SCREENING FOR ANTIBACTERIAL AND ANTIFUNGAL COMPOUNDS IN Bersama abyssinica FRESEN
ONG’ERA TABITHA NYANCHOKA (B.ED, SC.) (I56/CE/11221/2007)
A Research 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 Science, Kenyatta University
MAY 2016
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Declarations
I declare that this thesis is my original work and has not been previously presented for a degree in Kenyatta University or in any other University.
Ong’era Tabitha Nyanchoka
Reg. No. I56/CE/11221/07
Sign: ______Date ______
Department of Chemistry
We confirm that the work reported in this thesis was carried out by the candidate under our supervision.
Prof. Alex K. Machocho Department of Chemistry Kenyatta University
Sign: ______Date______
Prof. Nicholas K. Gikonyo Department of Pharmacy, Complementary and Alternative Medicine Kenyatta University
Sign: ______Date______
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Dedication To my daughter Flavian Bonareri, sons Adrian Nyaenya and Fabian Ong’era and my beloved husband Evans Nyaenya.
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Acknowledgements
I would like to display my sincere gratitude to the following persons for their support, assistance and encouragement throughout the course of my research and compilation of this thesis. My supervisors: Prof. Alex Machocho of the Department of Chemistry and Prof. Nicholas Gikonyo of the Department of Pharmacy, Complementary and Alternative medicine for their guidance, motivation, support and time invested in me. Also, Dr. Omari Amuka of Maseno University for assisting me in the plant collection exercise. Dr. Margret Ng’ang’a of the Department of
Chemistry for her encouragement and support throughout my research in the laboratory.
I would also like to thank the staff in the Chemistry laboratory, Kenyatta University for their assistance in the laboratory and the staff in the microbiology laboratory more especially Daniel
Ng’ang’a for providing expertise knowledge on in vitro antibacterial and antifungal assays. To my classmates and research partners; Regina Kihagi, Moses Oswago, Stephen Kamau, Ombuna
Naftal, Shylock Onduso and Nyaenya Evans for their valuable suggestions. In addition, my sincere gratitude goes to Keru Kamitha, University of Kwazulu Natal for assisting me in running the NMR of the isolated compounds and to the technical staff of Kenya Bureau of Standards for running GC-MS of the crude extracts.
Special thanks go to my husband and children, brothers, sisters and parents for their patience, support, encouragement, prayers and motivation when it was most needed and also to Vicres foundation for partially sponsoring my research work. Last but not least, God without whom nothing is possible.
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TABLE OF CONTENTS Declarations ...... ii Dedication ...... iii Acknowledgements …...... iv Table of contents…………………………………………………………………………………..v Abbreviations and Acronyms………………………………………………………………...... xiii Abstract ...... xvi
CHAPTER ONE ...... 1 INTRODUCTION ...... 1 1.1 Background ...... 1 1.2 Antibiotic resistance …………………………………………………………………………..2 1.3 Phytomedicine and Phytopharmaceutical agents ...... 5 1.4 Bacterial infections ...... 8 1.4.1 Staphylococcus aureus ...... 9 1.4.2 Escherichia coli ...... 9 1.4.3 Pseudomonas aeruginosa ...... 10 1.5 Antibacterial drugs ...... 11 1.5.1 Tetracyclines ...... 11 1.5.2 Penicillins ...... 12 1.5.3 Sulphonamides ...... 12 1.5.4 Flouroquinolones ...... 13 1.5.5 Quinolones ...... 14 1.6 Fungal infections ...... 14 1.7 Antifungal drugs...... 16 1.7.1 Flucytosine ...... 16 1.7.2 Amphotericin ...... 17 1.8 Statement of the problem ...... 18 1.9 Hypotheses…………………………………………………………………………………..19 1.10 Objectives of the study ...... 19 1.10.1 General objective ...... 19
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1.10.2 Specific objectives ...... 19 1.11 Justification and significance of the study ...... 20
