Phytochemical and Antimicrobial Investigation of Ochna Thomasiana Engl

PHYTOCHEMICAL AND ANTIMICROBIAL INVESTIGATION OF OCHNA THOMASIANA ENGL. & GILG

MBITHI JUSTUS MUEMA (B.ED, Sc) I56/10372/2007

A Thesis Submitted in Partial Fulfillment of the Requirements for the Award of the Degree of Master of Science in the School of Pure and Applied Sciences, Kenyatta University

JUNE, 2015

DECLARATION

DECLARATION BY CANDIDATE

This thesis is my original work and has not been presented for a degree in any other university or any other award.

Mbithi Justus Muema Reg No.I56/10372/2007

Signature Date

Department of Chemistry

DECLARATION BY SUPERVISORS

This thesis has been submitted in partial fulfillment of Master of Science degree of Kenyatta University with our approval as supervisors.

Prof. Alex K. Machocho Department of Chemistry Kenyatta University

Signature Date

Prof. Nicholas K. Gikonyo Department of Pharmacy and Complementary/Alternative Medicine Kenyatta University

Signature Date

DEDICATION

To my parents, my wife Merceline and children, Ndunge, Mwongeli and Wanza.

ACKNOWLEDGEMENTS

I wish to express my sincere gratitude to my supervisors, Prof. Alex K. Machocho and

Prof. Nicholas K. Gikonyo. Special thanks to Prof. Paul K. Tarus for initiating this project. I wish to thank Stephen Musyoka and Julius Mwenda who carried out the NMR analysis, Elias Maina the retired Chief Technician and Dennis Osoro the acting Chief

Technician for assisting with chemicals and glass ware, my classmates for encouraging me to continue with the course especially Ronald Okwemba, Mbarak Mohamed, Stanley

Tumbo, Philip Mayeku and the entire staff of Chemistry Department of Kenyatta

University.

I would like to extend my profound gratitude and appreciation to my wife Merceline, no words in this world can express my gratitude, my daughters Ndunge, Mwongeli and

Wanza, for the loneliness they suffered when they missed my company and care. The understanding was a special source of inspiration to me.

My heartfelt gratitude goes to my parents, sisters, brothers, Cousin David Mwania, Uncle

David Mutiso, in-law James Muya and friends for their encouragement and support both financially and otherwise.

My deepest appreciation goes to Higher Education Loans Board of Kenya and Ministry of

Education of Science and Technology for the financial support they gave me. I am also grateful to my employer, TSC for granting study leave to advance my education.

Last but not least, I am most indebted to God for giving me good health and strength during the entire period of this work. 5

TABLE OF CONTENTS

Page DECLARATION ii DEDICATION iii ACKNOWLEDGEMENT iv TABLE OF CONTENTS v LIST OF TABLES viii LIST OF PLATES ix LIST OF FIGURES x LIST OF SCHEMES xi LIST OF ABBREVIATIONS AND ACRONYMS xii ABSTRACT xiv

CHAPTER ONE INTRODUCTION 1 1.1 Background 1 1.2 Plants as a source of antimicrobials 2 1.3 Phytochemicals 3 1.4 Herbal medicine 3 1.5 Statement of the problem 5 1.6 Hypotheses 6 1.7 Objectives 6 1.7.1 General objective 6 1.7.2 Specific objectives 6 1.8 Justification and Significance 7

CHAPTER TWO LITERATURE REVIEW 8 2.1 Antimicrobial agents 8 2.1.1 Antibacterial agents 8 2.1.2 Antifungal agents 12 2.2 Antiviral agents 13 2.3 Flavonoids 16 2.4 Distribution of the Ochnaceae family 17 2.5 Economic importance of the Ochnaceae family 17 2.6 Medicinal uses of plants of Ochnaceae family 18 2.7 Medicinal uses of Ochna species 19 2.8 Phytochemical and Biological Activities of the Genus Ochna 22 2.9 Kenyan Ochna species 35

CHAPTER THREE METHODOLOGY 39 3.1 Plant collection and processing 39 3.2 Reagents 39 3.3 Cleaning procedures 39 3.4 Sequential extraction of root and stem barks 40 3.5 Thin layer chromatography 42 3.6 Melting point 43 3.7 Infrared (IR) spectroscopy 43 6

3.8 Ultraviolet (UV) spectroscopy 43 3.9 Nuclear magnetic resonance (NMR) spectroscopy 44 3.10 Mass Spectroscopy (MS) 45 3.11 Antibacterial tests 45 3.11.1 Bacterial strains tests 45 3.11.2 Preparation of media 46 3.11.3 Screening procedure 46 3.11.4 Minimum inhibitory concentrations (MIC) and MBC 47 3.11.5 Disc diffusion and MIC ratings of the extracts 48 3.12 Isolation of compounds from Ochna thomasiana 49 3.13 Physical and spectroscopic data of isolated compounds 54 3.13.1 Compound 18 54 3.13.2 Compound 20 54 3.13.3 Compound 17 55 3.13.4 Compound 23 55 3.13.4 Compound 74 56 3.13.5 Compound 75 56

CHAPTER FOUR RESULTS AND DISCUSSION 58 4.1 Plant material yield of extracts 58 4.2 Antibacterial disc diffusion screening test for the O. thomasiana extracts 59 4.2.1 The DCM extracts 59 4.2.2 The ethyl acetate extracts 59 4.2.3 The methanol extracts 59 4.2.4 MIC and MBC of O. thomasiana methanol extracts 60 4.3 Structure elucidation 61 4.3.1 Compound 18 61 4.3.2 Compound 20 66 4.3.3 Compound 17 69 4.3.4 Compound 23 and 74 71 4.3.5 Compound 75 75 4.4 Antibacterial activity test for the isolated compounds 77

CHAPTER FIVE CONCLUSIONS AND RECOMMENDATIONS 78 5.1 Conclusions from the study 78 5.2 Recommendations from the study 79 5.3 Suggestions for further research 79 REFERENCES 80 APPENDICES 92 1 Appendix 1a: H NMR (600 MHz CD3OD) of compound 18 92 13 Appendix 1b: C NMR (150 MHz CD3OD) of compound 18 93 13 Appendix 1c: C DEPT (CH) NMR (150 MHz CD3OD) of compound 18 94 13 Appendix 1d: C NMR APT (150 MHz CD3OD) of compound 18 95 1 1 Appendix 1e: H- H COSY (600 MHz CD3OD) of compound 18 96 13 Appendix 1f: C NMR HSQC (150 MHz CD3OD) of compound 18 97 13 Appendix 1g: C NMR HMBC (150 MHz CD3OD) of compound 18 98 Appendix 1h: IR of compound 18 99 Appendix 1i: UV of compound 18 100 7

1 Appendix 2a: H NMR (400 MHz CD3OD) of compound 20 101 13 Appendix 2b: C NMR (100 MHz CD3OD) of compound 20 102 Appendix 2c: IR of compound 20 103 1 Appendix 3a: H NMR (400 MHz CD3OD) of compound 17 104 13 Appendix 3b: C NMR (100 MHz CD3OD) of compound 17 105 Appendix 3c: IR of compound 17 106 13 Appendix 4a: C NMR (100 MHz CDCl3) of compound 23 and 74 107 13 Appendix 4b: C NMR (100 MHz CDCl3) of compound 23 and 74 108 Appendix 4c: IR of compound 23 and 74 109 Appendix 4d: MS of compound 23 and 74 110 1 Appendix 5a: H NMR (600 MHz CDCl3) of compound 75 111 1 Appendix 5b: H NMR (600 MHz CDCl3) of compound 75 112 1 Appendix 5c: Expanded part H NMR (600 MHz CDCl3) of compound 75 113 13 Appendix 5d: C NMR (150 MHz CDCl3) of compound 75 114 13 Appendix 5e: Part of C DEPT-135 (150 MHz CDCl3) of compound 75 115 13 Appendix 5f: Section of C NMR (150 MHz CDCl3) of compound 75 116 13 Appendix 5g: C NMR DEPT (150 MHz CDCl3) of compound 75 117 Appendix 5h: IR of compound 75 118 Appendix 5i: UV of compound 75 119

LIST OF TABLES Page Table 2.1: Medicinal uses of Ochna species 20 Table 4.1: Plant material yield of O. thomasiana extracts 58 Table 4.2: The inhibition zones (in mm) of crude extracts of O. thomasiana 60 Table 4.3: The MIC and MBC of crude extracts of O. thomasiana 60 1 Table 4.4: H NMR (600 MHz, CD3OD) and COSY for 18 63 13 Table 4.5: C NMR (150 MHz, CD3OD), DEPT, HSQC and HMBC 18 65 1 13 Table 4.6: H and C NMR (100 MHz, CD3OD) data for 20 68 1 13 Table 4.7: H and C NMR (100 MHz, CD3OD) data for 17 71 13 Table 4.8: C NMR DEPT (100 MHz, CDCl3) for 23 and 74 74 13 Table 4.9: C NMR (150 MHz, CDCl3) for 75 76 Table 4.10: Inhibition zones (in mm) of antibacterial activity of the compounds 77

LIST OF PLATES Page Plate 2.1: Map of distribution of Ochna species 18 Plate 2.2: Twig, leaves and fruits Ochna serrulata 20 Plate 2.3: Photograph of aerial part of Ochna thomasiana 38 Plate 2.4: Photograph of flowers and fruit of Ochna thomasiana 38 Plate 3.1: Photograph of Petri dish showing disk diffusion method 48 Plate 3.2: Test of bacterium 48

LIST OF FIGURES Page Figure 4.1: HMBC correlations in partial structure 18 62 Figure 4.2: HMBC correlations in the partial structure 18 64 Figure 4.3: Compound 18 66 Figure 4.4: Compound 20 69 Figure 4.5: Compound 17 70 Figure 4.6: Compound 23 73 Figure 4.7: Compound 74 73 Figure 4.8: Compound 75 76

LIST OF SCHEMES Page Scheme 3.1: Sequential extractions of O. thomasiana stem bark. 41 Scheme 3.2: Sequential extractions of O. thomasiana root bark. 42 Scheme 3.3: Chromatographic separation of O. thomasiana root bark extract. 52 Scheme 3.4: Chromatographic separation of O. thomasiana root bark extract. 53

ABBREVIATIONS AND ACRONYMS

¹³C NMR Carbon 13 Nuclear Magnetic Resonance ¹H NMR Proton Nuclear Magnetic Resonance 2D NMR Two Dimensional Nuclear Magnetic Resonance AA Antimicrobial Activity Ac Acetyl AIDS Acquired Immune Deficiency Syndrome APT Attached Proton Test ASFMT Arubuko Sokoke Forest Management Team ATCC American Type Culture Collection CC Column Chromatography

CD3OD Deuterated Methanol

CDCl3 Deuterated Chloroform COSY Correlation Spectroscopy d Doublet DCM Dichloromethane DEPT Distortionless Enhancement by Polarization Transfer DMSO Dimethyl Sulphoxide DR Democratic Republic DST Diagnostic Sensitivity Test Et Ethyl EtOAc Ethyl Acetate HETCOR Heteronuclear Correlation HIV Human Immunodeficiency Virus HMBC Heteronuclear Multiple Bond Coherence HPLC High Performance Liquid Chromatography HSQC Heteronuclear Single Quantam Coherence ICU Intensive Care Unit IR Infrared Jr Junior KEMRI Kenya Medical Research Institute KNPHL Kenya National Public Health Laboratories LD Lethal Dose 13 m Multiplet mb Millibar MBC Minimum Bactericidal Concentration MDRS Multi-Drug Resistance Strain MRSA Methicillin Resistant Staphylococcus Aureus MeOH Methanol MP Metalloporphyrin MIC Minimum Inhibitory Concentration MS Mass Spectroscopy NA Nutrient Agar NMR Nuclear Magnetic Resonance NOESY Nuclear Overhauser Enhancement Spectroscopy PDA Potatoes Dextrose Agar ppm Parts Per Million PTLC Preparative Thin Layer Chromatography s Singlet Sw Swahili

Rf Retention factor t Triplet TLC Thin Layer Chromatography TMS Tetramethylsilane µg Microgram UV Ultraviolet VLC Vacuum Liquid Chromatography WHO World Health Organization δ Chemical Shift

ABSTRACT

Infectious diseases are the leading cause of death world-wide despite the vigorous campaigns that have been made to combat them. This has been occasioned by drastic growth of drug resistant pathogens. Phytomedicines derived from plants have shown the ability to overcome resistance in some organisms and great promise in the treatment of intractable infectious diseases. Plant based antimicrobials represent a vast untapped source of medicines and a further exploration of plant antimicrobials is called for. Plant extracts have led to the discovery of many clinically useful drugs such as emetine, berberines and quinine. There is a continuous and urgent need to discover new antimicrobial compounds with diverse chemical structures and novel mechanisms of action for new and re-emerging infectious diseases. Therefore, researchers are increasingly turning their attention to folk medicine, looking for new leads to develop better drugs against microbial infections. The plant species O. thomasiana has been reported be used as a herbal remedy by the Mijikenda community traditional medicinal practitioners. The study aimed at the determination and evaluation of the biological activities of the plant Ochna thomasiana. In this study, the plant extracts were screened for their antibacterial activity against selected strains of bacteria, including Gram- negative Salmonella typhi (clinical isolate) and Pseudomonas aeruginosa, and Gram- positive Escherichia coli, Bacillus subtilis, Staphycoccus aureus. Various chromatographic techniques were utilized to separate and isolate the compounds. The purification of the extracts was done using silica gel, column chromatography (CC), Sephadex gel and preparative thin layer chromatography (PTLC). Structure characterization was carried out using standard spectroscopic methods: Infrared (IR), ultraviolet (UV) spectroscopy, mass spectroscopy (MS) and proton nuclear magnetic resonance (1H NMR), carbon-13 nuclear magnetic resonance (13C NMR), distortionless enhancement by polarization transfer (DEPT), coherence spectroscopy (COSY), heteronuclear multiple bond coherence (HMBC) and heteronuclear single quantam coherence (HSQC). Lophirone A (18), afzelone D dimethylether (20), calodenone (17), a mixture of stigmasterol (74) and β-sitosterol (23) and 3β-acetyl-24-ethylfriedelane (75) were identified. The stem and root bark methanol crude extracts showed high activity against the Gram-positive bacteria with zones of inhibition of 14, 15 and 20 mm against the same strains of bacteria. Lophirone A, afzelone D dimethylether and 3β-acetyl-24- ethylfriedelane showed high activity against the Gram-positive Staphylococcus aureus with zones of inhibition of 14, 16 and 18, respectively. The results of the study showed the root of O. thomasiana contains biflavonoids, and some sterols as its constituents and their antimicrobial activity is significant and is a lead towards the development antimicrobial agents. This indicated that this plant contains important bioactive compounds and further, the antimicrobial activity of the crude extracts of this plant confirms its use in traditional medicine. However, there is need for in vivo and in vitro evaluation of the crude extracts and isolated compounds. The plant species should be propagated using good agricultural practices for medicinal plants for future evaluation of their activity against pathogens.

CHAPTER ONE 16

INTRODUCTION

1.5 Background

The use of plants in indigenous cultures are multiple and very diverse. For many people they still form an important economic basis and are used as food, medicine, construction material, firewood, dyes, as ritual paraphernalia and ornaments. For thousands of years plants have been the foundation of traditional medicine systems where the knowledge on the plants has been passed on from generation to generation (Koehn and Carter, 2005).

The abundance of plants on the earth‟s surface has led to an increasing interest in the investigations of different extracts obtained from traditional medicinal plants as potential sources of new antimicrobials agents (Bonjar and Farrokhi, 2004). People have used plants for millennia and vast information of the medicinal uses of plants has therefore accumulated especially in the tropical parts of the world. In African, Indian and Chinese communities, plants have formed the main ingredient of traditional medicines (Gurib-

Fakim, 2006; Magassouba et al., 2007).

Medicinal plants are gifts of nature to cure limitless number of diseases among human beings (Bushra and Ganga, 2003). Over 66% of the population residing in developing countries was estimated by WHO in 2008 to be depending directly on plants for their primary medical requirements (WHO, 2008). This is attributed to the fact that plant- derived medicines can be easily accessed and are also cheap (Amin and Mousa, 2007;

Ramawat and Goyal, 2008; WHO, 2008). Even the communities of the developed world are also dependent directly or indirectly on plants for their health care. In the United

States, 11% of the prescriptions given from community pharmacies consisted of plant extracts or active ingredients of plant origin (Cragg and Newman, 2005). In Dar es

Salaam in Tanzania, 21% of patients who visited public hospitals had consulted a 17 traditional healer before they went to hospital (de Boer et al., 2005). Plant-derived medicines are taken in the form of tinctures, teas, poultices and powders, depending on the knowledge of the use and application method of a particular plant for a given ailment

(Fennell et al., 2004; Balunas and Kinghorn, 2005).

