity, butyrylcholinesterase activity, lipoxygenase activity, acetyl cholinesterase inhibitory activity. . The results of these activities and bioassays are briefly discussed blow.

4.4.1: Acetyl cholinesterase inhibition activities Conocarpus lancifolius extracts were examined for acetylcholinesterase inhibition activities by using the standard procedures. The IC50 value and percentage inhibitory concentration (0.5 mg/ml) of Conocarpus lancifolius extracts were measured and studied in comparison with standard drug (Eserine). The detailed procedure of activity is described in chapter 3. The detailed results are presented in table 4.3

Table 4.3: Result of acetylcholinesterase inhibition activity of Conocarpus lancifolius.

Extract / standard Percentage Inhibition at 0.5 IC50 value drug mg/ml ug/ml CLD 11.73±0.75 - CLM 87.48±0.09 0.072±0.11 Standard drug 82.81±1.09 (0.5 mM) 0.04±0.001 (µM) (Eserine)

The results of table 4.3 highlights that the methanol extract of Conocarpus lancifolius has significant acetylcholinesterase inhibition activity with percentage inhibitory value of 87.48 ± 0.09 at concentration of 0.5 mg/ml having 0.072 ± 0.11

IC50 value. The dichloromethane extract of Conocarpus lancifolius showed a little percentage inhibition value of 11.73±0.75.

140 4.4.2: Butyrylcholinesterase inhibition activities Conocarpus lancifolius extracts were examined for butyrylcholinesterase inhibition activities by using the standard procedures. The IC50 value and percentage inhibitory concentration (0.5 mg/ml) of Conocarpus lancifolius extracts were calculated and studied in comparison with standard drug (Eserine). The procedure of activity is briefly described in chapter 3. The detailed results are presented in table 4.4.

Table 4.4: Result of butyryl cholinesterase inhibition activity of Conocarpus lancifolius extracts.

Percentage Inhibition at IC50 value Extract / standard drug 0.5 mg/ml ug/ml CLD 89.52±0.39 55.25±0.07 CLM 89.06±0.89 52.31±0.11 Standard drug (Eserine) 82.81±1.09 (0.5 mM) 0.84±0.001(µM)

The results of table 4.4 highlights that dichloromethane and methanol extracts of Conocarpus lancifolius has significant butyryl cholinesterase inhibitory activity having percentage inhibitory value 88.51 ± 0.28 and 86.05 ± 0.79 with 55.25 ±

0.07 and 52.31 ± 0.11 IC50 values.

4.4.3: Lipoxygenase inhibition activities Conocarpus lancifolius extracts were examined for lipoxygenase inhibition activities by using the standard procedures. The IC50 value and percentage inhibitory concentration (0.5 mg/ml) of Conocarpus lancifolius extracts were calculated and studied in comparison with standard drug (Baicalein). The detailed procedure of activity is described in chapter 3. The detailed results are presented in table 4.5

Table 4.5: Results of lipoxygenase activity of Conocarpus lancifolius extracts.

Percentage Inhibition at IC50 value Extract / standard drug 0.5 mg/ml ug/ml CLD 65.97±0.55 158.61±0.08 CLM 63.89±0.02 186.31±0.04 Standard drug (Baicalein) 93.78±1.26 (0.5 mM) 22.42±1.3 (µM)

141

The results of table 4.5 highlights that the dichloromethane and methanol extract of Conocarpus lancifolius has significant lipoxygenase inhibitory activity having percentage inhibitory values 65.97 ± 0.55 and 63.89 ± 0.02 with 158.61 ± 0.08 and

186.31 ± 0.04 IC50 values.

4.4.4: Carbonic anhydrase inhibition activities Conocarpus lancifolius extracts were examined for carbonic anhydrase activities by using the standard procedures. The IC50 value and percentage of inhibitory concentration (0.5 mg/ml) of Conocarpus lancifolius extracts were measured and studied in comparison with standard drug (Acetazolsmide). Procedure of activity is briefly described in chapter 3. The detailed results are presented in table 4.6.