CHAPTER TWO ...... 21 LITERATURE REVIEW ...... 21 2.1 The family Melianthaceae ...... 21 2.1.1 Bersama yangambiensis Toussaint ...... 21 2.1.2 Bersama engleriana Gurke ...... 22 2.1.3 Bersama swinnyi Phil…………………………………………………………………23 2.1.4 Bersama abyssinica Fresen ...... 24 2.2 Biosynthesic pathway of anthraquinone ...... 27 2.3 Biosynthesis of lupeol ...... 29 2.4 Biosynthetic pathway of steroids ...... 30 2.5 Biosynthetic pathway of oleanolic acid……………………………………………………... 31
CHAPTER THREE …………………………………………………………………………….32 MATERIALS AND METHODS ...... 32 3.1 General procedures ...... 32 3.2 Chromatographic techniques ...... 33 3.2.1 Spray reagents ...... 33 3.2.3 Detection of terpenoids ...... 33 3.2.4 Column chromatography ...... 33 3.3 Plant material ...... 32 3.4 Bioactivity screening of the plant extracts...... 34 3.4.1 Anti-bacterial activity test ...... 34 3.4.1.1 Preparation of nutrient agar media and growing of bacteria cultures …………35 3.4.1.2 Introduction of the crude plant extract into inoculated Petri dishes.………..…36 3.5 Antifungal activity test ...... 36 3.5.1 Preparation of the drugs ...... 37 3.6 Extraction procedure for the stem bark of Bersama abyssinica ...... 38 3.7 Instrumentation………………………………………………………………………………39
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3.7.1 Melting point………………………………………………………………………….39 3.7.2 Ultraviolet (UV) ...... 39 3.7.3 Nuclear magnetic resonance (NMR) spectroscopy ...... 39 3.7.4 Gas chromatography linked with M/S ...... 40 3.8 Isolation of compounds from plant extracts ...... 40 3.9 Fractionation of the extract ...... 42 3.10 Physical and spectroscopic data ...... 45 3.10.1: Physical and spectral data of compound TN1 ...... 45 3.10.2 Physical and spectral data of compound TN2 ...... 45 3.10.3 Physical and spectral data of compound TN3 ...... 46 3.10.4 Physical and spectral data of compound TN4 ...... 46 3.10.5 Physical and spectral data of compound TN5 ...... 47 3.10.6 Physical and spectral data of compound TN6 ...... 47
CHAPTER FOUR ...... 48 RESULTS AND DISCUSSIONS ...... 48 4.1 Crude extract yields ...... 48 4.2 Bio activity tests…………………………………………………………..…………………48 4.2.1 Antibacterial assay for the extracts……………………………………………………48
4.2.2 Antifungal assays for the extracts………………………….…………………………..49
4.3 Structural elucidation of isolated compounds ...... 50 4.3.1 Structure of compound TN1 ...... 50 4.3.2 Structure of compound TN2 ...... 53 4.3.3 Structure of compound TN3 ...... 58 4.3.4 Structure of compound TN4 ...... 61 4.3.5 Structure of compound TN5 ...... 62 4.3.6 Structure of compound TN6 ...... 65 4.4 GC-MS data for the crude extracts of methanol and DCM...... 67 4.5 Antibacterial test assay for the isolated compounds ...... 68 4.6 Antifungal activities for isolated compounds ...... 69
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CHAPTER FIVE...... 70 CONCLUSION AND RECOMMENDATIONS ...... 71 5.1 Conclusion ...... 71 5.2 Recommendations ...... 72 5.2.1 Recommendations from the study……………………………………………………...72 5.2.2 Recommendations for further research…………………………………………………72 REFERENCES ...... ……………………………………………………………. 74
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LISTS OF TABLES
Table 3.1: Bacteria strains used in the bioassay ………………………………………...34
Table 3.2: Standard antibiotics used as reference drugs ………………………………………..35
Table 4.1: Yield of extract from 4kg of stem bark of B. abyssinica……………………………. 48
Table 4.2: Antibacterial activities of the crude extract and standard drug…...... ……………...49
Table 4.3: Antifungal activities of the crude extract and standard drug…...………………...... 50
Table 4.4: NMR spectral data for compound TN1………………………………………….. ….52
Table 4.5: 13C NMR data of the aglycone of compound TN2 and aglycone of sitosterol ……... 56
Table 4.6: NMR data for aglycone [Non-sugar part (sitosterol)] of compound TN2 ………….. 57
Table 4.7: NMR data for the sugar moiety ………………………………………………….…..58