1.6 Plants as a source of antimicrobials

Plants produce compounds of varying diversity as a means of defence against bacteria, fungi, pests and predators, hence the plants are efficient natural chemical factories, producing compounds of various structures that result in different physiological effects in the body once ingested (Edeoga et al., 2005). In 1971, substances isolated from plants were used as important drugs in one or more countries and that 60% of these compounds were discovered as a result of phytochemical studies on plants used for medicinal purposes (Farnsworth et al., 1985).

The relationship between man and plants has been very close throughout the development of almost all civilizations. The plant kingdom is abundant and natural products of higher plants may give a new source of antimicrobial agents with possibly novel mechanisms of action (Shahidi, 2004; Runyoro et al., 2006). The provision of safe and effective medicines could become a critical tool to increase access to health care (WHO, 2002;

Duraipandiyan and Ignacimuthu 2007). This has necessitated studies on other potential sources of effective, safe and cheap antimicrobial drugs, and plants have been indicated to be an alternative source (Thangadurai et al., 2004).

1.3 Phytochemicals 18

These are secondary metabolites that are temporarily biosynthesized and stored in the plant to protect against attack by microorganisms. These medicinal properties that the secondary metabolites could possess may be equal or superior to that found in synthetic drugs. Several of these compounds including the flavonoids, terpenoids, coumarins, saponins, alkaloids, iridoids and tannins have been identified (Jassim and Naji, 2003).

Antimicrobial activities exhibited by plants have been attributed mostly to the presence of one or more of these compounds (Asres et al., 2005).

1.4 Herbal medicine

Pharmaceutical drugs are seen increasingly as over prescribed, expensive and even toxic, while herbal remedies are seen as less expensive and less toxic. In the recent years there has been growing interest in therapeutic use of natural products, especially those derived from plants (Newman and Cragg, 2010). This interest in drugs of plant origin is due to several reasons, namely, convectional medicine have side effects and ineffective therapy.

A large percentage of world‟s population does not access to conventional pharmacological treatment, and folk medicine and ecological awareness suggest that

“natural” products are harmless (Adewole et al., 2004).

Historically, plants derived medicines have made large contribution to human health and well-being. Plants have provided a source of inspiration of novel drug compounds, as plant derived medicines have made large contributions to human health and well-being.

Their role is twofold namely; they provide key chemical structure for the development of new antimicrobial drugs and also as a phytomedicine to be used for the treatment of disease (Abukakar et al., 2008). Traditional medicine provides a reservoir of plants 19 materials that should be tapped. There is often (but by no means always) a correlation between village uses of plant and its activity in biological screening (Anokbongoo, 1992).

Many complementary medicines, particularly herbal medicines, have a long history of traditional use (Barnes, 2003). People all over the world have used plants as medicine for thousands of years. These drugs are usually prepared from roots, stems, leaves, seeds, tubers, or from exudates of plants (Mukherjee, 2002). There are various types of preparations including; concoctions, decoctions, infusions and powder. Latest developments show that because of commercialization and modern technology, products of herbal origin are available in forms of tablets, capsules, creams, ointments and injectable (Mukherjee, 2002). In the United Kingdom (UK), use of herbal medicine is a popular health care approach, and there are signs that the use of plant products is increasing (Barnes, 2003).

Plants have a long history of use on the African continent for the treatment of cancer and many people use plants for medicinal purposes as supplement to visiting western health care practitioners, and still continue to play an essential role in health care (Cragg and

Newman, 2005). Africa still boasts a wide variety of indigenous species, and if appropriate approach and investigations are put in place, very interesting biologically active compounds could be discovered from the traditional medicinal plants (Rosenthal,

2001). Up to 20% of modern drugs are derived from natural sources, using either the natural substance or a synthesized version (Jassim and Naji, 2003).

In an attempt to avoid huge hospital costs, unnecessary delay, and insufficient money to travel long distance for medical care and for buying drugs, the vast majority of the people 20 rely on traditional medicine, particularly herbal medicines (Nsowah-Nuamah et al.,

2005). The health facilities are inaccessible to most of the population, the ratio of doctor to patients is so high that it may take a very long time before a patient gets access to the doctor and in addition, inadequate resources and low wages offered by health institutions has made health workers poorly motivated. This has resulted in mass and continued exodus of health professionals to seek more lucrative opportunities in other countries

(Dovlo, 2003).

Based on the above reasons, Kenyans have not been left behind in the extraction and use of herbal remedies and mostly in crude form. Among the Kenyan plants that have also shown appreciable use in traditional medicine are members of the Ochnaceae family, especially the genus Ochna. There are eight Ochna species in Kenya on which phytochemical work has only been undertaken on Ochna holtzii (Mohamed, 2010). This research was directed specifically to investigate the Ochna thomasiana species which is widely distributed along the Coastal region of Kenya. It is used extensively in treating microbial infections in traditional therapy (Beentje, 2009).

1.5 Statement of the problem

Due to either limited availability or affordability of pharmaceutical medicine, the rural population in Coastal Kenyan communities and Africa as a whole rely on traditional herbal remedies for primary health care. The increase in infectious diseases and resistance to antimicrobial drugs has called for development of newer, safe and effective medicines.

This has necessitated studies on other potential sources of effective, safe and cheap antimicrobial drugs, and plants have been considered to be an alternative source. There is need for a reliable, bioassay guided fractionation which can detect a broad spectrum of 21 pharmacological activities in plants in order produce compounds of varying diversity. In

Coastal Kenyan communities, O. thomasiana is used in traditional medicine for management of microbial infection (Kokwaro, 2009). However, the antimicrobial activities have not been validated scientifically. Furthermore, safety of O. thomasiana to the patients using it is yet to be established.

1.6 Hypotheses

i. The root and stem bark of O. thomasiana do contain antimicrobial compounds.

ii. The antimicrobial compounds of the root and stem bark cannot be extracted,

isolated and identified.

iii. The antimicrobial compounds of the root and stem bark do not remain active

once isolated from the plant source.

1.7 Objectives

1.7.1 General objective

The study aims at phytochemical investigation of bioactive compounds with antimicrobial properties from the plant species O. thomasiana.

1.7.2 Specific objectives

i. To evaluate antimicrobial activities of the DCM, EtOAc and MeOH crude extracts

of O. thomasiana.

ii. To determine the structures of the isolated compounds from the acquired spectrum

using spectroscopic techniques (mp, IR, UV, 1D and 2D NMR, MS).

iii. To evaluate antimicrobial activities of isolated pure compounds from O.

thomasiana. 22

1.8 Justification and significance

The search for biologically active compounds extracted from traditionally used plants is relevant due the increasing resistance of bacteria to synthetic antibiotics and the occurrence of fatal opportunistic infections (Elgorashi et al., 2004). In Kenya, many plants used in traditional therapy have not been investigated systematically to determine their efficacy, safety and active principles. When the active components are known, they can be isolated and the application of structure activity modification studies can be done so as to synthesize more selective and potent derivatives. The bioactive compounds can be used as templates for synthetic drugs and as industrial raw materials.

Serious infections caused by bacteria that have become resistant to commonly used

st antibiotics have become a major global healthcare problem in the 21 century.

Microorganisms, especially bacteria, are becoming resistant to more and more antimicrobial agents. Persons infected with drug resistant strains take longer periods to recover and may even require treatment with second or third line drugs that may be effective but are more toxic or expensive. Generally there is lack of curative treatment for several chronic diseases and emerging infectious diseases. In Kenya, plants have a long history of use for the treatment of different diseases and complaints, and still continue to play an essential role in health care, with little or no scientific information on efficacy and side effects. The aim of the study was to determine the efficacy of the plant species

Ochna thomasiana, used in traditional medicine by the herbalists in coast region of Kenya with a view to ascertain their efficacy and possibly finding new effective antimicrobials

(Kokwaro, 2009).

CHAPTER TWO

LITERATURE REVIEW

2.1 Antimicrobial agents

Antimicrobial agents are drugs, chemicals, or other substances that either kill or slow the growth of microbes. Among the antimicrobial agents are antibacterial drugs, antiviral agents, antifungal agents, and antiparasitic drugs (Medical Dictionary, 2008).

Antimicrobial agents are among the most commonly used and misused of all drugs. The inevitable consequences of the wide spread use of anti-microbial agents has been the emergence of antibiotic resistant pathogens, fuelling an ever increasing need for new drugs (Chambers, 2006).

Herbal remedies used in traditional folk medicine provide an interesting and still largely unexplored source for the creation and development of potentially new drugs for chemotherapy which might help to overcome the growing problem of drug resistance and also the toxicity of currently available commercial antibiotics (Al-Wadh et al., 2001).

Antimicrobials can be synthesized by microorganism, plants or manufactured chemically, while some are semi-synthetic (Prescott, 2002).

2.1.1 Antibacterial agents

Antibacterial agents are either bacteriostatic (inhibiting growth of bacterial cells) or bactericidal (causing death of bacteria). Bactericidal drugs are usually independent in their actions while bacteriostatic ones are dependent on the host‟s defence mechanisms for the eventual elimination of pathogenic microorganisms (Grahame-Smith and Aronson,

1991). An important group is the sulphur drugs, which include the sulphonamides.

Sulphonamides inhibit both gram-positive and gram-negative bacteria, nocardia, 24

Chlamydia trachomatis and some protozoa. Sulfamethoxazole (1) is an important drug of sulphonamides group, and has been extensively employed in medicine in the treatment of pneumonia, staphylococci, gonococci, streptococcal infections, meningococcal meningitis, and in the treatment of open wounds to prevent gangrene (Garg et al., 1986).

NHSO2 N H3C O

NH2

However, some side effects have been observed during treatment with these drugs which include high fever, skin rashes, exfoliative dermatitis, photosensitivity, urticaria, nausea, vomiting, diarrhea, stomatitis, arthritis, hepatitis and hematopoietic disturbances and rarely, polyarteritis nodosa and psychosis. Resistance to sulphonamides is also a major problem (Mandell, 2003). Chloramphenicol (2) and tetracycline (3) are broad-spectrum bacteriostatic, broad-spectrum antimicrobials produced by species of Streptomyces venezuelae soil bacteria. They are useful against infections caused by many gram-positive and gram-negative bacteria. Chroramphenicol is a drug of choice for typhoid fever

(Wistreich and Lechtman, 1984).

Tigecycline (4) is the latest developed drug in this group. Tigecycline are also useful in mixed infections of the respiratory tract and in acne. It is effective for the treatment of skin and skin-structure infection and intra-abdominal infections. It is also active against wide range of multidrug-resistant nosocomial pathogens (for example, Staphylococcus aureus, β-lactamase-producing gram negatives and Acinetobacter species) (Hancock,

2005) infections. 25

H H3C N H3C OH OH

O2N O OH H H Cl NH2 N C C H OH O O O H CH2OH Cl OH OH

2 3

N(CH3)2 N(CH3)2 OH H O H3C NH2 H3C CH3 H HO O HO O O OH 4

However, some strains of organisms have become resistant to these agents and this has decreased their usefulness. Resistance is transmitted mainly by plasmids, and since the genes controlling resistance to tetracyclines are closely associated with gene resistance to other antibiotics, organisms may become resistant to many drugs simultaneously. The most adverse effects of tetracyclines include nausea, vomiting, diarrhea, intestinal functional disturbances, anal pruritus, vaginal or oral candidiasis, hepatic function impairment, may cause deformity or growth inhibition if taken for a long time, dizziness, vertigo, renal tubular acidosis and other renal injury (Speer et al., 2005).

Penicillins, like all β-lactam antibiotics, inhibit bacterial growth by interfering with the transpeptidation reaction of bacterial cell wall synthesis. Penicillins have the basic structure of thiazolidine ring attached to a β-lactam ring that carries a secondary amino group. Penicillin G (5) has the greatest activity against streptococci, meningococci, enterococci, non-β-lactamase producing staphylococci, Treponema pallidum and many other spirochetes, clostridium species, actinomyces and other Gram-positive rods and non-β-lactamase producing Gram-negative anaerobic organisms. Unfortunately, it has 26 little activity against Gram-negative rods and it is susceptible to hydrolysis by β- lactamases (Jacoby and Munoz-Price, 2005).

N H S CH3

CH3 N O O

The XF (dicationic porpyhrin derivatives) are a series of 19 antibacterial compounds that work very differently from antibiotics side stepping mechanism that bacteria defend themselves. XF-70 (6) and XF-73 (7) are novel porphyrin antibacterial drug candidates that rapidly kill exponential phase cultures of Staphylococcus aureus by interfering with the integrity of the cytoplasmic membrane (Ooi et al., 2009; Gonzales et al., 2010).

NH N NH N O O O O

HN N HN

+ N + N+ + _ _ N N Cl Cl Cl _ Cl _

6 7

The XF drugs kill bacteria extremely quickly than traditional antibiotics within biofilms

(the protective "jelly" bacteria produce around themselves, which acts as a protective barrier against antibiotics). Bacteria such as methicillin-resistant Staphylococcus aureus

(MRSA) seem unable to become resistant to XF/MP (Metalloporphyrin) drug action

(unlike many antibiotics, to which the super bug can rapidly become resistant). The MP drugs appear to share many of the same revolutionary and highly advantageous 27 antibacterial properties of the XF drugs (http://www.destinypharma.com/vision.shtml; Ooi et al., 2010).

2.1.2 Antifungal agents

The prolonged use of antifungal drugs in the treatment of chronic fungal infections has caused the emergence of amphotericin-B (8) and azole resistant candida species (Patel,

1998) Amphotericin B (8) is a potent antifungal drug which is active against most fungi and yeasts, both superficial (affecting the exposed organs) and systemic infections

(affecting deeper tissues and organs). This potent drug should not be used to treat noninvasive fungal infections, such as oral thrush, vaginal candidiasis, and esophageal candidiasis in patients with normal neutrophil counts. Amphotericin B may be useful in the treatment of American mucocutaneous leishmaniasis, but it is not the drug of choice as primary therapy (Bennett, 1996).

OH OH HO O OH

OH HO O OH OH OH OH O CH3 H O

H3C OH

O NH2

CH3 O OH 8

Azoles are another group of synthetic antifungal drugs. The azole antifungal agents in clinical use contain either two or three nitrogens in the azole ring and are thereby classified as imidazoles, ketoconazole (9) or triazoles, fluconazole (10), respectively

(Eicher and Hauptmann, 2003). With the exception of ketoconazole, use of the imidazoles is limited to the treatment of superficial mycoses, whereas the triazoles have a broad range of applications in the treatment of both superficial and systemic fungal infections. 28

Another advantage of the triazoles is their greater affinity for fungal rather than mammalian cytochrome P-230 enzymes, which contributes to an improved safety profile.

N N

N HO N N N O F O Cl O N

N N Cl F

9 10

The spectrum of azole medications is broad, ranging from many Candida species,

Cryptococcus neoformans, the endemic mycoses, the dermatophytes and even Aspergillus infections. They are also useful in the treatment of intrinsically amphotericin-resistant organisms such as Pseudallescheria boydii or Penicillium marneffei (Bennett, 1996).

Fluconazole (10) is the azole of choice in the treatment of primary and secondary prophylaxis of Cryptococcal meningitis and the most commonly used drug, especially in the treatment of Mucocutaneous candidiasis. Prophylactic use of fluconazole has been demonstrated to reduce fungal disease in bone marrow transplant recipients and in AIDS patients (Edwards et al., 1997). However, fluconazole displays no activity against

Aspergillus or other filamentous fungi. Moreover, its activity against dimorphic fungi is limited to coccidioidal disease. The emergence of fluconazole (10) resistant fungi has highly raised concerns about this indication though it has proved to be safer than both amphotericin B (6) and ketoconazole (9) (Sheehan et al., 1999).

2.2 Antiviral agents

Antiviral drugs are a class of medication used specifically for treating viral infections.