Table 4.6: Results of carbonic anhydrase activity of Conocarpus lancifolius extracts.

Extract / standard Concentration Percentage IC50 Value SEM drug (mM) Inhibition CLD 0.2 mg/ml 78.6 55.7 2.34 CLM Nil 47.5 Acetazolsmide 0.2 88.0 0.13 0.03 Standard Drug

The results of table 4.6 highlights that the dichloromethane extract of Conocarpus lancifolius has moderate carbonic anhydrase activity having percentage inhibitory value 78.6 at 0.2 mg/ml having 55.7 2.34 IC50 values. Methanol extract of Conocarpus lancifolius show no activity.

4.4.5: Xanthine oxidase inhibition activities Conocarpus lancifolius extracts were examined for xanthine oxidase inhibition activities by using the standard procedures. The IC50 value and percentage of inhibitory concentration (0.5 mg/ml) of Conocarpus lancifolius extracts were measured and studied in comparison with standard drug (Allopurinol). Procedure of activity is briefly described in chapter 3. The results are presented in table 4.7.

142 Table 4.7: Results of xanthine oxidase inhibition activity of Conocarpus lancifolius extracts.

Extract / Concentration Percentage IC50 Value SEM standard drug (mg/ml) Inhibition CLM 0.5 39.4 CLD 79.97 154.32 6.07 Allopurinol 98.8 0.273 0.01 Standard Drug

The results of table 4.7 highlights that the dichloromethane of Conocarpus lancifolius has significant xanthine oxidase inhibitory activity having percentage inhibitory value

79.97 at 0.5 mg/ml having 154.32 6.07 IC50 values. Methanol extract of Conocarpus lancifolius show no activity.

4.4.6: β-glucuronidase inhibition activities Conocarpus lancifolius extracts were examined for β-glucuronidase inhibition activities by using the standard procedures. The IC50 value and percentage inhibitory concentration (0.2 mg/ml) of Conocarpus lancifolius extracts were measured and studied in comparison with standard drug (Dsaccharic, 1, 4-lactone). Procedure of activity is briefly described in chapter 3. The results are presented in table 4.8

Table 4.8: Results of β-glucuronidase activity of Conocarpus lancifolius.extracts.

Extract / standard Conc. (mM) Percentage IC50 Value SEM drug Inhibition [µM] CLM 0.4 42.72 CLD 0.4 67.6 325.4 3.10 Standard (Dsaccharic, 1, 0.4 88.4 42.72 2.14 4 lactone)

The results of table 4.8 highlights that the dichloromethane extract of Conocarpus lancifolius has significant β-glucuronidase inhibitory activity having percentage inhibitory value 67.6 with 325.4 3.10 IC50 values. Moderate inhibitory value was observed by methanol extact of Conocarpus lancifolius having 42.72 persentage inhibition.

143 4.4.7: α-Chymotrypsin inhibition activities Conocarpus lancifolius extracts were examined for α- chymotrypsin inhibition activities by using the standard procedures. The IC50 value and percentage inhibitory concentration (0.5 mg/ml) of Conocarpus lancifolius extracts were measured and studied in comparison with standard drug PMSF (Phenyl methyl sulfonyl fluoride). Procedure of activity is briefly described in chapter 3. The detailed results are presented in table 4.9.

Table 4.9: Results of α-Chymotrypsin inhibition activities ofConocarpus lancifolius Percentage Inhibition at Extract / standard drug IC50 Value 0.5 mg/ml CLD 91.20±1.72 90.16±0.72 CLM 153.63±1.37 211.43±0.71 Standard drug ( PMSF) 97.71 ± 0.79 (0.5 mM) 47.71 ±0.13(µM)

The results of table 4.9 highlights that the dichloromethane extract of Conocarpus lancifolius has significant α- Chymotrypsin inhibitory activity having percentage inhibitory value 91.20 ± 1.72 and 86.05 ± 0.79 with 90.16 ± 0.72µg/ml IC50 values. Moderate inhibitory value was observed by methanol extact of Conocarpus lancifolius having 153.63 ± 1.37 persentage inhibitions. 4.4.8: Urease inhibition activities Conocarpus lancifolius extracts were examined for Urease inhibition activities by using the standard procedures. The IC50 value and percentage inhibitory concentration (0.5 mg/ml) of Conocarpus lancifolius extracts were measured and studied in comparison with standard drug (thiourea). Procedure of activity is briefly described in chapter 3. The results are given in table 4.10.