Table 4.8: 13C NMR data for compound TN3 and that reported for stigmasterol……………...60
Table 4.9: 13C-NMR data of compound TN4 and that reported for sitosterol……………….... 62
Table 4.10: 13C NMR data of compound TN5 compared to lupeol ……………………………..64
Table 4.11: 1H NMR and 13C NMR data of compound TN6 compared to oleanolic acid………66
Table 4.12: Antibacterial activities of isolated compounds…………………………………….. 68
Table 4.15: Antifungal activities of isolated compounds………………………………………. 69
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LIST OF FIGURES
Fig 2.1: Flowering branch of Bersama abyssinica………………………….………………….. 25
Fig 4.1: NOESY correlations of the sugar moiety...……………………………………………. 57
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LISTS OF SCHEMES
Scheme 2.1: Biosynthesis of anthraquinone……………………………………………...... ….. 28
Scheme 2.2: Biosynthesis of lupeol from squalene…………………………………………...... 29
Scheme 2.3: Biosynthesis of stigmasterol and sitosterol from squalene ………………...... 30
Scheme 2.4: Biosynthesis of oleanolic acid from squalene …………………………………..…31
Scheme 3.1: Sequential extraction of Bersama abyssinica Fresen ……………………………...38
Scheme 3.2: Chromatographic separation of DCM crude extract of the stem bark……………..43
Scheme 3.3: Chromatographic separation of the methanol crude extract of stem bark………....44
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LISTS OF APPENDICES
Appendix 1a: 1H NMR spectrum compound TN1……………………...……………………….80
Appendix 1b: 13C NMR spectrum of compound TN1…………………………………………...81
Appendix 1c: HMBC for compound TN1……………………………………………………….82
Appendix 1d: HSQC spectrum of compound TN1………………………………………………83
Appendix 1e: NOESY spectrum for compound TN1……………………………………………84
Appendix 2a: 1H NMR spectrum for compound TN2...... 85 Appendix 2b: 13C NMR spectrum for compound TN2...………………………………………..86
Appendix 2c: DEPT spectrum for compound TN2……………………………………………..87
Appendix 2d: HMBC spectrum for compound TN2…………………………………………….88
Appendix 2e: HSQC spectrum for compound TN2…………………………………………...... 89
Appendix 2f: NOESY spectrum for compound TN2...………………………………………….90
Appendix 2g: COSY spectrum for compound TN2……………………………..…………...... 91
Appendix 3a: 1H NMR spectrum for compound TN4...…………….………………………...... 92
Appendix 3b: 13C NMR spectrum for compound TN4……………...………………………...... 93
Appendix 4a: 1H NMR spectrum for compound TN5…..……..……………………..…….……94
Appendix 4b: 13C NMR spectrum for compound TN5 …………….………………….………...95
Appendix 5a: 13C NMR of compound TN3….…………………..……..………………………..96
Appendix 5b:1H NMR of compound TN3…...…….……………………………………………97
Appendix: 6a 1H NMR for compound TN6 …………………………....……………………….98
Appendix 6b:13C NMR for compound TN6………………………………………………….....99
Appendix 7a: GC-MS data for crude methanol extract ………………………………………..100
Appendix 7b: GC-MS data for the crude DCM extract …………………………..……………105
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ABBREVIATIONS AND ACRONYMS
AIDS Acquired Immune Deficiency Syndrome a.m.u Atomic mass unit
ATCC American Type Culture Collection
AQ Anthraquinones bAS Beta amyrin synthase
BCG Bacillus of Calmette and Guerin br d Broad doublet br s Broad singlet
CC Column chromatography
CDCl3 Deuterated chloroform
CNS Central Nervous System
CoA Coenzyme A
COSY Correlated Spectroscopy
CPR Cytochrome p450 reductase d Doublet dd Doublet of doublets
DCM Dichloromethane
DEPT Distortionless Enhancement by Polarization Transfer
DMSO Dimethyl sulfoxide
DMAPP 3,3 -Dimethylallyl diphosphate
DNA Deoxy ribonucleic acid
E-4-P Erythrose -4-phosphate
FAB Fast atomic bombardment
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FPP Farnesyl pyrophosphate
GC Gas chromatography
HIV Human Immunodeficiency Virus
HRMS High Resolution Mass Spectrometer
HMBC Heteronuclear Multiple Bond Correlation
HSQC Heteronuclear Single Quantum Correlation
Hz Hertz
IPP Isopentenyl diphosphate
J Coupling constant m Multiplet