Unlike most antibiotics, antiviral drugs do not destroy their target pathogens; they inhibit 29 their development. Ancistrocladus korupensis (Ancistrocladaceae), a plant native to

Korup National Park in Cameroon is the main source of michellamine B (11). The compound is an anti-HIV naphthalene-tetrahydroisoquinoline alkaloid and the leaves of this plant are the only known source of the compound. Michellamine B (11) is known to inhibit HIV-1 in its early T-lymphocyte viral infection phase and also inhibits HIV-2 in

MT-2 cells. The studies done on dogs showed that the effective anti-HIV in vivo concentration of the compound is reached close to its neurotoxic concentration level.

Thus, despite its in vitro activity against a wide range of HIV-1 and HIV-2 strains, the small difference between the toxic concentration and the concentration required for efficient antiviral activity has resulted in the discontinuation of further studies designed for its clinical development (McMahon et al., 1995; Yang et al., 2001; Singh et al., 2005;

Tandon and Chhor, 2005; Alves and Rosa, 2007; Cheng et al., 2008).

CH3 OH

H3C OH

H3C OH OCH3

OCH3 OH CH3

HO CH3

OH CH3 11

(+)-Calanolide A (12), an anti-HIV coumarin derivative, was isolated from the leaves and twigs of Calophyllum lanigerum Miq., a rare plant found in Sarawak, Malaysia. The compound act by inhibiting HIV-1 reverse transcriptase and has been chosen for preclinical trials by the US National Cancer Institute. However, the sources of 12 from plant material are problematic since the original population of plants was destroyed

(Flavin et al., 1996; Spino et al., 1998). 30

Other populations of the same species yielded only a small amount of (+)-Calanolide A

(12). However, the latex of another species, C. teysmanii with major anti-HIV activity, contained (–)-calanolide B (14) as the active ingredient (McKee et al., 1996). Although

14 was slightly less active than 12, it was regarded as a better alternative due to the availability of the plant and because the latex can easily be obtained by just making small slashes on the mature tree bark without harming the plant. The studies focused on the isolation of active compounds from Calophyllum species yielded two enantiomers of calanolide A (12 and 14), two enantiomers of calanolide B (13 and 15) and (-)- dihydrocalanolide B (16) (Yang et al., 2001; Singh et al., 2005; Tandon and Chhor,

2005).

CH3 CH3 CH3 H3C CH3 H3C CH3 H3C CH3

O O O

O O O O O O O O O

OH CH OH CH OH H3C 3 3

CH3 CH3 CH3

12 13 14

CH3 CH3 H3C CH3 H3C CH3

O O

O O O O O O

OH OH CH3 CH3

CH3 CH3

15 16

2.3 Flavonoids

Flavonoids are phenolic compounds which are derived from the parent substance chalcone. Phenolic compounds change colour when treated with a base. Flavonoids are generally present in plants bound to sugar as glycosides and in any one flavonoid, a glycone may occur in a single plant in several glycosidic combinations. Flavonoids are present in plants as mixture and very rare to find only a single flavonoid component in a plant tissue (Harborne, 1998). Flavonoids have been reported to have anticancer, antibacterial, antiviral, immunomodulatory agents, antimalarial and anti-HIV agents, analgesics, anti-inflammatory compounds and as insect antifeedants (Murakami et al.,

1971a; Murakami et al., 1971b; Murakami et al., 1992; Ichino et al., 2006; Reutrakul et al., 2007).

Anthocyanins are a special class of flavonoids. They are intensely coloured water soluble pigments that are responsible for nearly all the pink, scarlet, mauve, violet and blue colours in petals, leaves and all are derived from this pigment by addition or substraction of hydroxyl groups or by glycosylation (Harborne, 1998). The flavonoid chemistry has continued to be the fastest growing in terms of the number of new natural products reported.

Plants that have revealed the presence of flavonoids in their studies continue to play an important role in medicinal uses especially as antimicrobial agents. Flavonoids are particularly beneficial acting as antioxidants and giving protection from cardiovascular diseases, certain forms of cancer and age related degeneration of cell components

(Dewick, 2002).

2.4 Distribution of the Ochnaceae family

The family Ochnaceae is mainly comprised of trees and shrubs with about 16 genera and

230 species which are highly distributed around the globe, Tropical Africa, Asia,

Australia, Madagascar, the Mascarene islands and America as shown in the map (Plate

2.1), are the regions where these species are mostly found (Coates, 2002; Mabberley,

2008). Species of the family Ochnaceae flourish in areas ranging from open to semi-open and in evergreen forests; they are characterized by evergreen petiole leaves which sometimes becomes leathery, specifically for the genus O. lanceolata (Gardner et al.,

2000).

2.5 Economic importance of the species of the Ochnaceae family

Some species of the family Ochnaceae are known to have economic value. Ochna kirkii,

O. mossambicensis, O. schweinfurthiana, O. serrulata, and O. thomasiana are known to have very attractive yellow flowers and beautiful fruits, the genus have been cultivated for ornamental purposes (Starr et al., 2003). Members of the genus Sauvagesia are used for making tea in Lesser Antilles, whereas members of Ouratea and Lophira are used as a source of valuable oil, commonly called “meni oil”, from their seeds. “Meni oil” has gained popularity in Nigeria and some other West African countries where it is used for cooking, remedy for lice and as a hair lotion (Burkill, 2000). The seeds of these species also serve as food, regardless of their bitter and caustic taste. Lophira species found in

West Africa are an excellent source of commercially valuable timber known by different names such as African oak, “azobe”, “ekki”, “bongossi” or red ironwood (Burkill, 2000).

Plate 2.1: Map of distribution of Ochna species

(Map: http://www.discoverlife.org/mp/20q?search=Ochna & guide=Guianas_flora)

2.6 Medicinal uses of plants of Ochnaceae family

In various traditional medicine systems, members of the family Ochnaceae are popularly used as medicines for different ailments. The species of the genus Lophira also known as

“beung” in Chamba language and “namijin kadanya” in Hausa language of West Africa in North western Nigeria and in adjoining southern Niger, are known to be helpful to pregnant women during labour (Jiofack et al., 2010; Lohlum et al., 2010). The leaves of

Lophira plants are boiled in water and the resulting extract is used for drinking and washing for an easy labour. The inner bark of these plants is used for pain relieving purposes, especially for the treatment of headaches. The bark decoction is reported to be used to stop vaginal discharge and also for the treatment of diarrhoea, ovarian cyst and typhoid fever where one small cup is consumed three times a day (Lohlum et al., 2010). 34

The genus Ouratea is used in Brazil and many West African countries in traditional medicine systems, where the leaves extracts are used for the treatment of upper respiratory tract infections, dysentery, and diarrhoea and as pain-relieving agents, especially for treatment of tooth ache. The extracts from the epicarp fruit commonly called folha-de-serra (Brazil) are used for the treatment of liver and skin infections

(Pegnyemb et al., 2005; Brandão et al., 2011).

2.7 Medicinal uses of Ochna species

Among the plants often used in traditional medicine, Ochna species, which belong to the family Ochnaceae, play a vital role. The genus Ochna includes 85 species of evergreen trees, shrubs, and shrublets, and is distributed widely in tropical Asia, Africa, and

America (Rendle, 1952) of which eleven species occur in India (Kirtikar, 2012).

In Southern Africa Ochna species are found in South Africa (Limpopo, Mpumalanga,

KwaZulu-Natal and Eastern Cape provinces), Swaziland, Zimbabwe and Mozambique.

The genus was named by Linnaeus in 1541, the name Ochna originated from a Greek word, Ochne, which means “wild pear”. This is because the leaves of these species resemble those of the wild pear. Species of this genus are usually known as Ochnas or

Mickey Mouse plants, this name is a result of the appearance of their black drupelets fruits sitting on a red receptacle mimicking the face of Mickey Mouse as shown in Plate

2.2 (Coates, 2002; Mabberley, 2008). 35

Plate 2.2: Twig, leaves and fruits Ochna serrulata (Photo: http://www.biodiversityexplorer.org/plants/ochnaceae/ochna.htm)

Several members of this genus have long been used in folk medicine for treatment of various ailments as shown and summarized in table 2.1

Table 2.1: Medicinal uses of Ochna species

Plant species Common Region Medicinal use References name(s) O. squarrosa “Sunari” or “erra India Constipation, Imam et al., L. juvvi” ulcers, sores, 2003 cancer, epilepsy, lumbago, asthma, antidote to snake bites digestive tonic, lumbago and menstrual complaints. O. Lour Thailand, Digestive tonic, Perry, 1980; integerrima Indonesia antidysenteric, Likhitwitayawui (Lour.) Merr and Vietnam anthelmintic, d et al., 2001; antipyretic agent. Kaewamatawong et al., 2002 O. arborea Cape Plane Tree Swaziland Bone fractures, Schmidt et al., Burch. ex DC. or headaches, 2002 “sifubasenkhala” protective charm to in SiSwati drive off evil spirit. 36

Table 2.1: Continuued…………

Plant species Common Region Medicinal use References name(s) O. Bird's-eye Bush Mpumalanga Constipation, Coates, 2002 gamostigmata South Africa ulcers, sores O. holstii Oliv red ironwood or Mpumalang Headache pain Coates, 2002 “umthelelo South Africa O. natalitia Natal plane or Mpumalang Menstrual Coates, 2002 (Meisn.) Walp “lincedza” South Africa complaints O. serrulata “umvuma”, Mpumalanga Treatment of bone Hutchings and Tiegh. Ex “fynblaarrooihout South Africa diseases and Van Staden, Keay ” Umbomvane or gangrenous 1993; Hutchings “iliTye” proctitis et al., 1996 O. Central Stem bark is used Messanga et al., calodendron Africa for pain relieving 2001 Gilg. et purposes, liver Mildbr. infections and dysentery. O. pulchra “umnyelenyele” Zimbabwe, Diarrhaoea Sibanda et al., Hook. f., or Mashonaland 1993 „muparamhosva‟ O. afzelii adangme” Ghana Female sterility, Bouquet, 1969 R.Br. ex Oliv. menstrual complaints, jaundice, lumbago and dysentery “mandingbambar Senegal a” or “mananitiana” “Tem or “fanam” Togo O. lanceolata O. heyneana central and Stem bark is used Muthukumarasa Spreng. peninsular as an abortifacient, my et al., 2003. India treating gastric complaints and menstrual disorders. O. pumila “champa baha” Himalayas, Leaves used as Chopra et al., Buch.-Ham. Asia poultice in lumbago 1980 ex DC. and ulcers, root used as an antidote to snake bites, prevent miscarriage, threatened abortion, decoction used for constipation and asthma.

Table 2.1: Continuued………

Plant species Common Region Medicinal use References name(s) O. obtusata “Ramatana India Antidote, menstrual Chopra et al., DC champaka” complaints, asthma, 1980. “Sunari”, stem bark used for “Tammi, Erra” dry coughs, “Juvvi” or infusion for “Kukkamovi” bronchitis, dysentery, cholera, inflammations, fever and pain O. macrocalyx “Mchimchim” Tanzania Washambaas use it Schlage et al., Oliv. (“Pogoro”) to treat, 2000 dysmenorrhoea, diarrhea, hemorrhoids, and stomach disorders

2.8 Phytochemical and Biological Activities of the Genus Ochna

From the previous phytochemical studies on the genus Ochna, it has been revealed that the genus is rich in phenolic biflavonoids (Likhitwitayawuid et al., 2001; Pegnyemb et al., 2001, 2003b; Khalivulla et al., 2008). The rearranged biflavonoids, calodenone (17), lophirone A (18), afzelone D (19) and afzelone D dimethyether (20) were isolated for the first time from the methanol extracts of the stem bark of O. calodendron, Lophira lanceolata and O. afzelii (Ghogomu et al., 1989; Messanga et al., 1994; Pegnyemb et al.,

2003a). The compounds represent a very unusual skeleton of a biflavonoid (Ghogomu et al., 1987).

To date there are many reports on the isolation of lophirone A (18) from Ochna species

(Kaewamatawong et al., 2002; Pegnyemb et al., 2003b; Kittisak et al., 2005; Anuradha et al., 2006). There are no reports on the biological activities of calodenone (17), afzelone D

(19) and afzelone D dimethyether (20). Plants in this genus are known to be rich in 38 biflavonoids, anthranoids and isoflavonoids (Kamil et al., 1983; Sibanda et al., 1993;

Messanga et al., 1994; Rao et al., 1997). Preliminary phytochemical studies on the

Ochnaceae members in Cameroon, especially those of Lophira and Ochna, have revealed the occurrence of constituents representing biflavonoids (Ghogomu et al., 1987).

R O O 3 O OR H 4

O OR1

H R2O OR2

17 R1= R2= R3= R 4 =H R2= Me

18 R 1 = R2= R3= R4= H

19 R 2 = Me R1 = R3= R4= H

20 R1 =R2 = Me R3=R4= H

Ochna pretoriensis occurs in South Africa (Germishuizen and Meyer, 2003) and two biflavonoids, Ochnaflavone (21), 2, 3-dihydroochnaflavone 7-O-methyl ether (22) and a

β-sitosterol (23), a common plant sterol have been reported from plant species

(Makhafola et al., 2009).

OH HO O O

OH O O OH

O OH

21 39

H3C OH MeO O H3C CH3 O CH3 CH3 O OH OH O CH3

O OH HO

22 23

Ochna integerrima occurs in Thailand (Smitinand and Larsen, 1981) and two biflavonoids, 2,3-dihydroochnaflavone (24), 2,3-dihydroochnaflavone 7-O-methyl ether (25) and a flavonoid glucoside: 6-,-dimethylallyl taxifolin 7-O--D-glucoside (26) have been reported from the leaves O. integerrima (Kaewamatawong et al., 2002).

OH HO O O O OR OH O

O OH

24 R=H 25 R=CH3

OH OH

O HO O O OH OH

OH O OH

Further investigations on the leaves of O. integerrima, led to the isolation of two ochnaflavones (27) and (28) (Likhitwitayawuid et al., 2003). 40

OH HO O O O OR OH O

O OH

27 R=H 28 R=CH3

From the stem bark of O. integerrima, the flavonoids 6-hydroxylophirone B (29) and

6-hydroxylophirone B 4-O--glucoside (30) were reported by Pegnyemb et al. (1998).

HO OH

O OH O O

HO R

29 R = OH 30 R = OGlu

Ochnaflavone (21) has a broad range of biological/pharmacological activities including anti-inflammatory, anticancer, anti-HIV and anti-atherogenic activities (Suh et al., 2006a; b; c). Reutrakul and group reported the isolation and anti-HIV activity of the derivatives of (21) viz. 7''-methoxyochnaflavone (31) and 7”-methoxy-2'',3''-dihydroochnaflavone

(32). The compounds were isolated from the leaves of O. integerrima and were found to exhibit a potent anti-HIV activity with EC30 values of 2.0 and 0.9 μg/ml, respectively

(Reutrakul et al., 2007). The two compounds 31 and 32 were also found to inhibit the

HIV-1 reverse transcriptase (RT) enzyme with IC30 values of 2.0 and 2.4 μg/ml, respectively (Reutrakul et al., 2007). 41

OH OH

HO O HO O O O

OH O OH O

O O

O O H CO OH 3 H3CO OH

31 32

Ochna beddomei is an important Indian medicinal plant (Chopra et al., 1980).

Phytochemical investigations on the leaves of O. beddomei led to the isolation of a biflavanone, 7-O-methyltetrahydroochnaflavone (33), together with nine known flavonoids: afrormosin (34), 2,3-dihydroochnaflavone 7-O-methyl ether (22), 2,3- dihydroochnaflavone (35), ochnaflavone (21), kaempferol (36), kaempferol 3-O- glucoside (37), kaempferol 3-O-rhamnoside (38), taxifolin 3-O-rhamnoside (39) and (-)- epicatechin (40) as reported by Jayaprakasam et al. (2000).

MeO O O HO O O OH OH O MeO O OH O OMe

33 34

OH HO O O

OH O O OH

O OH

35 42

Afrormosin (34) has shown strong anticancer inhibitory effects on the mouse lymphoma cell line (Gyemant et al., 2005). Kaempferol (36) has proven to be a potent antioxidant. It is also reported to offer protection against heart disease. It also acts as chemopreventive, which means that it inhibits the formation of cancer cells (Ray, 2007). (-)-Epicatechin is reported to have insulin mimetic action with protective effects on erythrocytes in a manner similar to insulin. It is also used as antioxidant and as well inhibits platelet aggregation (Khalivulla et al., 2008).