Table.4.10: Results of Urease inhibition activity of Conocarpus lancifolius extracts.

Extract/ standard Concentration Percentage IC50Value± drug ( mM) inhibition SEM(µM) CLM 0.25 51.7±1.91 229±2.11 CLM 0.5 81.1±1.82 49.1± 1.31 CLD 0.25 Inactive

Standred drug 0.5 95.9 21.9±1.62 (Thiourea)

144 The results of table 4.10 highlights that the methanol extract of Conocarpus lancifolius has significant antiurease activity having percentage inhibitory value

81.1±1.82 with 49.1± 1.31 IC50 values. No percentage inhibition of the enzyme was observed by dichloromethane extract.

4.4.9: α- glucosidase inhibitory activities Conocarpus lancifolius extracts were examined for α-glucosidase inhibition activities by using the standard procedures. The IC50 value and percentage of inhibitory concentrations of Conocarpus lancifolius extracts were calculated and studied in comparison with standard drug (Acorbose). Procedure of activity is briefly described in chapter 3. The results are given in table 4.11.

Table 4.11: Results of α- glucosidase inhibitory activities of Conocarpus lancifolius extracts.

Conc.Of Inhibition Inhibition IC50 (μg/ml) CLM(mg/ml) %age(exp.1) %age(exp.2) 0.5 99.11 99.54 166.78±1.74 0.25 98.65 97.91 1.85±0.98. 0.125 99.85 98.1 2.11±0.032. 0.0625 98.92 98.92 4.32±0.021 0.03125 98.08 99.08 8.91±0.045. 0.0156 99.08 99.08 5.66±0.095. 0.0078 99.03 98.31 7.1±0.12 0.0039 98.46 79.56 5.59±0.06 Std .(Acorbose) 92.23 38.25±0.12 (µM)

The results of table 4.11 highlights that the methanol extract of Conocarpus lancifolius has very significant α-glucosidase inhibitory activity having percentage inhibitory value 79.56 with 5.59±0.06µg/ml IC50 values at even very low concentration (0.0039 mg/ml) of extract.

145 4.4.10: Blood glucose level

Table 4.12 highlights the effect of Conocarpus lancifolius on blood glucose level treated (100mg/kg and 200mg/kg) in comparison with treated with 80 mg/kg diamicron as standard drug. Table 4.12: Result of Conocarpus lancifolius extract on glucose level (blood) in diabetic rabbits. Diabetic (with Diabetic (with No of Diabetic ( with Control Diabetic 100mg/kg C. 200mg/kg C. days diamicron 80mg/kg ) lancifolius) lancifolius) 270.40  3 0 day 93  5.15 270 5.45 269 4.75 272 5.90 .30

268.25 4 7 day 91 4.30 155 5.10 144.5 5.5 232.15 3.25 .10

14 day 93 5.11 271 6.33 146.2 3.75 138.25 5.60 230.3 4.32

21 day 97 4.35 271 4.40 134.75 4.20 130.85 4.4 222.4 5.45

28 day 92 4.45 265 5.60 130.3 5.15 124.6 3.45 220.35 4.45

Rabbits taken in each group = 6 All values presented as mean ± SD

4.4.11: Hypolipidemic effects Table 4.13: Results of Conocarpus lancifolius extract on total cholesterol level in diabetic rabbits. Diabetic (with Diabetic (with No of Diabetic (with Control Diabetic 100mg/kg 200mg/kg days diamicran 80mg/kg ) C. lancifolius) C.lancifolius)