MEP Methyl-D-erythritol 4-phosphate
Mm Millimole
MS Mass Spectroscopy
MVA Mevalonic acid
NMR Nuclear Magnetic Resonance
NOESY Nuclear Overhauser Enhancement Spectroscopy
OSC Oxidosqualene cyclases
PDA Potato Dextrose Agar
PEP Phosphoenol pyruvate ppm Parts per million q Quartet
RT Retention time
s Singlet
SQS Squalene synthase t Triplet
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TCA Tricarboxylic acid
TLC Thin Layer Chromatography
UV Ultra violet
VLC Vacuum liquid chromatography
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ABSTRACT
Medicinal plants contain a wide range of substances that can be used to treat chronic illness as well as infectious diseases. A vast knowledge of how to use the plants against different illnesses may be expected to have accumulated in areas where plants are commonly used. Conventional medicine has created problems due to microbial resistance. This has enhanced the interest in search for natural products with medicinal property. Information on the chemical constituents in herbs aid in discovering new therapeutic drugs. Bersama abyssinica was selected for this study because of its uses in traditional medicine as an antimicrobial agent. For example, the bark, root, and leaf decoctions are taken to treat a range of stomach disorders such as abdominal pain, colic, diarrhoea, intestinal worms and amoebiasis. A stem bark decoction is drunk to cure cancer and rheumatism. The overall objective of this study was to extract, isolate and characterize bioactive compounds with antibacterial and antifungal activities in Bersama abyssinica. Crude extracts of the stem bark was bioassayed for antibacterial and antifungal activities. Column chromatography, thin layer chromatography and vacuum liquid chromatography were used for separation, isolation and purification of the extracts. Spectroscopic techniques were used to elucidate their structures. Six compounds were isolated which include: β-sitosterol (TN4), β- stigmasterol (TN3), β-sitosterol glycoside (TN2), lupeol (TN5), oleanolic acid (TN6) and an anthraquinone [Bersamanone] (TN1). From the isolated compounds, five have been previously reported in literature while Bersamanone is reported for the first time from this plant species. The bacteria used to test for antibacterial activities included Salmonella typhi, Shigella dysentriae, Vibrio cholerae, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis while the antifungal test was carried out against Candida albicans and Penicillium notatum. Dichloromethane extract of the stem bark had mild activity of 8 and 9 mm on Bacillus subtilis and Staphyloccocus aureus, respectively. Ethyl acetate extract had moderate activity of 10 and 10 mm on Staphyloccocus aureus and Bacillus subtilis. It had mild activity on the other strains of bacteria. Antifungal activities of this extracts were also mild on Candida albicans and Penicillium notatum. Methanol extract had the highest activity of 16 and 16 mm on Klebsiella pneumoniae and Pseudomonas aeruginosa, respectively. Moderate activity was shown in Vibrio cholerae, Escherichia coli, Bacillus subtilis and Staphylococcus aureus. Among the compounds isolated β-sitosterol glucoside had activity against all the strains of bacteria and fungi the highest being that of B. subtilis and E. coli with inhibition zones of 14 and 15 mm, respectively. The anthraquinone had activity against Bacillus subtilis, Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli with inhibition zones of 14, 9, 14 and 10 mm, respectively. Lupeol had moderate activity against Bacillus subtilis, Staphylococcus aureus and Escherichia coli with inhibition zones of 11, 14 and 8 mm, respectively. The study showed that the studied plant species contain compounds that showed varied bioactivities on the test bacteria and fungi and can be used in treatment of the diseases caused by respective pathogens used in this study once cytotoxicity tests are done.