OH HO OH OH HO O O OH OH O H H OH OH OH O OH

36 37

OH OH HO O HO O OH OH OH O O O O OH O H H OH O H H H H H H H OH H OH

38 39

OH OH HO O H OH H OH

A biflavonoid, 2,3-dihydroochnaflavone 7,4‟,7"-tri-O-methyl ether (41) together with two known biflavonoids 2,3-dihydroochnaflavone (35) and ochnaflavone (21) were isolated from the stem bark of O. beddomei, as reported by Jayakrishna et al. (2003).

OMe MeO O O

O OMe OH O

O OH 41

A phytochemical study of Ochna pumila Buch, an Asian species found mainly in

Himalayas, led to the isolation of tetrahydroamentoflavone (42), 7"-O- methyltetrahydroamentoflavone (43), ochnaflavone (21) and 7"-O-methyl ochnaflavone

(44) (Kamil et al., 1983). Ochna pumila is also alleged to have antitubercular activities

(Chopra et al., 1980).

OH HO O

OH OH O R O

OH O

42 R = OH 43 R = OCH3 OH HO O O O OH O OCH3

O OH

Phytochemical studies on the leaf extracts of O. japotapita yielded three glycoflavones: vitexin (apigenin-8-C-glucoside) (45), orientin (luteolin-8-C-glucoside) (46) and isoorientin (luteolin-6-C-glucoside) (47) (Nair et al., 1975). Vitexin has been reported to be a potent antioxidant. It is also used as potent hypotensive, anti-inflammatory and possesses anti-spasmodic properties (Nguyen et al., 2006). 44

HO OH

OH Glu HO O OH OH HO O HO O OH OH O OH O

45 46

OH OH HO OH O O OH OH HO OH O

Phytochemical studies on the root extracts of O. squarrosa revealed the presence of an isoflavone, squarrosin (48). Squarrosin has been reported to be a strong analgesic and has anti-inflammatory effects (Nia and Gunasekar, 1992).

O O OMe O O OMe OMe

The compounds reported to be isolated from the root bark of O. obtusata include ochnaflavone flavonoids. Further investigation of the leaves of this plant yielded two biflavonoids: 2,3-dihydro ochnaflavone 7-O-methyl ether (49) and 2,3- dihydroochnaflavone (35) (Rao et al., 1997). 45

OR2

R1O O O

O OR2 O OR2

O OR2

49 R1 = Me R2 = H

Phytochemical studies on the root bark of O. pulchra led to the isolation of vismiones D

(50), L (51), F (52) and M (53) along with acetylvismione D (54) and 3-O- geranylemodine anthrone (55) (Sibanda et al., 1993).

O OH OH O OH OH

O OMe HO HO

50 51

OH O OH O OH OH

OH HO O

52 53

O OH OH OH O OH

O AcO O

54 55 46

Six biflavonoids have been reported from root bark Ochna macrocalyx Oliv., a medicinal plant commonly used by the Washambaas in Western Tanzania (Schlage et al., 2000).

They include, dehydroxy hexaspermone C (56), biisoflavanones, hexaspermone C (57), tetrahydrofuran derivative ochnone (58), furobenzopyran derivative, cordigol (59), and biflavonoids calodenin A (58) and dihydrocalodenin B (59) (Mabry et al., 1970). These compounds showed cytotoxity activity against breast cancer cells using the MIT reduction assay method and also showed strong antibacterial activity (Tang et al., 2003).

The ethanolic extract of the bark also showed NF-αB inhibitory activity.

HO HO O OH O OH H H H H MeO O MeO O O OMe O OMe H H H H OH O OMe O OMe

56 57

OH HO OH OH O H O OH OH O O HO H O O OH OH HO HO OH

58 59 47

O OH

HO O OH O OH HO O

OH O OH OH OH O

HO HO 60 61

During the phytochemical studies of O. lanceolata, two biflavonoids: 7,4‟,7",4"'- tetramethylisochamaejasmin (62) and 2,3–dihydro ochnaflavone 7"–O–methyl ether (63), together with six known flavonoids were isolated from the stem bark (Reddy et al., 2008).

OMe OH O HO O O OH O O O OMe OH O O OMe MeO O OH

62 63

From Ochna afzelii, a prominent Cameroonian medicinal plant (Bouqoet, 1969), it has been reported that the stem bark of plant when extracted with methanol yielded the biflavonoids, lophirone A (18), lophirone C (64) and isolophirone C (65) (Pegnyemb et al., 2001). The biflavonoid isolated from this plant is afzelone D (19), which is derivative of lophirone A, and was reported by Pegnyemb et al. (2003a). Pharmacological studies with the isolated major compound lophirone A (18) showed anti-tumor promoting activities (Murakami et al., 1971a). 48

OH OH HO O HO O

O O OH O OH O OH OH

OH HO

64 65

Further studies on root bark of O. afzelii yielded three biflavonoids afzelones A (66), B

(67), C (68) and a known calodenin B (60), as reported by Pegnyemb et al. (2003b).

HO O OH OCH3 O O O

O OH

O OH OH O

OH HO HO

66 67

HO O OH

OH O

Phytochemical investigation of O. calodendron stem bark, a common Cameroonian medicinal plant used as a fungicide, yielded two biflavonoids: lophirone K (69) and 49 hexaspermone C (57) and a known biflavonoid, calodenin B (60) by Messanga et al.

(1994).

OH HO O OH OH O O HO OH

Further investigation of the O. calodendron leaves, led to the isolation and identification of an interesting triflavonoid constituent, namely caloflavan A (70) together with irilone

(71), 3'-methoxyirilone (72) and prunetin (73) (Messanga et al., 2001).

HO HO

O OH OH OH

HO HO O O O O

OH O OH OH O OH OH

70 71

O O H3CO O

OCH O 3

OH O OH O OH OH

72 73

2.9 Kenyan Ochna species

Kenya boasts of eight Ochna species namely O. holtzii Gilg. O. holstii Engl., O. inermis

(Forssk) Schweinf, O. insculpta Sleumer, O. kirkii Oliv., O. ovata F. Hoffm., O. mossambicensis Klotzsch and O. thomasiana Engl. & Gilg. (Beentje, 2009). Several members of this genus are cultivated as decorative plants due to their colorful flowers and unusual fruits for instance O. kirkii, O. mossambicensis, and O. thomasiana

(Oppenheimer, 2003).

The wood of Ochna holstii Engl also called “Mkamachuma” in Digo vernacular language is used for joinery, furniture, domestic utensils and tool handles. It is suitable for heavy construction, heavy flooring, interior trim, ship building, sporting goods, toys, novelties, musical instruments, agricultural implements, carvings, pattern making, sliced veneer and plywood. It is also used as firewood and for charcoal production. The tree is used as ornamental shade tree. The bark produces a yellow dye. In Kenya, root extracts are taken against kidney and stomach problems (Verdcourt, 2005b).

Ochna ovata F. Hoffm., called “buttercup bush”, is a deciduous shrub or small tree up to

(9–15m) tall, occurring in dry forest and bush land in Kenya. Its wood is similar to that of

O. holstii and is used for construction, walking sticks, arrow shafts and pegs, and as firewood. Its leaf sap is applied as an eye medicine and root extracts against stomach- ache with diarrhea (Starr et al., 2003).

Ochna inermis (Forssk) Schweinf is mostly a rounded shrub, rarely a small tree. Branches greyish to whitish, covered in white lenticels. Leaves broadly elliptic to almost round, up to 0.5 cm long; margin finely but sharply toothed. It grows in dry Acacia, Commiphora or

Mopane scrub, among rocks or in sandy or rocky soils. Found in northern Kenya and 51

Machakos County, 217 km from Mombasa on Nairobi road, near Kenani (Verdcourt,

2005b).

Ochna kirkii Oliv. native to Kenya, Tanzania and Mozambique is elsewhere cultivated as ornamental plant. Ochna kirkii is a shrub or small tree growing up to 6 m tall. The glabrous leaves colourful generally have elliptic or oblong leaf blades with slightly cordate bases. Short bristles also line the leaf margins. The bisexual flowers develop on a raceme and bear five yellow petals. Upon fertilisation, the sepals turn from green-yellow to bright red when ripe and which encircle the black druplets on a pink receptable (Keng,

2003).

O. mossambicensis Klotzsch, shrub, small tree or rhizomatous sub shrub, 0.05–5(–9) m tall, bark pale grey or brown, smooth or rather rough and fissured; branches rather thick.

Leaves coriaceous, obovate to oblanceolate or oblong, (3.5–) 5.5–22.5 cm long, (1.5–) 2–

8.4 cm wide, obtuse to broadly rounded at the apex, less often subacute with a short mucro, cuneate to subtruncate at the base, margin densely serrulate; lateral veins 20–11, prominent on both surfaces; tertiary venation finely reticulate, more prominent above than beneath; petiole ± stout, 1.5–8 mm long (Verdcourt, 2005b).

Flowers numerous in branched panicles terminating lateral shoots; pedicels 1.5–3 cm long, jointed 4–8 mm from base, the joints forming characteristic tufts when flowers have fallen. It is found in Kwale District: 36 km from Mombasa on main Nairobi road,

Mombasa District: Nyali Bridge, mainland, Tana River District: 28 km South of Garsen

(Verdcourt, 2005b). 52

Almost all Kenyan species have not been studied phytochemically, thus the need to investigate them and establish the structures of their compounds and their bioactivity and phytochemical properties. Out of the eight Kenyan species, it is only O. holtzii Gilg which has been investigated from a phytochemical point of view (Mohamed, 2010).

Common uses include treatment of high fever, headache and coughs, and relieving persistent backaches especially in the old age. It is also used extensively as a mixture with other plant extracts in treating microbial infections. The compounds isolated from the

Ochna species from other parts of the world have been observed to possess very unique structures that have effective antimicrobial activity (Mohamed, 2010).

This study therefore aims at reporting the isolation, structural elucidation as well as biological activities of the compounds from O. thomasiana which is distributed at the

Kenyan coast in Kilifi and Kwale Counties (Verdcourt, 2005b). Ochna thomasiana (Plate.

2.3) is shrub or small tree 1.5–8.4(–9.6) m. Leaves simple, alternate, blade elliptic, 3-12 cm long (1¼-5 in), glabrous, with bristle-toothed margins. Flowers intermittently during the year and several, borne in auxiliary and terminal clusters. Corolla of five free, obviate,

3 yellow petals 1.8-2.5 cm long ( /4-1 in), with many yellow stamens in the centre. Fruit

3 composed of one to five black ovoid drupelets 7-10 mm long (¼- /8 in) borne on a fleshy red disk around the long, red style, with a red, persistent calyx (Starr et al., 2003;

Verdcourt, 2005b).

Ochna thomasiana also called “mvuapweza” (digo), “bird‟s eye plant” or “mickey mouse plant” occurs in evergreen bush land and coastal forest in Kenya. Its brown durable wood is used for door frames. Ochna thomasiana is cultivated as ornamental and sometimes naturalized outside its natural distribution area, like in DR Congo, Hawaii and India (Starr 53 et al., 2003; Pakia, 2005). Ochna thomasiana is used by the Mijikenda community for treatment of pneumonia especially in children, hypertension, ulcers and also rheumatism

(Kokwaro, 2009). The sticks of the O. thomasiana are also used in octopus fishing (Pakia,

2005).

Plate 2.3: Aerial part of Ochna thomasiana

Plate 2.4: Flowers and Fruit of Ochna thomasiana (http://www.starrenvironmental.com/images/image/?q=010700-0062&o=plants

CHAPTER THREE

METHODOLOGY

3.1 Plant collection and processing

Plant samples were collected from Arabuko-Sokoke, forest in Malindi district, Kilifi

County of Kenyas‟ coast province. The forest is situated about 120 Km north of

Mombasa town and transverses Kilifi County at latitude 3 20S and longitude 39 50E

(ASFMT, 2002). The root and stem barks were collected and plant identity was authenticated by a taxonomist, Mr Lucas Karimi of Kenyatta University, School of

Medicine, Department of Pharmacy and Complementary/Alternative Medicine, and confirmed at the herbarium of the Kenya National Museums where Voucher specimen

(MM/OH/001/06) was deposited at Kenyatta University Botany Herbarium. The samples were air-dried in the shade at room temperature (25C  3C) away from direct sun light for two weeks. The dry material was ground into a fine powder in the Kenyatta University

Chemistry research laboratory using a KHD Humboldt Wedag AG Electric grinding mill of Ollmann and Company KG of Germany and weighed.

3.2 Reagents

The general purpose reagent grade organic solvents like acetone, n-hexane, dichloromethane, ethyl acetate and methanol were purchased from Kobian, Nairobi,

Kenya. All solvents used for the extraction, fractionation and crystallization were distilled twice before use. The distilled solvents were then kept in amber bottles.

3.3 Cleaning procedures

All glassware were soaked in chromic acid for 24 hours then washed in hot water with liquid detergent and rinsed with tap water. A final rinse was done with distilled water 55 followed by acetone and finally kept in the oven to dry at temperature of 110C. They were then allowed to cool slowly to room temperature before use.

3.4 Sequential extraction of root and stem barks

6.43 kg of powdered material of O. thomasiana stem barks was soaked in 10 litres DCM for 3 days. Enough solvent was added until the plant material was fully covered for thorough extraction. Sequential extraction was repeated with solvents of increasing polarity starting with 8 litres ethylacetate (EtOAc) and finally 7 litres methanol (MeOH) for 3 days each with occasional swirling to ensure thorough extraction according to the

National Cancer Institute (NCI) protocol extraction of bioactive molecules (McCloud et al., 1998). Sequential extraction of 4.01 kg of powdered material of O. thomasiana root barks was also done using 8 litres DCM, 7 litres EtOAc and finally 6 litres MeOH for 3 days each.

The extracts were decanted and filtered using a buchner funnel under reduced pressure of a vacuum pump through 40 mm Whatman filter paper and the residue soaked in solvent again for 3 days. The extraction process was repeated 3 times until a clear extract was obtained. Plant residue (material) was allowed to dry before the next solvent was used.

The filtrates were combined and concentrated using rotary evaporator under reduced

o pressure at a temperature of 45 C to recover back solvents. The crude extracts were kept in the decicator to remove the excess solvent and thereafter in a deep freezer wrapped in aluminum foil to avoid decomposition of compounds. A portion of 2 g each crude extract was used for bioassay.

Residue 6.43 kg

DCM

DCM extracts Residue 70.59 g 6.359 kg

EtOAc

EtOAc3 days x 3 Residue Extract 100 g 6.259 kg

MeOH

MeOH extract Residue 740.8 g 5.519 kg

Scheme 3.1: Sequential extractions of O. thomasiana stem bark

Root bark 4.01 kg

DCM

DCM extracts Residue 31.5 g 3.979 kg

EtOAc

EtOAc3 daysextract x 3 Residue 167 g 3.812 kg

MeOH

MeOH extract Residue 631.3 g 3.181 kg

Scheme 3.2: Sequential extractions of O. thomasiana root bark

3.5 Thin layer chromatography (TLC)

Analytical TLC was performed on Silica gel 60 F254 (Macherey-Nagel) plates of 0.25 mm thickness. The spots were viewed on the chromatograms using a multi-band ultraviolet

(UV) light (254/365 nm) lamp (UV GL-58). The TLC plates were sprayed with p- anisaldehyde reagent kept in the oven at 110C until the spots appeared. Anisaldehyde, the spraying reagent (a solution made of 0.5 ml p-anisaldehyde with 10ml glacial acetic acid 85 ml chilled methanol or ethanol and 5 ml of 98% sulphuric acid) was used as a 58 developing reagent (Randerath et al., 1968). Plants extracts were tested for steroids/terpenes and flavonoids. The TLC plates were sprayed with p-anisaldehyde reagent kept in the oven at 110C until the spots appeared. Visualization of spots on a developed TLC plates was done using long and short wavelengths (λ) (365 and 254 nm respectively) on an ENF-240 C/F UV lamp (Spectronics Co., Westbury, UK).

3.6 Melting point

Uncorrected melting points were recorded using open capillary tubes using Gallenkamp melting point apparatus (Sanyo, West Sussex, and UK).

3.7 Infrared (IR) spectroscopy

IR spectra were recorded on a Shimadzu (FTIR 8400S, Fourier transform Infrared spectrophotometer (Japan make) and measured using potassium bromide (KBr) pellets in

Chemistry research laboratory of Jomo Kenyatta University of Agriculture and

Technology (JKUAT). The sample (2 mg) was mixed with KBr (100 mg) in a mortar and the mixture ground with a pestle to a fine powder until the sample was uniformly mixed with KBr. The mixture was then pressed in an evacuable die to produce a transparent disk

(pellet). The sample was then placed in a sample holder that was inserted in the spectrophotometer.