0 day 41.2 3.55 64 4.75 64.35 2.92 66.15 3.10 66.95 3.95

7 day 45.3 2.65 66 5.15 54.5 3.6 52.15 2.75 58.3 4.35

14 65.5 5.55 day 43.15 2.3 53.4 3.15 52 3.1 55 3.75

21 64.2 5.20 day 42 3.45 52 2.3 51 3.5 55.1 3.8

28 65 5.50 day 43.75 3.1 52.15 2.35 51.3 3.5 54.2 3.65 Number of rabbits in each group=6 Values are expressed as mean  SD

146 Table 4.14: Result of Conocarpus lancifolius extract on Triglyceride (mg/dl) in alloxan induced diabetic rabbits.

Diabetic (with Diabetic (with Diabetic (with No of Control Diabetic 100mg/kg C. 200mg/kg C. diamicran days lancifolius lancifolius 80mg/kg )

0 day 57.2 8.3 139 9.11 140.4 11.5 135.15 13.3 137.95 9.3

7 day 58.1 8.5 135.4 8.5 104 9.3 99.3 12.2 104 9.5

14 136.10 9.89 57.5 9.1 96.5 10.2 99.5 10.35 100 12.25 day

21 136 7.50 57.4 12 74 8.5 80 8.7 93 13.10 day

28 139 11 59 10.1 74 9.3 74.2 8.5 81.1 11.5 day

Rabbits taken in each group=6 All values presented as mean ± SD

Table 4.15: Results of Conocarpus lancifolius extract on HDL and LDL(mg/dl). HDL (mg/dl) LDL (mg/dl) Diabetic Diabetic Diabetic ( Diabetic Diabetic Diabetic ( with No of (with with (with (with (with diamicr Days Control Diabetic 200mg/kg diamicra 100mg/kg 100mg/kg 200mg/kg an C. n C.lancifolis C.lancifolis C.lancifolius 80mg/k lancifolius 80mg/kg ) g ) 28.1  3. 34.20 35.3 2. 36.1 3 35.15 8.95 2. 8.9 3 0 day 9.75 3.1 5 2.02 5 .1 2.50 5 .5

35.40 34.25 34.55 7.75 7 day 28 3.15 36 3.1 7.3 3.1 7.25 3.1 2.75 2.75 3 3.25

29.4 3. 34.5 3. 36.2 3 7.1 2.7 7.50 14 day 34 2.3 34 2.5 7.15 2.4 5 0 15 .2 5 2.57

28.2 2. 34.5 3 34.75 33.75 34.5 3 6.95 3. 7.95 21 day 6.75 3.1 5 3 2.2 .1 5 3.1

27.5 3. 35 2.8 36.2 3. 36.95 33.5 2 7.2 4 28 day 7.2 3.2 7.31 3.1 1 0 75 2.9 .75 .1 Rabbits taken in each group=6 All values presented as mean ± SD

The results of table 4.15 highlights that the Conocarpus lancifolius extract increase Triglyceride, Total Cholesterol and LDL significantly. It represents that Conocarpus lancifolius extract reduces the side effects which are due to diabetes and decreases the blood glucose level.

147 4.4.12: Effect on liver function tests (SGPT & SGOT values) Table 4.16: Resuts of Conocarpus lancifolius extract on serum SGPT and SGOT (IU/L) in diabetic rabbits.