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CHAPTER ONE
INTRODUCTION 1.1 Background
Man has used plants as medicine since time immemorial. Evidence of these is thousands of years old traditions and records of healing using herbal medicine. To this age of great development and progress in the field of chemistry, pharmaceutics and medicine, drugs of plant origin have lost none of their importance. Over the years, scientific research has expanded and made more precise the knowledge on chemical composition and their bioactivity. It is thus possible to know more about their action and be more exact in prescribing their use in treatment of various diseases (Diallo et al., 1999; Kokwaro, 2009). Medicinal plants have become of chief interest and chemists have succeeded in isolating the pure active substances contained in plant parts. The organic chemicals from crude drugs also provide a model which can be copied or modified by organic chemists to produce more potent drugs or better drugs with fewer side effects. Examples of these latter groups are the drugs used as local anesthetics which are based on the artificially modified chemical structure of cocaine. Other examples can be found in penicillin drugs, many of which are semi-synthetic but all based on the molecular configuration first isolated from
Penicillium fungus. Drugs from medicinal plants are also basic materials for making herbal tea mixtures, taken in the form of a decoction according to the disease they are intended to treat
(Diallo et al., 1999).
The Maasai community in Kenya for example, uses variety of plant preparations to treat different kinds of microbial and non microbial diseases (Miaron, 2003). One of the microbial diseases is diarrhoea which causes morbidity and mortality in developing countries. Diarrhoea is caused by 2
enteric bacteria pathogens. The major bacteria pathogens include; Vibrio cholera, Shigella spp,
Salmonella spp, Campylobacter spp, Yesinia enterocolitica, and several strains of diarrheagenic
Escherichia coli. The bacteria pathogens are mainly associated with diarrhoea diseases but cause other infections such as pneumonia and septicaemia (Samie et al., 2005).
1.2 Antibiotic resistance
Antibiotic resistance is a worldwide problem (Lessa et al., 2012). Many forms of resistance spread with remarkable speed. World health leaders have described antibiotic-resistant microorganisms as “nightmare bacteria” that “pose a catastrophic threat” to people in every country in the world. Among all of the bacterial resistance problems, Gram-negative pathogens are particularly worrisome, because they are becoming resistant to nearly all drugs that would be considered for treatment (Roberts et al., 2009). This is true as well, but not to the same extent, for some of the Gram-positive pathogens for example Staphylococcus and Enterococcus. The most serious Gram-negative infections are healthcare-associated, and the most common pathogens are Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter. Treating infections of Gram-negative microorganisms is an increasingly common challenge in many hospitals (Kochanek et al., 2009).
As antibiotic resistance grows, the antibiotics used to treat infections do not work well at all. The loss of effective antibiotic treatments will not only cripple the ability to fight routine infectious diseases but will also undermine treatment of infectious complications in patients with other diseases. Many of the advances in medical treatment for example joint replacements, organ transplants, cancer therapy and treatment of chronic diseases are dependent on the ability to fight
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infections with antibiotics. If that ability of the antibiotic is lost, the ability to save and improve peoples’ lives will be lost. Resistant bacteria can contaminate foods that come from animals, and people who consume these foods can develop antibiotic-resistant infections (Sievert et al., 2013).