3.8 Ultraviolet (UV) spectroscopy

The UV spectra were recorded on an ENF-240 C/F UV lamp (Spectronics Co., Westbury,

UK) in the Kenyatta University research laboratory instrument room. A very small amount of the sample was dissolved in MeOH and chloroform, respectively for only those samples that were soluble in them. This spectroscopic technique is ratio recording; where 59

the ratio between reference beam and the sample beam intensities (I0/I) is fed to a pen recorder. The recorder trace is invariably absorbance (A) against wavelength (λ).

3.9 Nuclear magnetic resonance (NMR) spectroscopy

1 13 H (1D, 2D COSY) and C spectra were recorded in CDCl3 and CD3OD as solvent with

TMS (Tetramethylsilane) as internal standard on a Bruker (400 MHz and 600 MHz) spectrometer operating at 400 and 600 MHz (1H-NMR), 100 and 150 MHz (13C NMR) machine at Kwazulu Natal University, South Africa and Nagasaki University, Japan

Chemistry research laboratories, respectively. Peaks on 1H NMR were recorded as singlet

(s), doublet (d), doublet of doublet (dd), triplet (t), quartet (q), multiplet (m) and (or) broad (b) using TMS as reference. The 13C NMR multiplicity was determined by DEPT experiments, which gave chemical shift values for assignment. Chemical shifts were recorded in δ (ppm) and coupling constants, J, in hertz (Hz). A known weight of the sample was dissolved in CD3OD in a 5 mm NMR tube and mixed thoroughly. The solution was transferred into NMR tube and the spectrum recorded.

Two-dimensional (2D) NMR techniques may be used to access the correlations between nuclear in a compound. J-resolved 2D NMR experiments are useful in the measurement of 13C_1H coupling in complex spin systems since they have the ability to separate overlapping multiplets (Abraham et al., 2003). Correlated 2D experiments applied to homonuclear spin systems include COSY (Correlated spectroscopy) while NOESY

(Nuclear overhauser enhancement spectroscopy) is used in the analysis of NMR spectra of large molecules (Abraham et al., 2003). HETCOR (Heteronuclear correlation) and

HMQC (Heteronuclear multiple quantum coherence) show all the protons to which carbon atoms are attached although HMQC has higher sensitivity. On one axis the 1H 60

NMR spectrum is displayed while the other axis has the 13C NMR spectrum and thus signals indicate a direct coupling of the protons with the carbons (Silverstein et al., 2005).

HMBC (Heteronuclear Multiple Bond Correlation) is a long range correlation experiment which provides information about carbons bonded to proton which are 2-3 bonds away.

HMBC has applications in every field for identifying and characterizing the structure of compound (Vasavi et al., 2011).

3.10 Mass Spectroscopy (MS)

Mass spectrometers use the difference in mass to charge ratio (m/z) of ionized atoms or molecules to separate them. Mass spectroscopy allows quantisation of atoms or molecules and provides structural information by the identification of distinctive fragmentation patterns (Uggerud et al., 2003). The analysis was carried out at Chemistry research laboratory of Jomo Kenyatta University of Agriculture and Technology (JKUAT) using

Shimadzu Gas Chromatography Mass Spectrometry (GCMS). A little amount of a compound, typically one micromole/ microgram or less was injected in the spectrometer and evaporated with vapour leaking into the ionization chamber where a pressure is maintained of about 10-7 mb.

3.11 Antibacterial tests

3.11.1 Bacterial strains tests

Plants extracts were screened in Mycology laboratory centre for microbiology research at

Kenya Medical Research Institute (KEMRI) against two standard strains of bacteria obtained from the American Type Culture Collection (ATCC) and two local clinical isolates from patients. The strains used were obtained from Kenya National Public Health

Laboratories (KNPHL), (KEMRI) and Plant and Microbial laboratory of Kenyatta 61

University. Strains that were used included: two Gram-positive bacteria, Staphylococcus aureus, (ATCC 20572), a reference strain, and Bacillus subtilis (Type K [11]), clinical isolate. Two Gram-negative bacteria, Pseudomonas aeruginosa (ATCC 27853), a reference strain, Salmonella typhi (Type K [I]) clinical isolate was also used. These microorganisms were grown in diagnostic sensitivity test agar (DST) and Muller–Hinton broth (MHB-Merck Germany).

3.11.2 Preparation of media

DST agar was prepared by weighing the quantities recommended by the manufacturer and dissolving in recommended quantities of distilled water. MHB was prepared by using

28 g of the media, dissolved in 1 litre of distilled water and boiled. The media were sterised separately by autoclaving at 15 atmospheres pressures and 121C for 15 minutes, before used.

3.11.3 Screening procedure

The disc diffusion method was used for initial activity of the tests on the plant extracts at concentrations of 450 g/disc. Concentrations of sample solutions were prepared by dissolving 1g of extract in 1ml of the solvent that had been used in their extractions followed by appropriate dilutions to the required concentrations. This was then used to prepare the discs by soaking method. Each disc was inoculated with 0.1 ml of bacterial and yeast culture directly from the 24 h broth culture diluted to match 0.5 and 1.0

MacFarlands standard, respectively (108 Colony Forming Units (CFU)/ ml) and fungi diluted to match 1.0 MacFarland standard (108 spores/ml). The discs loaded with the extracts were then placed onto the seeded plates.

The bacterial and yeast cultures were incubated at 37 °C for 24 and 48 hours, respectively, while fungi were incubated at 25 °C for 5 days. After the incubation period the zones of inhibition were measured and recorded in mm as described by Elgayyar et al.

(2000). Negative control plates had discs with sterile distilled water and methanol.

Sterility of media and growth of the organism was controlled by use of broth only in negative control tubes and broth plus microorganism in question in positive control tubes.

Tetracycline was used as the standard for positive control. All the controls were subjected to the same conditions as the tests.

3.11.4 Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC)

Minimum inhibitory concentrations (MICs) are defined as the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation, and minimum bactericidal concentrations (MBCs) as the lowest concentration of antimicrobial that will prevent the growth of an organism after subculture on to antibiotic free media (Andrews, 2001).

The active extracts from the antimicrobial screening were tested for minimum inhibitory concentrations (MICs) and minimum bactericidal (MBCs). The MICs were determined using a two-fold serial dilution method in a peptone water solution for bacterial of the active extracts to give a final extract with concentration of between 1.74 and 8000 μg/ml.

Each tube was then inoculated with 0.1 ml of standardized bacterial suspension (1×108

CFU/ml). The cultures were incubated at 37°C for 24 hrs for bacteria. The first tube showing no growth was taken as the MICs. MBCs were determined by sub-culturing 0.1 ml of all the tubes showing no growth on Nutrient Agar (NA) for bacteria. The first plate 63 showing no growth was considered as the MBC after 48 hrs at 37°C (Michael et al.,

2003).

3.11.5 Disc diffusion and MIC ratings of the extracts

The negative controls of the disc diffusion testing was done by use of MeOH that showed no inhibition, while positive control was done by use of standard antibiotic discs (Oxoid).

The average zone of inhibition was calculated for the 3 replicates. A clearing zone of 9 mm for Gram-positive and gram-negative bacteria or greater was used as the criterion for designating significant antibacterial (Faizi et al., 2003). The extracts that displayed MIC lower than 100 μg/ml, were considered as having very high antimicrobial activity (AA); from 100-500 μg/ml, high AA; 500-1000 μg/ml, moderate ; 1000-4000 μg/ml, low AA and anything above this, the extracts were considered inactive for bacteria.

Plate 3.1: Photograph of petri dish showing disk diffusion method

Plate 3.2: Test of bacterium 64

3.12 Isolation of compounds from Ochna thomasiana

A combination of chromatographic techniques: column chromatography (CC), thin layer chromatography (TLC), sephadex and prep-TLC were used for purification of dichloromethane (DCM) and ethyl acetate (EtOAc) root bark extracts. The DCM and

EtOAc root bark extracts were separately fractioned by CC of 4.75 cm and 5.75 cm in diameter, respectively with each having a height of 70 cm. Further purification was achieved by use of smaller CC of 2.75 cm in diameter and a height of 40 cm on silica gel

60 mesh 0.6 nm (230-400) Merck-Germany and eluted with varying concentrations of hexane (Hex), DCM, EtOAc and methanol (MeOH) mixtures until 100 % of each solvent was achieved.

Packing of both types of columns was done using slurry method with silica gel suspended in the least polar solvent in the solvent system. The mass of the silica gel packed was in the ratio of 30:1 to the mass of the extract to be loaded. The sample (extract) was dissolved in the minimum possible solvent that dissolved it, mixed with an equal amount of the silica gel used in the CC, ground thoroughly into fine powder form and made more homogenous and dry by evaporating in a vacuum evaporator to remove the excess solvent. This powder was then loaded at the top of the column, covered by a small amount of dry silica gel and then covered with a piece of cotton wool to minimize the disturbance of the sample when eluting solvent is added. The eluting solvent or solvent mixture was allowed to trickle down drop wise from a separating funnel to the column to avoid pouring of the solvents directly into the column.

Analytical TLC 60 F254 plates were used throughout the purification process. These were mainly for establishment of optimum solvent systems for the separations and for 65 purification of isolated compounds. Spots on the chromatograms were detected under UV light at λ 254 and 365 nm for UV active compounds and visualized upon development by separately spraying p-anisaldehyde and heating for about 10 min at 110 °C in an oven.

Fractions that had same Rf were combined and concentrated together to give pure compounds or semi-pure compounds for further purification. Centrifuging, recrystalization, sephadex column and PTLC were used particularly for further purifications. The sephadex (LH-20) columns were run using 1:1 (DCM:MeOH). The specific chromatographic techniques and solvent systems used are outlined in Schemes

3.3 and 3.4.

DCM root extract (9.65 g) of the O. thomasiana subjected to fractionation by column chromatography on silica gel with a Hex:DCM-DCM:MeOH gradient (100:0-0:100) to yield 209 x 25 ml fractions. Three fractions were obtained based on Rf portrayed on the

TLC profiles. Fractions 1-167 were pooled together because they gave a purple colour on spraying with p-anisaldehyde followed by heating in the oven for 10 minutes at 110 oC.

These fractions were produced at different solvent mixtures of hexane and DCM ranging from 4:1 (Hex:DCM) to 100% DCM. Fractions 168-181 were pooled together and was obtained at 19:1 (DCM:MeOH). This fraction had purple and red brown pigmentation after spraying with spraying reagent.

Fractions 182-209 were pooled together to form the third fraction which had yellow and red brown pigmentation after spraying the TLC plate with p-anisaldehyde and heating in the oven for 5-10 minutes. This fraction was obtained at 9:1 (DCM:MeOH). The first fraction (5-167) was washed with MeOH to remove brown colouration obtained from the column followed by hexane to make the crystals clean. Three other fractions were 66 obtained but they all had two spots each based on the TLC profile. The white crystals obtained were dissolved in chloroform and recrystallised. Decanting was done leaving behind the white sharp crystals of compound of MJ/RD/OT01 (75) of 3 g. PTLC was done on one of the resulting three fractions to isolate 14.5 mg of MJ/RD/OT010 (mixture of 23 and 74) at 100% DCM. The other two fractions were separated further using

Sephadex (L-20) column 1:1 (DCM:MeOH) and obtained fractions 30 and 40, respectively. A PTLC was done on these combined fractions from the Sephadex column using 10% MeOH/DCM and 11.7 mg of cream white compound MJ/RD/OT02 (17) was obtained.

Ethyl acetate extract (18.6 g) of the O. thomasiana root bark was subjected to normal CC on silica gel with a DCM:EtOAc:MeOH gradient (100:0-0:100) to yield 43 x 200 ml fractions. The fractions 1-5 obtained at 43:7 (DCM:EtOAc), 6-9 obtained at 5:3

(DCM:EtOAc), 10 obtained at 11:9 (DCM:EtOAc) , 11-15 obtained at 23:27

(DCM:EtOAc) and 16-43 were ignored because of tailing produced in the TLC profiles.

Again the pigmentation produced after spraying with the p-anisaldehyde was dark brown.

The fraction 11-15 of 9.36 g was subjected to a smaller CC to yield four other smaller fractions. The fractions 1-4 obtained at 19:1 (DCM:EtOAc), fraction 5-11 obtained at 7:3

(DCM:EtOAc), fraction 12-15 obtained at 5:3 (DCM:EtOAc), was separated further using

Sephadex (L-20) column 1:1 (DCM:MeOH) to obtain 21 fractions which were of the same Rf. A PTLC was developed for the combined fraction from the Sephadex using 9:1

(DCM:MeOH) and 15.3 mg of compound 18 (MJ/RE/OT06) was obtained. The same procedure above was used for fractions 6-9 to obtain 13.2 mg of compound 20

(MJ/RE/0T05) and 14.5 mg compound 17 (MJ/RE/0T02). Compound 17 was isolated from both DCM and EtOAc root bark extracts. 67

DCM extract 9.65 g

CC solvent system as below

1:1 H:D 2:1 D:H 9:1 D:M

Fraction 5-149 Fraction 149-167 Fraction 168-182 5g 1.376g 310.73mg

Washing with Sephadex 1:1 Sephadex 1:1 MeOH/hexane D:M D:M

Recrystalisation Fraction 8-20 Fraction 22-40 Fraction 20-25 with chloroform 55mg 50mg 45mg

Compound PTLC 100% PTLC 100% PTLC 10:1 MJ/OT01 200mg DCM 30 mg DCM 30 mg D:M 30 mg

Compound Compound Compound MJ/RD/OT01 MJ/RD/OT010 MJ/RD/OT02 18mg 14.5mg 11.7mg

Key: H -Hexane D -Dichloromethane (DCM) M -Methanol

Scheme 3.3: Chromatographic separation of O. thomasiana DCM root bark extract. 68

EtOAc extract 186g

CC solvent system as below

5:3 D: EtOAc 11:9 D: EtOAc 23:27 D: EtOAc

Fraction 6-9 Fraction 10 Fraction 11-15 3.198g 1.729g 9.364g

Sephadex 1:1 Sephadex 1:1 CC D: M D: M

Fraction 12-15 1.814g Fraction 5-9 Fraction 2-5 F 6-15 F 6-15 50mg 50mg 45mg 45mg Sephadex 1:1 D:M PTLC 9:1 PTLC 24:1 PTLC 19:1 D:M D:M PTLC 19:1 D:M 30 mg D:M 30 mg PTLC 9:1 D:M 30 mg Compound Compound MJ/RD/OT0 MJ/RE/OT0 2 5 Compound Compound 13.2mg 14.5mg Compound MJ/OT0 MJ/OT04 9 MJ/RE/OT0 4.6mg 11.6mg 6 15.3mg

Key: D -DCM M -MeOH

Scheme 3.4: Chromatographic separation of O. thomasiana EtOAc root bark extract.

3.13 Physical and spectroscopic data of isolated compounds

3.13.1 Compound 18; Lophirone A

Cream white crystals (MeOH), Mp 211-213°C; UV (MeOH) max 237, 335 nm; IR (KBr)

-1 1 max 3751, 3368, 2728, 1626, 1510, 1454, 1364, 1238, 831 cm ; H NMR (600 MHz,

MeOD): δ 8.25 (1H, s, β1),, 6.68 (1H, d, J= 2.2, B1H3), 6.83 (1H, dd, J=2.2, 8.9, B1H5),

7.87 (1H, d, J=8.9Hz, B1H6), 5.97 (1H, d, J= 12.16, H-α1), 4.67 (1H, d, J=12.16, H-β1),

7.16 (4H, d, J=8.4, H-C2, A1 C6, A2), 6.59 (4H, d, J=8.4, H-C3, A1C5, A2), 6.12 (1H, d,

13 J=2.3, B2H3), 6.33 (1H, dd, J=2.3, 9.0, B2H5), 8.14 (1H, d, J=9.0, B2H6). C NMR (150

MHz, MeOD): δ 157.4 (β1), 122.6 (α1), 177.1 (s, C1), 117.1 (s, B1C1), 159.4 (s, B1C6),

103.5 (d, B2C3), 164.7 (B1C4), 116.5 (d, A1C5), 128.2 (d, A1C2), 44.7 (α2), 54.3 (d, β1),

204.8 (s, C2), 135.9 (s, A1C1), 129.8 (d, A2C2), 116.0 (d, A2C3), 156.7 (s, A1C4), 134.9 (s,

A2C1), 130.5 (d, A2C6), 116.1 (d, A2C5), 159.4 (B1C6), 114.3 (s, B2C1), 166.9 (s, B2C2),

103.5 (d, B2C3), 166.8 (s, B2C4), 109.2 (d, B2C5), 134.4 (d, A2C1) (Tables 4.4 and 4.5).