Serum SGPT (IU/L) Serum SGOT (IU/L) Diabetic Diabetic Diabetic with Diabetic (with Diabetic ( (with (with diamicra No of (with 100mg/k with 100mg/kg 200mg/ n Days Control Diabetic 200mg/kg g diamicran C.lancifoli kg 80mg/kg C.lancifoli C.lancifo 80mg/kg ) us C.lancif us) lius) olius

0 day 202  206 1 206 1 203.5 204 13. 197 13. 201 203 1 51.1 5.50 1.5 12.1 5 5 5.1 6.4

203 203.5 188.3 183.3 189.3 1 176 180.3 7 day 172 5.3 12.5 12.1 25.3 11.5 5.3 36.3 9.5

14 200 204 9 186 1 185.3 185.3 5 172 178.5 160 3.3 day 13.5 .8 3.5 15.1 .1 12.55 3.5

176.3 21 204 202 1 178 1 180 1 186 13. 170 12. 168 12.1 day 15.11 3.30 1.1 3.3 5 5 13.2 5

28 201 205 2 178.9 180.5 188.3 3 165 176 1 171 3.2 day 13.5 5.10 15.1 23.3 .5 16.1 3.5

Rabbits taken in each group=6 All values presented as mean ± SD

The results of table 4.16 highlights that the Conocarpus lancifolius extract decreases SGOT and SGPT significantly while SGOT and SGPT increased significantly (p  0.05) in diabetic rabbits.

4.4.13: In vitro screening on isolated tissue for spasmolytic assay 4.4.13.1: Conocarpus lancifolius arial The aqueous ethanol extract (30:70) of Conocarpus lancifolius was subjected to rabbit jejunum which relaxed the spontaneous contractions. It relaxes at tissue bath concentrations (0.01-3 mg/ml) in dose dependant manner. Rabbit jejunum preparation relax K+ (80 mM) indicates the relaxant affect and it may be mediated by blocking of voltage dependant Ca+2 channels. It also relaxes trachea in dose dependant method (0.1-3 mg/ml) by carbacchol (1μM).

148 4.4.13.2: Conocarpus lancifolius root The aqueous ethanol extract (30:70) of Conocarpus lancifolius was subjected to rabbit jejunum which relaxed the spontaneous contractions. It relaxeses at tissue bath concentrations (0.01-3 mg/ml) in dose dependant manner. Rabbit jejunum preparation relaxe K+ (80 mM) indicates the relaxant affect and it may be mediated by blocking of voltage dependant Ca+2 channels. It also relaxses trachea in dose dependant method (0.1-3 mg/ml) by carbacchol (1μM).

4.4.14: Antimicrobial activity Conocarpus lancifolius extracts were screened against microbs (P. aeruginosa, B. subtilis, S. aureus, S. typhi, E. coli,and S. flexinari) by using the standard procedures.

The IC50 value and percentage of inhibitory concentration (0.5 mg/ml) of Conocarpus lancifolius extracts were measured and studied in comparison with standard drug (Amphotericin-B). Same procedure was adopted for antifungal activity by using standard drug (miconazole). Procedure of activity is briefly described in chapter 3. The detailed results are given in table 4.17, 4.18 and 4.19.

Table 4.17: Results of antibacterial activities of different extracts of Conocarpus lancifolius

E. coli S. typhi B. spizizenii S. aureus S. epidermidis Plant Extract D D D D D D D D D D D 1 2 3 4 1 2 3 4 1 2 3 D4 D1 D2 D3 D4 D1 D2 D3 D4

1 1 1 1 1 2 1 1 2 2 1 CLD 5 5 4 4 9 0 2 2 1 0 6 15 22 20 14 15 16 34 30 20 2 2 2 2 2 2 2 2 4 3 3 10 CLM 0 7 4 3 6 4 1 4 0 0 3 35 14 .5 0 0 30 16 16 15

Erythro mycin 15 13 16.3 15.6 15.3

149 4.4.15: antifungal activity Table 4.18: Antifungal assay results of Conocarpus lancifolius extracts. Extract/ standard drug Fungal growth inhibition (%) after 24 hours A. nigar A. flavus F. solani A. fumigates Mucor CLD 43.3886 87.8254 64.87871 19.697 49.1356 CLM 87.3146 77.5843 55.31123 12.3348 55.0768 Positive (Control) 116.439 114.876 103.6815 108.421 107.459 Negative (Control) 26.7136 23.7008 13.34094 19.9233 13.6487