Antibiotics must be used judiciously in humans and animals because both uses contribute to not only the emergence, but also the persistence and spread of antibiotic-resistant bacteria (Roberts et al., 2009). Scientists around the world have provided strong evidence that antibiotic use in food-producing animals can harm public health through the following sequence of events; Use of antibiotics in food producing animals allows antibiotic-resistant bacteria to thrive while susceptible bacteria are suppressed or die, resistant bacteria can be transmitted from food producing animals to humans through the food supply and resistant bacteria can cause infections in humans (Scallan et al., 2011).
Opportunistic pathogens and nosocomial infections are important causes of infection in burn wounds due to the compromised skin barrier in burn injuries (Sokmen et al., 1999). According to the WHO antibiotic resistant bacteria are responsible for up to 60% of hospital-acquired infections in the United States (World Health Report, 2000), with the burns and trauma departments reported as some of the most common sites for the emergence of resistance.
Antibiotic resistance is considered a global health concern and has been termed one of the world’s most pressing public health problems.
Some of the bacteria are resistant to many different drugs, raising the concern of a post-antibiotic era (Kochanek et al., 2009). Current trends suggest that no effective therapies will be available for treating some diseases within the next ten years. The rates of some communicable diseases
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have started to increase again as a result of the rise in antibiotic resistance (WHO, 2014). Various facets contribute to the occurrence and spread of antimicrobial resistance. The uncontrolled and inappropriate use of antibiotics today may reduce future effectiveness of the antibiotics. For example, people who are repeatedly medicating acne with antibiotics in a household may raise the concentration of antibiotic resistant bacteria on the skin of family members (WHO, 2014).
Antimicrobial soaps and detergents as well as the agricultural use of antibiotics as growth factors, increase the pressure on wild bacteria to evolve resistance (WHO, 2014).
Resistant bacteria have various mechanisms to disable the harmful actions of certain antibiotics, ensuring bacterial survival, such as: production of enzymes that destroy the active antibiotics, changing cell wall permeability to antibiotics, rapid discharge of antibiotics from the interior of the bacteria and developing structural alteration in the attachment site for antibiotics (Fansworth,
1996).
The search for new effective antimicrobial agents may alleviate the difficulties associated with patient outcome and treatment of antibiotic resistant infections. The investigation and discovery of novel effective antimicrobial agents should be accompanied with an appreciation and rational use of current antibiotics. Scientific investigation of traditionally used medicinal plants for antimicrobial properties may serve as effective agents for the treatment of antibiotic resistant infections. Fansworth (1996) suggested that antimicrobial agents originating from plants might use a different mechanism to inhibit microorganisms and resistant pathogens. Implementation of simple infection control practices such as hand washing, use of protective clothing and aseptic techniques may limit the spread of resistant microbes, especially in hospitals. The WHO
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launched a global strategy in 2001 for combating antimicrobial resistance which is aimed at slowing the emergence and reducing the spread of resistance (WHO, 2001). Antibiotic resistance is inevitable, but measures such as infection control, development of new antimicrobial agents and rational use of effective antimicrobial agents may slow resistance (Fansworth, 1996).
1.3 Phytomedicine and Phytopharmaceutical agents
Apart from being the major components of ethno-medicines, medicinal plants have also been major sources of drugs used in the orthodox medical practice. It is reported that about 74% of the plant derived drugs which are currently in the market were actually derived from the indigenous knowledge of traditional people on ethno-medicines (Gurib-Farkim, 2006; Kokwaro, 2009). The first generation of plant medicine was simple botanical materials employed in more or less crude form. These medicines from Cinchona, Opium, Belladonna and Aloe species of plants were selected based on empirical evidence as gathered by traditional practitioners (Iwu et al., 1999).