3.13.2 Compound 20; Afzelone D dimethylether

Yellow crystals (MeOH), Mp 172-175°C; Formula: C33H28O8, IR (KBr) max 3855, 3443,

2933, 1611, 1510, 1452, 1365, 1256, 1026, 831 cm-1; 1H NMR (400 MHz, MeOD): δ

8.22 (1H, s, β1), 6.69 (1H, d, J= 2.2, B1H3), 6.83 (1H, dd, J=2.2, 8.9, B1H5), 7.85 (1H, d,

J=8.9 Hz, B1H6), 6.02 (1H, d, J= 12.2, H-α1), 4.70 (1H, d, J=12.2, H-β1), 7.10 (4H, d,

J=8.4, H-C2, A1C6, A2), 6.56 (4H, d, J=8.4, H-C3, A1C5, A2), 6.28 (1H, d, J=2.3, B2H3),

6.44 (1H, dd, J=2.3, 9.0, B2H5), 8.14 (1H, d, J=9.0, B2H6), 3.76 (3H, A1C4, OMe), 3.75

13 (3H, A2C4, OMe), 3.59 (3H, OMe B2C4). C NMR (100 MHz, MeOD): δ 157.4 (β1),

122.6 (α1), 177.1 (s, C1), 117.1 (s, B1C-1), 159.4 (s, B1C6), 103.5 (d, B2C3), 164.7 (B1C4),

116.5 (d, A1C5), 128.2 (d, A1C2), 44.7 (α2), 54.3 (d, Cβ1), 204.8 (s, C2), 135.9 (s, A1C1),

129.8 (d, A2C2), 116.0 (d, A2C3), 156.7 (s, A1C4), 134.9 (s, A2C1), 130.5 (d, A2C6), 116.1 70

(d, A2C5), 159.4 (B1C6), 114.3 (s, B2C1), 166.9 (s, B2C2), 103.5 (d, B2C3), 166.8 (s, B2C4),

109.2 (d, B2C5), 134.4 (d, A2C1), 56.0 (q, A2C4 OMe), 56.1 (q, A1C4 OMe), 55.5 (q, B2C4

OMe) (Table 4.6).

3.13.3 Compound 17; Calodenone

Cream white crystals (MeOH), Mp 172-175°C; Formula: C31H24O8, IR (KBr) max 3753,

3423, 2747, 1626, 1510, 1454, 1369,1253, 1024, 749, 829 cm-1; 1H NMR (400 MHz,

MeOD): δ 8.22 (1H, s, β1), 6.83(1H, d, J= 2.2, B1H3), 6.72 (1H, dd, J=2.2, 8.9, B1H5),

7.87 (1H, d, J=8.9Hz, B1H2), 6.03 (1H, d, J= 12.2, H-α2), 4.67 (1H, d, J=12.2, H-β2), 7.15

(4H, d, J=8.4, H-C2, A1C6, A2), 6.55 (4H, d, J=8.4, H-C3, A1C5, A2), 6.29 (1H, d, J=2.3,

B2H3), 6.60 (1H, dd, J=2.3, 9.0, A1H5), 8.21 (1H, d, J=9.0, B2H6), 3.76 (3H, OMe A2C4).

13 C NMR (100 MHz, MeOD): δ 157.4 (β1), 122.6 (α1), 177.1 (s, C1), 117.1 (s, B1C1),

159.4 (s, B1C6), 103.5 (d, B2C3), 164.7 (B1C4), 116.5 (d, A1C5), 128.2 (d, A1C2), 44.7 (α2),

54.3 (d, Cβ1), 204.8 (s, C2), 135.9 (s, A1C1), 129.8 (d, A2C2), 116.0 (d, A2C3), 156.7 (s,

A1C4), 134.9 (s, A2C1), 130.5 (d, A2C6), 116.1 (d, A2C5), 159.4 (B1C6), 114.3 (s, B2C1),

166.9 (s, B2C2), 103.5 (d, B2C3), 166.8 (s, B2C4), 109.2 (d, B2C5), 134.4 (d, A2C1)., 56.1

(q, A1C4 OMe) (Table 4.7).

3.13.4 Compound 23; β-sitosterol

White amorphous solid (CHCl3) Mp 130-134°C; Mol. Wts: 415 and Mol. Formula: C29H50O

-1 -1 - (CHCl3). IR (KBr) max: 3400 cm (OH- stretching), 2899cm (CH-stretching), 1679 cm

1, 1460 cm-1 C=C stretching, 1041 cm-1 C-O-C stretching. Mass spectra: m/z: 415 (M+,

1 C20H50O), 399 (M+-CH3), 376 (M+ H2O), 381 (M+ CH, OH), 320, 303, 273, 255. H

NMR: (CDCl3, 400 MHz) 0.68 ppm (3H, s, C18H), 0.85 (H26), 0.80 (H27), 0.81 (H29), 1.01

(H19), 1.02 (3H, s, C21H), 3.53 (1H, m, C3H), 5.36 (1H, t, J=6 Hz, C6H). Other peaks are 71

13 13 observed at δ 0.80‐ δ 2.4. C NMR (CDCl3, 100 MHz) of 23: C NMR has given signal at 140.8 (C5), 121.7(C6), 33.7 (C22), 26.1 (C23), 56.9 (C14), 71.8 (C3), 42.3 (C14),

56.1(C17), 50.2 (C9), 36.2 (C20), 39.8 (C12), 42.3 (C13), 42.3 (C4), 37.2 (C1), 30.5 (C10),

31.9 (C8), 36.2 (C20), 33.7 (C22), 31.7 (C7), 31.9 (C8), 29.2 (C25), 28.2 (C16), 31.7 (C2),

24.3 (C15), 23.1 (C28), 21.1 (C11), 21.2, 19.0 (C27), 19.4 (C19), 18.8 (C21), 11.9, 12.0 (C18),

12.0, 11.7 (C29) (Table 4.8).

3.13.5 Compound 74; Stigmasterol

White amorphous solid (CHCl3) Mp 130-134°C; Mol. Wts: 413 and Mol. Formula: C29H48O

-1 -1 - (CHCl3). IR (KBr) max: 3400 cm (OH- stretching), 2899cm (CH-stretching), 1679 cm

1, 1460 cm-1 C=C stretching, 1041 cm-1 C-O-C stretching. Mass spectra: m/z: 414 (M+,

1 C20H50O), 399 (M+-CH3), 376 (M+ H2O), 381 (M+ CH, OH), 320, 303, 273, 255. H

NMR: (CDCl3, 400 MHz) 0.68 ppm (3H, s, C18H), 0.85 (H26), 0.80 (H27), 0.81 (H29), 1.01

(H19), 1.02 (3H, s, C21H), 3.53 (1H, m, C3H), 5.36 (1H, t, J=6 Hz, C6H), 5.02 (1H, m,

13 H23), 5.15 (1H, m, H22). Other peaks are observed at δ 0.80‐ δ 2.4. C NMR (CDCl3, 100

13 MHz) of 74: C NMR has given signal at 140.8 (C5), 121.7(C6), 129.3(C23), 56.8(C14)

71.8 (C3), 42.3 (C14), 56.1(C17), 50.2 (C9), 40.5 (C20), 39.7 (C12), 42.3 (C13), 42.3 (C4),

37.2 (C1), 30.5 (C10), 31.9 (C8), 40.5 (C20), 138.3(C22), 31.7 (C7), 31.9 (C8), 31.9 (C25),

28.7 (C16), 31.7 (C2), 24.3 (C15), 25.4 (C28), 21.1 (C11), 21.2 (C27), 19.4 (C19), 21.1 (C21),

11.9 (C18), 12.0 (C29) (Table 4.8).

3.13.6 Compound 75; 3β-acetyl-24-ethylfriedelane

o White amorphous solid (CHCl3), Mp 266–267 C; IR ((KBr) max: 3619, 3471, 2725,

2869, 1448, 1384, 1360, 1172, 1089, 1020, 1000, 979, and 720; 1H-NMR (600 MHz;

1 CDCl3; ppm) δ 3.70 (l s; H-3; 1H) and 0.83-0.97 (superposed signals) H NMR (CDCl3, 72

600 MHz), 0.82 (3H, s, CH3-25), 0.86 (3H, s, CH3-24), 0.93 (3H, s, CH3-28), 0.97 (3H, s,

CH3-32), 1.00 (3H, s, CH3-23), 1.03 (6H, s, CH3-27, CH3-28), 1.23 (3H, s, CH3-30), 2.00

13 (3H, s, CH3), 4.10, (1H, d,14.0, H3). C-NMR (150 MHz; CDCl3; ppm) δ 72.8 (C3), 61.3

(C10), 53.2 (C8), 37.1 (C4), 42.8 (C18), 41.7 (C6), 39.7 (C14), 39.3 (C22), 14.2 (31), 38.4

(C13), 60.4 (C5), 37.1 (C32), 37.8(C9), 35.2 (C2), 35.6 (C16), 35.3 (C11), 35.2 (C19), 35.0

(C30), 32.8 (C21), 32.3 (C15), 32.1 (C28), 31.8 (C29), 30.6 (C12), 30.0 (C17), 28.2 (C20), 20.1

(C26), 18.6 (C27), 18.2 (C25), 17.5 (C7), 16.4 (C24), 15.8 (C1), and 11.6 (C23) (Table 4.9).

CHAPTER FOUR

RESULTS AND DISCUSSION

4.1 Plant material yield of extracts

The finely ground O. thomasiana stem and root barks (6.43 and 4.01 kg, respectively) were extracted sequentially using DCM, EtOAc and finally MeOH. The excess solvents were then evaporated using a vacuum evaporator at reduced pressure and a temperature of

45°C to obtain the extracts. The MeOH extracts of root and stem recorded the highest yield. The yields were recorded and tabulated as shown in table 4.1.

Table 4.1: Plant material yield of O. thomasiana extracts

Plant part Extract Mass of extract(g) % yield DCM 71 1.10 Stem bark EtOAc 100 165 MeOH 741 19.7 DCM 46 1.15 Root bark EtOAc 167 6.65 MeOH 631 270

The percentage yields indicated that the root bark of O. thomasiana was richer in metabolites that were soluble in the solvents used in the extraction more so MeOH solvent compared DCM and EtOAc solvents, respectively. It also showed that the percentage yield increased with increased polarity, as in the case of the root bark extract which increased from 1.15 % yield with DCM as solvent to 27.0 % with MeOH. This implied that both the stem and root bark of MeOH extracts contain more polar compounds as compared to the non-polar compounds. These extracts were subjected to various antibacterial tests before embarking on fractionation of the crude extracts. Total 74 yield of the extract from the stem bark was 22.45 % while that of the root bark was 34.6

4.2 Antibacterial disc diffusion screening test for the O. thomasiana extracts

4.2.1 The DCM extracts

The stem DCM extracts of the plant did not show any activity against S. typhi, P. aeruginosa and S. aureus. The root DCM extracts of the plant showed slight activity against B. subtilis (8 mm), but no activity on S. typhi, P. aeruginosa and S. aureus as shown in table 4.2.

4.2.2 The ethyl acetate extracts

Both the root and the stem bark extracts of O. thomasiana showed moderate activity against S. aureus (9 mm) and B. subtilis (11 mm) as observed in table 4.2. There was no antibacterial activity observed for the stem and the root ethyl acetate extracts of the plant against the Gram-negative bacteria S. typhi and P. aeruginosa.

4.2.3 The methanol extracts

There was high activity against B. subtilis (20 mm) and S. aureus (15 mm) for the root bark extracts of O. thomasiana (Table 4.2). The zones of inhibition for the stem bark extracts against the same bacteria were 14 mm and 10 mm. The root and stem methanol extracts for plant were highly active against B. subtilis. Methanol extracts showed broad spectra of activity against the tested organisms, except for S. typhi and P. aeruginosa. The

MeOH root extract of the plant generally had moderate activity at the concentrations tested with mean zone of inhibition diameter greater than or equal to 15 mm.

Table 4.2: The inhibition zones (in mm) of crude extracts of O. thomasiana

Extract S. aureus B. subtilis P. aeruginosa S. typhi EtOAc stem 9 11 6 6 MeOH stem 10 14 6 6 EtOAc root 9 11 6 6 MeOH root 15 20 6 6 Control experiment (+ve) 20 (Tet) 18 (Tet) 19 (Tet) 15 (Tet)

Key: Staphylococcus aureus ATCC 25724, Bacillus subtilis ATCC 25726, Pseudomonas aeruginosa ATCC 25723, Salmonella typhi (clinical isolate), Tet = Tetracycline.

The selective effects of the extracts on gram-positive (S. aureus and B. subtilis) suggest that the extracts may serve as source of compounds that can be used to combat infection caused by these organisms (Tania et al., 2000).

4.2.4 Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of O. thomasiana

The MIC of the extracts that showed antimicrobial activity in the screening stage was carried out. The MIC and MBC results are also given in table 4.3.

Table 4.3: The MIC and MBC (µg/ml) of crude extracts of O. thomasiana

Extract µg/ml S. aureus B. subtilis MIC 1000 1000 EtOAc stem MBC 8000 8000 MIC 1000 1000 MeOH stem MBC 8000 8000 MIC 1000 1000 EtOAc root MBC 8000 8000 MIC 500 500 MeOH root MBC 8000 8000

The lowest MIC recorded was that of MeOH root bark of O. thomasiana of 500 μg/ml against both B. subtilis and S. aureus. The highest MIC of 1000 μg/ml was observed with both the stem and root extracts of EtOAc and MeOH stem respectively against B. subtilis.

The activity against all the Gram-positive bacteria tested was high in the stem and root extracts as all of them had MICs of 500-1000 μg/ml. The MBCs for all extracts were observed to be about 8000 μg/ml; hence a confirmation that O. thomasiana had appreciable bactericidal activity against gram-positive tested microorganisms. The EtOAc and MeOH extract showed more activity against the tested organisms as indicated by the susceptibility, MIC and MBC tests suggesting that more of the bio-active chemical constituents were extracted by methanol. These could probably be polar or moderately- polar compounds such as flavonoids. All the EtOAc and MeOH extracts of O. thomasiana had no activity against all the Gram-negative bacteria tested.

4.3 Structure elucidation The structure elucidation of compounds 17 to 75 was done by interpretive NMR, IR spectroscopic and mass spectrometric spectral data obtained for the purified compounds in comparison with literature values.

4.3.1 Compound 18

Compound 18 was isolated as a cream white amorphous solid (15.3 mg) from a MeOH soluble fraction of EtOAc root bark extract (MeOH/DCM; 1:9) and with a mp of 211-

213°C. It was fluorescing under UV-light (254 nm) and produced a characteristic red coloured spot when sprayed with p-anisaldehyde followed by heating at 110oC in the oven for 5-10 minutes. The UV in MeOH showed absorptions 237 and 335 nm (Appendix

1i) suggestive of an isoflavonoid nucleus (Enas et al., 2012). The IR spectrum (KBr), comprised of absorption bands at 3252 cm-1 (C-H aromatic stretching), 831-1 (out of plane 77 bend), 1625 cm-1 (aromatic C=C). The IR spectrum exhibited a broad O-H absorption band at 3368 cm-1 and two carbonyl bands at 1625 cm-1.

The 1H, 1H COSY (Appendix 1e) and the 1H NMR spectra (Appendix 1a), showed the presence of two p-substituted aromatic rings and two trisubstituted phenyl ring systems.

A singlet at δ 8.25 is characteristic of H-β1 of a 3-substituted benzopyran- 4-one moiety

(such as an isoflavone) and the peaks at δ 6.01 and 4.67 revealed the presence of an AX system. The large coupling (J=12.2 Hz) observed between the latter two protons suggested that they are in an antiperiplanar as shown in table 4.4 (Mabry et al., 1970;

Pegnyemb et al., 2003).