Table 4.19: Antifungal assay results of Conocarpus lancifolius extracts. Extract/ standard drug Fungal growth inhibition (%) after 48 hours A. nigar A. flavus F. solani A. fumigates Mucor CLD 35.5345 67.2816 30.98592 0 24.4344 CLM 59.1236 51.3158 18.46154 0 22.8374 Positive (Control) 103.149 95.7429 88.93437 98.1518 95.9871 Negative (Control) 13.6284 5.36326 0 9.53857 5.98236

The results of table 4.18 highlights that the methanolic extract of Conocarpus lancifolius has significant antifungal activity having percentage inhibitory value 30% against Microsporum canis. The results of table 4.19 highlights that the dichloromethane and methanolic extract of Conocarpus lancifolius has significant antifungal activity having percentage inhibitory value 90% against F. solani.

4.4.16: Phytotoxic assay Conocarpus lancifolius extracts were screened for in vitro phytotoxic activity by using the standard procedures. The percentage of growth regulation was examined at concentration (0.015µg/ml.) of Conocarpus lancifolius extracts were measured and studied in comparison with standard drug (Paraquat). Procedure of activity is briefly described in chapter 3. The detailed results are presented in table 4.20.

150 Table 4.20: Results of phytotoxic assay of extract of Conocarpus lancifolius Sample Plant Total Fronds Percentage Standard Extracts conc. Name Growth conc. (µg/ml) (µg/ml) Sample Control 1000 02 90 CLM 100 20 20 20 Lemna 10 20 20 0.015 minor 1000 09 55 CLD 20 100 20 0

Standard drug = Paraquat(0.015 (µg/ml) Incubation Condition = 28o 2Co

4.4.17: Brine shrimp lethality assay Conocarpus lancifolius extracts were examined for brine shrimp lethality assay by using the standard procedures. The LD50 value and percentage of inhibitory concentration (1000, 100 and 10 ppm.) of Conocarpus lancifolius extracts were measured and studied in comparison with standard drug (Etoposide). Procedure of activity is briefly described in chapter 3. The detailed results are presented in table 4.21.

Table 4.21: Brine shrimp lethality assay results of Conocarpus lancifolius Dose LD LD Total shrimps 50 50 Extracts Shrimps survive Extract Standrad used (µg/ml) (µg/ml) (µg/ml) 1000 30 14

CLD 100 30 25 857.73

10 30 29

7.4625 1000 30 24

CLM 100 30 28 45456.4

10 30 29

Standard drug: Etoposide

The results of table 4.21 highlights that dichloromethane extract of Conocarpus lancifolius have moderate Brine shrimp lethality activity only at high dose of 1000 µg/ml. At low dose of dichloromethane extract and methanol extract show no activity.

151 4.4.18: Antileishmanial activity Conocarpus lancifolius extracts were screened for in vitro antileishmanial activity by using the standard procedures. L. major was used as test organism. The IC50 value and percentage of inhibitory concentration (0.5 mg/ml) of Conocarpus lancifolius extracts were measured and studied in comparison with standard drugs (amphotericin- B and pentamidine). Procedure of activity is briefly described in chapter 3. The detailed results are presented in table 4.22.

Table 4.22: Result of antileishmanicidal activity of Conocarpus lancifolius extracts.

IC50Value (ug/ml) ± S.D CLD CLM Amphotericin B Pentamidine >100 >100 0.29 ± 0.05 5.09 ± 0.09

The results of table 4.22 highlight that dichloromethane and methanol extracts of Conocarpus lancifolius have very little antileishmanicidal activity.

4.4.19: Antioxidant activity Conocarpus lancifolius extracts were examined for in vitro free radical scavenging activities by using the standard procedures. The IC50 value and percentage of inhibitory concentration (0.5 mg/ml) of Conocarpus lancifolius extracts were calculated and studied in comparison with standard drug (quercetin). Procedure of activity is briefly described in chapter 3. The detailed results are given in table 4.23.