The second generation of phytopharmaceutical agents were pure molecules whose compounds differ from the synthetic therapeutic agent only in their origin, for example taxol from Taxus spp., quinine from Cinchona spp and reserpine from Rauwolfia spp (Iwu et al., 1999). In the development of third generation of plant medicine, the formulation is based on well controlled clinical and toxicological studies of phytomedicine to improve the quality, efficacy, stability and the safety of the preparations (Heinrich et al., 2004).
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Numerous secondary metabolites have been isolated from different plants and have provided chemical templates for developing more potent analogs (Cowan, 1999). A good example is the anti-malarial artemisinin (1) which was isolated from Artemisia annua L. (Huang, 1999).
Artemisinin enabled the synthesis of more potent and water soluble artenilic acid (2), artemotil
(3) and artesunate (4) (Babu et al., 2003). Quinine (5) was first isolated from Cinchona officialis
L. barks in 1820 by Polletier and Caventou. This drug together with cinchonine (6) has been in use for many years as antimalarials (Ma et al., 2006). Afterwards derivatives were synthesized and gave birth to more potent drugs such as mefloquine (7) and lumefantrine (8) (Vennerstrom and Bergie, 2004).
H O OO O O O O O O O H O H O O H 1 H O O 3 2
O OH
O O HO N O O O 4 OH MeO O O N
5
7
CF 3 CF N 3 H HN OH HO N HC-OH CH2NHC(CH3)3
N H C NH Cl 8 N 6 7
Africa is one of the main world producers of medicinal plant (Elujoba et al., 2005). Yohimbine
(9), an indole alkaloid isolated from Corynanthe pachyceras K.Schum native to Ghana is a stimulant, an antiviral and is used to treat male impotence. The isoquinoline alkaloid, michellamine B (10) which was isolated from Ancistrocladus abbreviates Airy shaw from
Cameroon and Ghana has anti-HIV properties (Elujoba et al., 2005). Tanzanian’s export plant
Agave sisalana P, is used in manufacturing of steroidal drugs like corticosteroids and oral contraceptive. Prunus Africana Hook. F is exported by Cameroon, Kenya and Madagascar is used for prostate gland hyperthrophy (Elujoba et al., 2005).
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OH H N H N Me OH N H Me OH H H OMe O OH 9 O OMe OH Me
HO Me
N H 10 HO Me
1.4 Bacterial infections
Bacteria may be classified as either Gram-negative or Gram-positive according to a staining technique devised by Christian Gram in 1884 (Newton et al., 2002). On staining, those that retain the primary dye color are Gram-positive while those that change the color are Gram- negative. Some examples of Gram-positive are Staphylococcus aureus, Staphylococcus epidermics, Streptococcus pyogennes and Actinomyces odontolyticu. Examples of Gram-negative bacteria include; Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Vibrio cholerae and Bordetella pertussis. The mammalian body provides a favourable environment for growth of numerous microorganisms which results into unfavourable interaction, usually causing diseases
(Newton et al., 2002).
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1.4.1 Staphylococcus aureus
Staphylococcus aureus is a facultative anaerobic, Gram-positive bacterium which is found living as a commensal on skin and often in the nose of healthy people. However, it is one of the commonest causes of acute pyogenic infections in man including boils, carbuncles and abscesses. It is also responsible for highly infectious type of bronchopneumonia (Roberts et al.,
2009). It can also cause food poisoning and its invasion into blood causes septicemia. Although several anti-staphylococcal agents have been discovered, the organism is still a major threat to human health (Newton et al., 2002).
Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium responsible for difficult-to- treat infections in humans. MRSA is by definition a strain of Staphylococcus aureus that is resistant to a large group of antibiotics called the beta-lactams, including methicillin, dicloxacillin and oxacillin (UK Office for National Statistics Online, 2007). This is due to the altered penicillin-binding proteins (Barnes, 2000). MRSA is especially troublesome in hospital- associated (nosocomial) infections. In hospitals, patients with open wounds, invasive devices, and weakened immune systems are at greater risk for infection than the general public (UK
Office for National Statistics Online, 2007).