R  R H

H 

3 A 3 1 6 6 A2 HO OH

Figure 4.1: HMBC correlations in partial structure 18

In the 13C NMR spectrum (Appendix 1b) there are 22 peaks corresponding to 26 carbon atoms with chemical shifts between δ 100 to 170 implying the presence of four aromatic ring systems in addition to the pyran-4-one ring (Markham et al., 1978). The methine carbon atoms at δ 54.3 and 44.7 were correlated to protons at δ 4.67 and 6.01, respectively, in the HSQC spectrum (Appendix 1f).The two carbonyl carbon atoms 78 resonated at δ 204.6 and 177.1 and the downfield resonance of the later carbonyl carbon atom suggested that it is part of a 4-pyrone system.

1 Table 4.4: H NMR (600 MHz, CD3OD) and COSY for compound 18

Position δObs Multiplicity JObs(Hz) COSY δ Lit.

β1 8.25 s 8.27

B13 6.85 dd 8.9, 2.2 6.72

B15 6.72 d 2.2 6.96

B16 7.89 d 8.9 B2H3 7.74

α2 6.01 d 12.2 α2 6.01

B23 6.14 d 2.3 6.19

B25 6.34 dd 9.0, 2.2 6.44

B26 8.15 d 8.9 8.34

β2 4.67 d 12.2 4.79

A12 7.15 dd 19.5, 8.4 A1H3 7.26

A13 6.55 d 3.1 6.65

A15 6.60 d 8.5 A1H6 6.65

A16 7.15 dd 19.5, 8.4 7.26

A22 7.15 dd 19.5, 8.4 A2H3 7.26

A23 6.60 dd 9.0, 2.3 6.61

A25 6.60 dd 9.0, 2.3 6.61

A26 7.15 dd 19.5, 8.4 7.26

Literature data (400 MHz CD3OD) derived from Ghogomu et al. (1987)

The position of the two p-substituted aromatic ring systems (AA‟XX') was unambiguously assigned by the long-range heteronuclear correlation [HMBC spectrum

(Appendix 1g)] between H-β2 at δ 4.67 and A2C1 and A1C1 at δ 134.9 and 129.7 of ring

A1 and A2, respectively as shown in table 4.6, indicative of the connection of both rings to

C-β2, therefore, validating partial structure compound 18 (Figure 4.1). The downfield resonance of B1H6 at δ 7.89 shows its close proximity to the carbonyl and therefore, the second trisubstituted aromatic ring (ring B1) is not substituted at C5 (Abraham et al.,

2003). 79

The presence of a singlet at around δ 8.25 (H-β1) evidenced that C2 of the benzopyran moiety is not substituted. The long-range correlation between H-α2 (at δ 6.01) and C-α1

(at δ 122.6) confirms that C-α2 is linked to the benzopyran moiety through C-α1. The position of the first trisubstituted aromatic system was confirmed by the long-range correlations between B1H6 (at δ 7.89) and the carbonyl at δ 177.1 suggesting that this particular ring B1 is part of a benzopyran moiety and therefore forms ring A of a flavonoid skeleton. Long-range correlations between B2H6 (δ 8.15) and a carbonyl at δ

204.7 (C2) suggested the connectivity of the second trisubstituted aromatic ring to this carbonyl, which in turn was also correlated to H-α2 as shown in partial structure and table

4.5.

HO O H  O OH C B1

  C  C2 1 H B2 H O H OH R

Figure 4.2: HMBC correlations in the partial structure compound 18

The assignment of compound 18 was with an agreement with molecular formula of

C30H22O8. The compound was previously isolated from the stem bark of Lophira lanceolata (Ochnaceae) and the NMR data of compound 18 was in agreement with those reported for the compound (Ghogomu et al., 1987). The compound was confirmed to be a biflavonoid; Lophirone A and with the same structure (Figure 4.3) as compound 18 based 80 on the analysis of data above, and it is the first time the compound was isolated from this plant.

13 Table 4.5: C NMR (150 MHz, CD3OD), DEPT, HSQC and HMBC of 18

Position δObs DEPT HSQC(δH) HMBC δ Lit.

β1 157.4 CH 8.25 156.4

α1 122.6 C α2H 122.1

C1 177.1 C α2H 175.4

B11 117.0 C 117.2

B12 159.2 C B1H2 158.5

B13 116.0 CH 6.85 115.9

B14 164.7 C B1H2 163.4

B15 103.3 CH 6.72 103.2

B16 128.2 CH 7.89 128.2

α2 44.7 CH 6.01 β2H 43.9

C2 204.6 C α2H 204.5

B21 114.3 C 114.1

B22 166.9 C 166.8

B23 103.5 CH 6.14 103.5

B24 166.8 C 166.1

B25 109.2 CH 6.34 109.0

B26 134.4 CH 8.15 134.4

β2 54.3 CH 4.67 α2H 53.4

A21 134.9 C 134.6

A22 130.5 CH 7.15 130.0

A23 116.1 CH 6.60 115.8

A24 156.7 C 156.4

A25 116.1 CH 6.60 115.8

A26 130.5 CH 7.15 130.0

A11 129.7 C A1H3, A1H5 129.6

A12 129.8 CH 7.15 A1H6 129.4

A13 116.1 CH 6.55 115.9

A14 156.7 C A1H6 156.5

A15 116.5 CH 6.60 115.9

A16 129.8 CH 7.15 129.4

Literature data derived from Ghogomu et al. (1987); Pegnyemb et al. (2003); Anuradha et al. (2006)

     C    C C  2 1     

    

Figure 4.3 Structure for Lophirone A (18)

4.3.2 Compound 20

Compound 20 was obtained as an amorphous cream white powder from MeOH soluble fraction of EtOAc root bark. It gave a red colour when sprayed with p-anisaldehyde and heated to a temperature of 110 oC for 5 to 10 minutes and fluoresced under UV-light (254 nm) suggesting the structure of a biflavonoid. Its mp was 172-176 oC.

-1 -1 The IR spectrum (KBr), Vmax cm comprised of absorption bands at 3026 cm (C-H aromatic ring str), 957 cm-1 (out of plane bend), 1510 cm-1 and 1452 cm-1 (aromatic C=C-

C str). Absence of absorption band at 1620 cm-1 to 1670 cm-1 suggests compound as an isoflavonoid or a chalconoid (Mabry et al., 1970; Peng et al., 2006). The IR spectrum exhibited a broad O-H absorption band at 3256 cm-1 and aromatic ring stretch at 1611 cm-

1 -1 -1 . It also exhibited an OCH3 group and vicinal C-H bands at 2841 cm and 2715 cm , respectively. The IR spectrum of 18 and 20 are similar in almost all aspects.

The 1H-NMR spectrum exhibited signals typical of a 1,2,4-trisubstituted benzene ring indicated by a set of meta-coupled proton at δ 6.82 (dd, J=2.2 Hz, B1H3), δ 6.66 (dd, 82

J=2.2 Hz, 8.58 Hz, B1H5) and an ortho-coupled proton downfield at δ 7.86, (d, J=8.94), representing B1H6 (Mabry et al., 1970). The proton resonance which appeared as a singlet further downfield at δ 8.22 presumably due to the influence of a keto group suggests a H-

β1 proton as in an isoflavone system (Mabry et al., 1970; Pegnyemb et al., 2003). Another set of 1,2,4-trisubstituted benzene ring system is represented by 1H NMR resonances at δ

6.27 (d, J=2.5 Hz, B2H3), δ 6.43 (dd, J=2.5 Hz, 9.1 Hz, B2H5) and an ortho-coupled proton downfield at δ 7.74 (d, J=9.1, B2H6) (Pegnyemb et al., 2003a; Anuradha et al.,

2006). This ring sub structure is in close proximity with a carbonyl group similar to

Lophirone A (18). The 1H NMR spectrum also exhibited two closely para overlapping

1,4-disubstituted benzene rings. The protons integrated for 8 hydrogens and these 2

(AA‟XX') systems comprise of ortho-coupled protons at δ 7.21, (d, J=9.1, 2H) assignable to A1H2 and A1H6 and δ 6.60 (d, J=2.4, 2H) assignable to A1H3 and A1H5 as well as δ

7.15 (d, J=8.4, 2H) assignable to A2H2 and A2H6 and δ 6.60 (d, J=8.4, 2H) assignable to

A2H3 and A2H5 of ring A2, respectively.

The 1H NMR spectrum of compound 20 was very similar to that of Lophirone A (18) and

Afzelone D (19) (Pegnyemb et al., 2003a), in that it showed the same signal for protons on all the rings present as well as the ring system of two aliphatic protons. Differences noted between compound 18 (Lophirone A) and 20 included modification of the chemical shifts of the ring A1, A2 and B2 protons and the presence of two OMe signals at δ 3.76 (a long singlet peak, double integrated, 6H) and one OMe signal at δ 3.59 (3H, s) in compound 20 (Table 4.6).

1 13 Table 4.6: H (400MHz) and C NMR data for 20 (100 MHz, CD3OD)

δ Position δObs H (ppm), m (J (Hz)) δ Lit.

β1 157.3 8.22 (1H, s) 156.4

α1 122.4 122.1

C1 177.0 175.4

B11 117.0 117.2

B12 128.2 128.2

B13 116.0 6.82 (1H, dd, J= 2.2 Hz) 115.9

B14 164.7 163.4

B15 103.3 6.66 (1H, dd, J= 2.2, 8.6 Hz) 103.2

B16 159.4 7.86 (1H, d, J= 8.7 Hz) 158.5

α2 44.6 6.04 (1H, d, J= 12.2 Hz) 43.9

C2 205.2 204.5

B21 114.3 114.1

B22 166.9 166.8

B23 103.5 6.27 (2H, d, J=8.4 Hz) 103.5

B24 166.8 166.1

B25 109.2 6.43 (dd, J = 2.5 Hz, 9.1 Hz) 109.0

B26 134.4 7.74 (4H, d, J= 9.1 Hz) 134.4

β2 54.3 4.69 (2H, d, J= 12.2 Hz) 53.4

A21 134.9 134.6

A22 130.5 7.15 (2H, d, J=8.4 Hz) 130.0

A23 116.1 6.56 (4H, d, J=8.4 Hz) 115.8

A24 156.7 156.4

A25 116.1 6.60 (4H, d, J=8.4 Hz) 115.8

A26 130.5 7.15 (2H, d, J=8.4 Hz) 130.0

A11 129.7 129.6

A12 129.8 7.21 (1H, d, J=9.1 Hz) 129.4

A13 116.1 6.59 (1H, d, J=2.4 Hz) 115.9

A14 156.7 156.5

A15 116.5 6.60 (1H, dd, J=2.4 Hz, 9.1 Hz) 115.9

A16 129.8 7.21 (1H, d, J=9.1 Hz) 129.4

A1-4 - OCH3 56.1 3.76 (3H, s) 56.0

A2-4 - OCH3 56.0 3.75 (3H, s) 56.4

B2-4 - OCH3 55.5 3.59 (3H, s) 55.5

Literature data derived from Messanga et al. (2001); Pegnyemb et al. (2003); Abdullahi et al. (2014)

The double integrated singlet peak indicated the presence of two symmetrically positioned methoxyl groups. These results strongly suggested that compound 20 is a derivative of compounds 18 and 19 (Ghogomu et al., 1987; Messanga et al., 2001;

Pegnyemb, 2003a; Anuradha et al., 2006; Abdullahi et al., 2014). The 13C NMR spectrum 84

exhibited signals for 33 carbon atoms, including the intensely overlapping signals at δ

129.8 (A1C2 and A1C6), 130.5 (A2C2 and A2C6), 116.1 (A1C3 and A1C5) and 116.1 (A2C3 and A2C5) (Markham et al., 1978). The carbon chemical shift values showed 8 of the carbon atoms are oxygenated. The 3 singlet signals at δ 56.1, 56.0 and 55.5 were assignable to methoxy (OCH3) group attached to aromatic ring system. The summary of the 1D of compound 20 in comparison with 1D and 2D results of Lophirone A (18) established and characterized the structure of compound 20 as Afzelone D dimethylether

(Trimethoxy Lophirone A) (20) as shown in figure 4.4.

     C  

    OMe 

   MeO OMe

Figure 4.4: Structure for compound 20

4.3.3 Compound 17

Compound 17 was obtained as an amorphous cream white powder from MeOH soluble fraction of both DCM and EtOAc root bark. It gave a red colour when sprayed with p- anisaldehyde and heated to a temperature of 110oC for 5 to 10 minutes and fluoresced under UV-light (254 nm) suggesting the structure of a biflavonoid. The IR spectrum exhibited a broad O-H absorption band at 3423 cm-1 and two conjugated carbonyl groups at 1626 cm1 due to aromatic rings and conjugated double bonds. It also exhibited an O-

-1 CH3 group and vicinal C-H absorption bands at 2850 and 2757 cm , respectively. 85

The 1H NMR spectrum of compound 17 was almost similar in many aspects to that of

Lophirone A (18), Afzelone D (19) and Afzelone D dimethyether (20) (Pegnyemb et al.,

2003a), in that it showed the same signal for protons on all the rings present as well as the ring system of two aliphatic protons. The only difference noted arises in modification of the chemical shifts of the ring A protons and the presence of one OMe signal at δ 3.76 integrating for 3H (s) in compound 17 (Table 4.7). The OMe group can be assigned equally to positions A1C4 and A2C4 because its protons are equivalent to those of ring A of compound 20.

These results strongly suggests that compound 17 is a derivative of Lophirone A and

Afzelone D (Ghogomu et al., 1987; Pegnyemb et al., 2003a). The spectral data of compound MJ/RE/OT02 were similar to those reported for Calodenone (17) (Messanga et al., 1992; Pegnyemb et al., 2003a; Anuradha et al., 2006), a compound isolated from the stem bark of Ochna calodendron which is a derivative of lophirone A (Anuradha et al., 2006). Compound 17 was therefore confirmed to be Calodenone (17) corresponding to the molecular formula C31H24O8 and a molecular weight of 524 (Figure 4.5) and it is the first time to be reported from this plant.

     C  

    OH 

    e

Figure 4.5: Structure of compound 17 86

1 13 Table 4.7: H (400MHz) and C NMR data for 17 (100 MHz, CD3OD)

δ Position δObs H (ppm), m (J (Hz)) HMBC δ Lit.

β1 157.3 8.22 (1H, s) 156.4 α1 122.4 α2H 122.1 C1 177.1 α2H 175.4 B11 117.0 117.2 B12 128.2 7.87 (1H, d, J=8.7 Hz) 128.2 B13 116.0 6.83 (1H, dd, J=2.1 Hz) 115.9 B14 164.7 B1H2 163.4 B15 103.3 6.72 (1H, dd, J=2.1, 8.8 Hz) 103.2 B16 159.4 B1H2 158.5 α2 44.6 6.03 (1H, d, J=12.2 Hz) β2H 43.9 C2 205.3 α2H 204.5 B21 114.3 114.1 B22 166.9 166.8 B23 103.5 6.14 (2H, d, J=8.4 Hz) 103.5 B24 166.8 166.1 B25 109.2 6.29 (d, J = 2.1Hz) 109.0 B26 134.4 8.21 (4H, d, J=9.0Hz) 134.4 β2 54.3 4.67 (1H, d, J=12.2 Hz) α2H 53.4 A21 134.9 134.6 A22 130.5 7.15 (2H, d, J=8.4 Hz) 130.0 A23 116.1 6.58 (4H, d, J=8.4 Hz) 115.8 A24 156.7 156.4 A25 116.1 6.60 (4H, d, J=8.4 Hz) 115.8 A26 130.5 7.15 (2H, d, J=8.4 Hz) 130.0 A11 129.7 A1H3, A1H5 129.6 A12 129.8 7.15 A1H6 129.4 A13 116.1 6.55 (1H, d, J=2.4 Hz) 115.9 A14 156.7 A1H6 156.5 A15 116.5 6.60 (1H, dd, J=2.4, 9.1 Hz) 115.9 A16 129.8 7.15 (1H, d, J=9.1 Hz) 129.4

A14 - OCH3 56.1 3.76 (3H, s) 56.0

Literature data derived from Messanga et al., (1992); Pegnyemb et al. (2003); Anuradha et al. (2006)

4.3.4 Compound 23 and 74

This compound was isolated as white amorphous solid in CHCl3 soluble fraction of DCM root bark with a melting point 130-134oC. It gave purple colour on spraying in p-anisaldehyde and did not fluoresce in UV suggesting that the compound had steroidal structure. The mass spectra showed a molecular ion peaks at m/z 415 [M+H] + and m/z 413 [M+H] +, 87

respectively which corresponded to the molecular formula C29H50O and C29H48O, respectively. The other prominent fragments showed at m/z 396, 351, 300, 271 and 255

(Appendix 4d).