Table 4.23: Result of antioxidant activities of Conocarpus lancifolius extracts. Part Extracts Conc. % inhibition % inhibition by % inhibition by (mg/ml) by DPPH NO scavenging FRAP method method method

CLD 0.5 28.1 ± 0.98 34.35 ± 0.46 42.06 ± 0.64 Arial CLM 0.5 92.1 ± 0.11 93.35 ± 0.41 93.2 ± 1.02

Std. Quercetin 0.1 92.12 ± 0.49 99.34 ± 0.48 98.12 ± 0.46

The results of table 4.23 highlight that dichloromethane and methanol extract of Conocarpus lancifolius has significant antioxidant activity. Documented shows that the methanol extracts have secondary metabolites like flavonoids and other phenolic compounds that have antioxidant activity.

152 4.5: Total Flavonoid Contents Dichloromethane, methanol and water extracts conforms the flavonoid contents of Conocarpus lancifolius. Quercetin is used as standard (990.17 ± 2.84). The outcome describe that dichloromethane extract of aerial part of Conocarpus lancifolius have flavonoid contents maximally (629.4 ± 1.57) and water extract of Conocarpus lancifolius shows flavonoid contents least (104.2 ± 0.06).

1200

990.17

1000

g/ml) µ

800 629.4 600 549.49 438.6

400 306.1 256.61

200 104.2 TOTAL FLAVONOIDS CONTENTS( FLAVONOIDS TOTAL

0 STD CLD CLM CLW CLSD CLSM CLSW

Figure 4.61: Total Flavonoids contents of Conocarpus lancifolius.

4.6: Quantification of phenolic contents by HPLC Conocarpus lancifolius methanol extract was analysed for phenolic contents by using HPLC. Outcome indicates that Conocarpus lancifolius methanol extract have chlorogenic acid, quercetin, ferulic acid, gallic acid and 4-OH 3-methoxy benzoic acid in concentration of 70.304, 45.772, , 74.93, 9 and 57.80 ppm. While stem part methanol extract of Conocarpus lancifolius contained M.coumeric acid, quercetin, chlorogenic acid, gallic acid, sinapic acid and ferulic acid in concentration of 15.772, 14.0149 , 10.356 , 37.108, 9.0325, 7.82, 32.4786 and 15.330 ppm respectively.

153 Table 4.24: Results phenolic contents by HPLC of Conocarpus lancifolius. N Compounds Conocarpus lancifolius o Names Arial Stem . Retention Area(mV.s) Concentratio Retention Area(mV.s) Concentration time n ppm time ppm 1 Quercetin 2.753 497.586 45.772 2.742 297.586 15.772 2 Gallic acid 4.227 271.652 9.779 4.325 360.740 14.0149 3 Vitamin c - - - 23.667 517.799 10.356 4 Chlorogenic 15.673 901.329 70.304 15.593 475.745 37.108 acid 5 M.Coumeric - - - 19.627 247.604 9.0325 acid 6 Sinapicacid - - - 26.580 601.639 7.82 7 Ferulic acid 22.587 1046.64 74.93 22.587 451.092 32.4786 0 8 4-OH 3- 14.387 545.298 57.80 - - - methoxy benzoic acid 9 Caffeic acid - - - 12.993 333.265 15.330

Figure 4.62: Calibration curve for quantification of phenolic contents of Conocarpus lancifolius.

4.7: Screening of biological activities of isolated compounds

4.7.1: DPPH radical scavenging, acetylchohlinestrase, butylchohlinestrase, α-glucosidase and Urease inhibition activities of isolated compounds A-G Compounds A-G was screened for biological activities such as acetylchohlinestrase butylchohlinestrase, free radical scavenging, and α- glucosidase inhibition activities by using the standard procedures. The percentage inhibition at 0.5 mM and IC50 value

(μM) values was measured. Procedures of activities are briefly described in chapter 3. The detailed results are presented in table 4.25and 4.26.