On subjecting to IR spectroscopic analysis, the observed absorption bands are 3459.1 cm‐1 that is characteristic of O‐H stretching and tri-substituted double bonds (2939, 1647 and 1053 cm-1). Absorption at 2939.3 cm‐1and 2734.9 cm‐1 is due aliphatic C‐H stretching. Other absorption frequencies include 1647.1 cm‐1 as a result C=C stretching

‐1 however this band is weak. At 1463.9 cm is a bending frequency for cyclic (CH2)n and

‐1 ‐1 1381.6 cm is for –CH2(CH3)2. The absorption frequency at 1053.1 cm signifies cycloalkane. The out of plane C‐H vibration of unsaturated part was observed at 881.4 cm‐1 (Appendix 4c).

1 The H NMR of compounds 23 and 74 revealed signals for two singlet methyls at δ 1.01

(H-19) and 0.68 (H-18), four doublet methyls at δ 1.02 (d, J = 6.63 Hz, H-21), 0.85 (d, J

= 6.41 Hz, H-26), 0.81 (d, J = 7.55 Hz, H-29) and 0.80 (d, J = 6.41 Hz, H-27). There were three vinylic proton signals at δ 5.30 (2H, m, H-6), 5.15 (1H, dd, J = 15.1, 8.7 Hz,

H-22) and 5.02 (1H, dd, J = 15.1, 8.7 Hz, H-23) and an OH proton signal at δ 3.53 (1H, m, H-3). The remaining proton signals were at δ 0.8-2.4 (Appendix 4a).

13 Direct comparison of the C NMR data of 23 and 74 with those reported in a literature

(Kovganko et al., 1999; Subhadhirasakul and Pechpongs, 2005; Kamboj and Saluja,

2011) (Table 4.9) showed they were identical. Therefore, 23 and 74 was identified as a mixture of two identical compounds. From the 1H NMR spectra, integration of proton signals at δ 5.30 (H-6), 5.15 (H-22), 5.02 (H-23) and 3.53 (H-3) were in the ratio 2:1:1:2. 88

Thus, Compounds 23 and 74 was confirmed to be a mixture of two of β-sitosterol (23) and Stigmasterol (74) (Figures 4.6 and 4.7, respectively) and approximately in the ratio

1:1.

The 13C NMR spectrum showed a total of 47 carbon signals, among them four olefinic carbon signals (δ 140.8, 138.3, 129.3 and 121.7) and one monoxygenated carbon signal (δ

13 71.8) were observed. The C NMR gave a signal at 140.8 and 121.7 ppm for C5=C6 double bond and 138.3 and 129.3 ppm for C22=C23 double bond, respectively for compound 74 and at 140.8 and 121.7 ppm for C5=C6 double bond for compound 23

(Table 4.8) which showed high level of saturation in the carbon-proton linkages in its structures. The remaining carbons showed signals having chemical shifts between 11 and

57 ppm (Appendix 4b). From the above spectroscopic data, it seemed to be a mixture of almost related compounds.

H3C H3C

CH3 CH3 CH3 CH3

H3C H3C CH3 CH3

HO HO

Figure 4.6: β-sitosterol (23) Figure 4.7: Stigmasterol (74)

Structures for compounds 23 and 74

13 Table 4.8: C NMR (100 MHz, CDCl3), of compounds 23 and 74

1 Position Group δObs (74) δObs (23) δLit. (74 ; 23) Major δ H

1 CH2 37.2 37.2 37.3 2 CH2 31.7 31.7 31.6 3 CH 71.8 71.8 71.8 3.53 (m, H3) 4 CH2 42.3 42.3 42.3 5 C 140.8 140.8 140.1 6 CH 121.7 121.7 121.7 5.30 (d, H6) 7 CH2 31.7 31.7 31.9, 31.6 8 CH 31.9 31.9 31.9 9 CH 50.2 50.2 50.1, 50.1 10 C 30.5 30.5 30.5 11 CH2 21.1 21.1 21.1 12 CH2 39.7 39.8 39.7, 39.8 13 C 42.3 42.3 42.3 14 CH 56.8 56.9 56.7, 56.9 15 CH 24.3 24.3 24.3, 24.3 16 CH2 28.7 28.2 28.7, 28.2 17 CH 56.1 56.1 56.7, 56.1 18 CH3 11.9 12.0 11.8, 12.0 0.68 CH3 19 CH3 19.4 19.4 19.4 1.01 CH3 20 CH 40.5 36.2 40.5, 36.1 21 CH3 21.1 18.8 21.1, 18.8 1.02 CH3 22 CH, CH2 138.3 33.7 137.3, 33.7 5.15 23 CH, CH2 129.3 26.1 129.3, 26.1 5.02 24 CH 51.2 45.8 51.2, 45.8 25 CH2 31.9 29.2 31.9, 29.2 26 CH3 20.0 19.8 19.0, 19.8 0.85 CH3 27 CH2 21.2 19.0 21.2, 19.0 28 CH3 25.4 23.1 25.4, 23.1 0.81 CH3 29 CH3 12.0 11.7 12.0, 12.0 0.86 CH3

Literature data derived from Kovganko et al. (1999); Subhadhirasakul and Pechpongs (2005)

4.3.5 Compound 75

This compound was isolated as white amorphous solid in CHCl3 soluble fraction of DCM root bark with a melting point 266-267 oC. It gave purple colour on spraying in p-anisaldehyde and did not fluoresce in UV suggesting that the compound had triterpenoid skeleton. The

UV in CDCl3 showed one absorption band at 231.5 nm (Appendix 5i) suggestive of a triterpenoid nucleus (Enas et al., 2012). The calculated molecular mass was 500.4 which 90

corresponded to the molecular formula C34H60O2. IR (KBr) spectrum peak shows a strong

-1 -1 C-H stretch at 2936 cm , 2866 cm shows the presence of –CH3 and –CH2, CH antisymmetric / symmetric stretch. The C-O-C stretching frequency was attributed to

-1 1226 cm . At δ 171.2 an acetyl group is attached at C3. This was supported by the appearance of a peak in the IR spectrum at 1707 cm-1 as a result ester carbonyl stretch

(Houghton and Lian, 1986).

Structural determination of 75 was achieved by careful analysis of 1D (1H, 13C and

DEPT) NMR spectra. The 1H NMR spectrum of compound 75 displayed singlet signals for nine methyl groups of a pentacyclic triterpenoid and a signal at δ 2.00 (s, 3H) is characteristic of an acetyl group. The 1H NMR data of 75 (Appendix 5a & 5b) showed methyl protons at δ 0.835-0.982, one C3 protons at δ 3.70 (1H, s) are typical of ester protons and δ 4.08 (d, J = 13 Hz) are typical of two methylene hydrogens. The 1H NMR data did not allow much analysis of the hindered signals, however, a doublet (J = 7.8 Hz) centred at δ 0.835 ppm characteristic of methyl group at position C23 of friedelane compounds was observed (Lopez Perez et al., 2007).

Comparison of the 1H and 13C NMR data of compound 75 with that of 3β,24- diacetylfriedelane (76) indicated that compound 75 was similar to 76 in rings A, B C and

D suggesting that the acetyl group is attached to C3, which was supported by the HMBC correlations of the latter (Mahato and Kundu 1994; Carvalho et al., 1995). The DEPT 13C

NMR spectra (Appendix 5c ) was used to recognize the signals corresponding to six quaternary carbons, one monoxygenated carbon at δ 72.8 (C3) besides signals of nine methyl, twelve methylene, six methyne groups and one acetyl group (δ 171.2, 21.1). From 91 the DEPT spectra (Appendix 5c) the compound has 34 carbons of which 27 are protonated. Compound 75 was assigned the name 3β-acetyl-24-ethylfriedelane.

13 Table 4.9: C NMR (150 MHz, CDCl3), of compound 75

Position Group δObs δLit. Position Group δObs δLit.

1 CH2 15.8 15.8 18 CH 42.8 42.9 2 CH2 35.2 35.2 19 CH2 35.3 35.6 3 CH 72.8 72.8 20 C 28.2 28.2 4 CH 49.2 49.1 21 CH2 32.8 32.9 5 C 60.4 59.0 22 CH2 39.3 39.3 6 CH2 42.8 41.8 23 CH3 11.6 11.6 7 CH2 17.5 17.5 24 CH3 16.4 16.4 8 CH 53.2 53.1 25 CH3 18.2 18.2 9 C 37.8 37.8 26 CH3 18.7 18.6 10 CH 61.3 61.3 27 CH3 20.1 20.1 11 CH2 35.6 35.6 28 CH3 32.1 32.1 12 CH2 32.8 32.9 29 CH 31.8 31.8 13 C 38.3 38.2 30 CH3 35.0 35.0 14 C 39.7 39.7 31 CH2 14.2 14.1 15 CH2 30.6 30.6 32 CH3 37.1 37.1 16 CH2 36.1 36.1 OCOCH3 - 171.2 171.1 17 C 30.0 29.3 OCOCH3 - 21.1 21.4

Assignments according to Aragao et al. (1990); Costa and Carvalho (2003)

R1O R1O

R2 OR2

R1 = Ac R2 = Et R1 = R2 = Ac

Figure 4.8: structure for 75 76

4.4 Antibacterial activity test for the isolated compounds

Table 4.10: Inhibition zones (in mm) of antibacterial activity of isolated compounds

Compound S. aureus E. coli S. typhi Compound 75 16 6 12 Compound 17 12 6 14 Compound 20 14 7 14 Compound 18 18 6 12 Compound 23 and 74 10 6 6 Chloramphenicol (+ve) 25 24 23 DMSO (-ve) 6 6 6

Key: Staphylococcus aureus ATCC 25724, Escherichia coli ATCC 25723, Salmonella. typhi (clinical isolate)

The activity of isolated compounds 17 to 75 was carried out using the same procedure as discussed for the crude extracts, following the method used by Chhabra and Usio (1991).

The inhibition zone was measured as in the crude extracts using the same pathogens except E. coli. An isolated compound with a clear zone of 9 mm or greater was considered to have significant activity. The results of antimicrobial investigations suggest the compounds have activity against S. aureus and S. typhi, but were not active, at the tested dose of E. coli (Table 4.10). The isolated compounds with zones of inhibition below 9 mm were considered to be inactive as described by Faizi et al. (2003). There was some activity shown by all the compounds against the tested bacteria but compound mixture 23 and 74 had moderate activity as presented in table 4.10.

CHAPTER FIVE

CONCLUSIONS AND RECOMMENDATIONS

5.1 Conclusions from the study

The study supports the finding that biologically-active compounds and potential pharmaceuticals can be isolated from medicinal plants. As was found from other

Ochnaceae species, the majority of the compounds isolated from the plant were biflavonoids. This project aimed at investigating and establishing the presence of antimicrobial activity in Ochna thomasiana on the basis of its use in traditional medicine against microbial infections. The following was established:-

i. The plant crude extracts had appreciable yields especially the methanolic

extracts.

ii. There was notable activity for the root and stem bark methanolic extracts with

MICs against the tested Gram-positive bacteria ranging between 500 and 1000

μg/ml.

iii. The methanolic root bark extract recorded the highest activity against the tested

Gram-positive bacteria ranging between 17 and 20 mm.

iv. All the extracts had no activity against the Gram-negative bacteria strains.

v. A total of six compounds (three biflavonoids and three sterols) were isolated and

characterized.

vi. Compounds 18, 20, and 75 from the plant had fairly noticeable activity against

Salmonella typhi and Staphylococcus aureus ranging between 12-18 mm.

However, all the isolated compounds had no activity against Escherichia coli.

5.2 Recommendations from the study

This study demonstrated that there is need for further investigation and bioassay guided isolation of pure compounds from the leaf, stem and root of O. thomasiana, in addition to the following recommendations:-

i. The methanolic root bark with an activity of 20 mm against B. subtilis should be

packaged for use to combat the spread of the pathogen.

ii. The pharmaceutical companies should evaluate whether singly or in combination

compound 18, 20 and 75 or compound 18 with 20 and 18 with 75 is a more

potent antibacterial drug. iii. Due to the many benefits derived from the plant species, it should be propagated

using good agricultural practices for medicinal plants.

5.3 Suggestions for further research

i. Crude methanolic extracts stem and root and ethyl acetate extracts stem need to

be fractionally separated and purified for chemical analysis.

ii. The leaves of Ochna thomasiana should be screened for antimicrobial activity

since they were not worked on.

iii. Studies should be extended to other Kenyan related species of the plants to

evaluate their activity against pathogens.

iv. There is need for in vivo and in vitro evaluation of the crude extracts and

isolated compounds.

v. Blending compounds with significant good activity should be tried followed by

screening for both in vivo and in vitro to check for activity enhancement.

vi. Further tests on other microbial strains including antifungal activity should also

be done. 95

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1 Appendix 1a: H NMR (600 MHz CD3OD) of compound 18





 



  C 







 

  

 

C2 

  

 

13 Appendix 1b: C NMR (150 MHz CD3OD) of compound 18

108





 



  C 







 

  

 

C2 

  

 

13 Appendix 1c: C DEPT (CH) NMR (150 MHz CD3OD) of compound 18

109





 



  C 







 

  

 

C2 

  

 

13 Appendix 1d: C NMR APT (150 MHz CD3OD) of compound 18

110





 



  C 







 

  

 

C2 

  

 

1 1 Appendix 1e: H- H COSY (600 MHz CD3OD) of compound 18

111

OH 

  C 







 

  

 

C2 

   HO HO

13 Appendix 1f: C NMR HSQC (150 MHz CD3OD) of compound 18

112





 



  C 







 

  

 

C2 

  

 

13 Appendix 1g: C NMR HMBC (150 MHz CD3OD) of compound 18 113





 



  C 







 

  

 

C2 

  

  Appendix 1h: IR of compound 18 114





 



  C 







 

  

 

C2 

  

 

Appendix 1i: UV of compound 18

115





 



  C 







 

  

 

C2 

  

 

116

1 Appendix 2a: H NMR (400 MHz CD3OD) of compound 20





e O 

  C 







 

  

  

   MeO

OMe

13 Appendix 2b: C NMR (100 MHz CD3OD) of compound 20 117





e O 

  C 







 

  

  

  

OMe OMe

Appendix 2c: IR of compound 20 118





e O 

  C 







 

  

  

  

OMe OMe

1 Appendix 3a: H NMR (400 MHz CD3OD) of compound 17 119



HO 

  C 







 

  

  

   MeO HO

13 Appendix 3b: C NMR (100 MHz CD3OD) of compound 17

120



HO 

  C 







 

  

  

   MeO HO

Appendix 3c: IR of compound 17

121



HO 

  C 







 

  

  

   MeO HO

1 Appendix 4a: H NMR (100 MHz CDCl3) of compound 23 and 74 122

CH3

CH3 CH3

CH3

H3C

CH3

CH3 CH3

CH3

H3C

CH3

13 Appendix 4b: C NMR (100 MHz CDCl3) of compound 23 and 74

123

CH3

CH3 CH3

CH3

H3C

CH3

CH3 CH3

CH3

H3C

CH3

Appendix 4c: IR of compound 23 and 74 124

CH3

CH3 CH3

CH3

H3C

CH3

CH3 CH3

CH3

H3C

CH3

Appendix 4d: MS of compound 23 and 74 125

HO HO

CH3 CH3

CH3 CH3 CH3 CH3

CH3 CH3

H3C H3C

CH3 CH3

1 Appendix 5a: H NMR (600 MHz CDCl3) of compound 75 126

R1O

1 Appendix 5b: H NMR (600 MHz CDCl3) of compound 75 127

R1O

1 Appendix 5c: Expanded part H NMR (600 MHz CDCl3) of compound 75 128

R1O

13 Appendix 5d: C NMR (150 MHz CDCl3) of compound 75 129

R1O

13 Appendix 5e: Section of C DEPT-135 (150 MHz CDCl3) of compound 75 130

R1O

13 Appendix 5f: Section of C NMR (150 MHz CDCl3) of compound 75 131

R1O

13 Appendix 5g: C NMR DEPT-135 (150 MHz CDCl3) of compound 75

132

R1O

Appendix 5h: IR of compound 75

133

R1O

Appendix 5i: UV of compound 75

134

R1O