154

Table 4.25: Results of DPPH radical scavenging, AChE, BChE inhibition activities of compounds A-G

DPPH activity AChE BChE

Percentage IC Percentage IC Percentage Compound 50 50 IC value inhibition value inhibition at value inhibition 50 (μM) at 0.5 mM (μM) 0.5 mM (μM) at 0.5 Mm A 42.14±0.13 - 36.41±0.19 _ 32.54±0.17 _

B 16.23±0.15 - 48.43±0.14 _ 15.54±0.14 _ 0.08±0 C 16.55±0.13 - 71.62±0.16 45.62±0.13 _ .0004 0.07±0 D 17.15±0.16 - 65.91±0.17 48.52±0.18 _ .0003 E 13.36±0.17 - 46.35±0.13 _ 46.58±0.16 _ 94.24± F 83.68±0.18 45.91±0.14 _ 17.83±0.15 _ 0.13 184.32 G 68.43±0.17 35.24±0.16 _ 24.38±0.12 _ ±0.14 Quercetin Eserine Standard 16.96± Standard 0.04±0 0.85±0.00 93.21±0.97 91.27±1.17 81.82±1.07 control 0.14 control .0001 1

The results of table 4.25 highlights that F and G compounds show antioxidant activity which is significant, when calculated by DPPH method having percentage inhibition of 83.68±0.18 and 68.43±0.17 at 0.5 mM concentration. A, B, C, D and E compounds represents moderate antioxidant activity by DPPH method having percentage inhibition of 42.14±0.13, 16.23±0.15, 16.55±0.13, 17.15±0.16 and 13.36±0.17. Quercetin is used as standard control. Acetylchohline esterase inhibitory activity of compounds C and D was significant having percentage inhibition of 71.62±0.16 and 65.91±0.17 at 0.5 mM concentration. The other compounds A, B, E, F, and G showed little percentage inhibition of 36.41±0.19, 48.43±0.14, 46.35±0.13, 45.91±0.14 and 35.24±0.16 respectively. Butyl cholinesterase activity of all the compounds (A-G) showed little percentage inhibition of 32.54±0.17, 15.54±0.14, 45.62±0.13, 48.52±0.18, 46.58±0.16, 17.83±0.15 and 24.38±0.12 at 0.5 mM concentration. Eserine is used as standard control.

155

Table 4.26: Results of urease and α- glucosidase inhibition activities of isolated Compounds A-G α- glucosidase inhibition Urease inhibition activity Activity

Percentage Percentage Compound IC value IC value inhibition at 50 inhibition at 50 (μM) (μM) 0.5 mM 0.5 mM A 45.91±0.15 - 17.74±0.17 -

B 48.85±0.16 - 64.25±0.24 321.84±0.17

C 39.75±0.14 - 92.83±0.18 87.46±0.12

D 35.96±0.15 - 42.24±0.16 -

E 38.64±0.12 - 32.64±0.14 -

F 40.79±0.14 - 72.26±0.18 94.23±0.16

G 66.54±0.26 174.97±0.14 92.58±0.22 93.54±0.13

Standard control Standard control 98.45±0.87 21.25±0.15 93.22±0.16 37.24±0.14 Thiourea Acarbose

The results of table 4.26 highlights that C, F and G compounds have significant α- glucosidase inhibition activity having percentage inhibition of 92.83±0.18, 72.26±0.18 and 92.58±0.2. B compound showed a moderate inhibition having percentage inhibition 64.25±0.24. A, D and E compounds have little activity with percent inhibitory values of 17.74±0.17, 42.24±0.16 and 32.64±0.14. Acarbose was uesd as standard drug. Urease inhibitory activity of G compound was significant with percentage inhibition 66.54±0.26. A, B, C, D, E and F compounds have little percentage inhibition of 45.91±0.15, 48.85±0.16, 39.75±0.14, 35.96±0.15, 38.64±0.12 and 40.79±0.14. Thiourea was used as standard control.

156

CHAPTER 5

157 5 REFERENCES

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