1.4.2 Escherichia coli
Escherichia coli is Gram-negative bacterium that is commonly found in lower intestine of warm- blooded animals. Most E. coli strains are harmless, but some, such as serotype O157:H7 can cause serious food poisoning in humans. The optimal growth of E. coli occurs at 37oC, but some laboratory strains can multiply at temperatures of up to 49o C (Hudault, 2001). E. coli are not
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always confined to the intestine, and their ability to survive for brief periods outside the body makes them an ideal indicator organism to test environmental samples for faecal contamination
(Hudault, 2001). As Gram-negative organisms, E. coli are resistant to many antibiotics that are effective against Gram-positive organisms. Resistance to beta-lactam antibiotics has become a particular problem in recent decades, as strains of bacteria that produce extended spectrum beta- lactamases have become more common. These beta-lactamase enzymes make many if not all of the penicillins and cephalosporins ineffective as therapy (Paterson and Bonomo, 2005).
1.4.3 Pseudomonas aeruginosa
Pseudomonas aeruginosa is a Gram-negative rod. It is a common bacterium which can cause disease in animals and humans. It is found in soil, water and most man-made environments throughout the world. It thrives not only in normal atmospheres, but also with little oxygen, and has thus colonized many natural and artificial environments. It uses a wide range of organic material for food, in animals; the versatility enables the organism to infect damaged tissues or people with reduced immunity (Cornelis, 2008). Biofilms of P. aeruginosa can cause chronic opportunistic infections. These kinds of infections are a serious problem for medical care in industrialized societies, especially for immune compromised patients and the elderly (Cornelis,
2008).
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1.5 Antibacterial drugs
An antibacterial is an agent that interferes with the growth and reproduction of bacteria.
Antibacterial agents consist of antibiotics and non-antibiotic synthetic compounds such as sulphonamides and quinolones. Antibiotics inhibit growth and survival of microorganisms in small doses.
1.5.1 Tetracyclines
Tetracyclines (11-16) are a group of broad spectrum orally active antibiotics produced by cultures of streptomyces species. Tetracyclines act by inhibiting protein synthesis after uptake by active transport. However, many strains of organisms have become resistant to these agents hence decreasing their usefulness. Tetracyclines are known to chelate calcium and depositing in growing bones and teeth causing staining and sometimes dental hypoplasia. High doses of tetracyclines are also known to decrease protein synthesis in host cells (Barnes, 2000).
3 2 R R N(CH ) 1 3 2 R4 R OH
CONH O O 2 OH HO OH
1 4 2 3 11 Tetracycline R =R =H,R =OH,R =Me 1 2 4 3 12 Doxytetracycline R =OH,R =R =H,R =Me 13 Methacycline R1=OH,R2=H,R3=Me,R4=H 14 Chlorotetracycline R1=H,R2=OH,R3=Me, R4=Cl 15 Oxytetracycline R1=R2=OH,R3=Me,R4=H 1 3 2 4 16 Demecycline R =R =H,R =OH,R =Cl
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1.5.2 Penicillins
Penicillins (17-22) are β-lactam natural antibiotics which are mainly produced by Penicillium notatum and P. chrysogem. The basic nucleus of penicillin is 6-aminopenicillanic acid, where R can be substituted by different groups to give different properties. This is very important in the various staphylococcal strains where their resistance has spread progressively. In developing countries at least 80% of Staphylococci strains now produce β-lactamase (Paterson and Bonomo,
2005). The drug has low permeability of the outer membrane of the organism hence reducing the ability of the drug to reach the target site.
H R N S O N O COOH
Chemical name Other names R
17 Pent-2-enyl penicilln Penicillin I or F -CH2CH CHCH2CH3 18 Benzyl penicillin Penicillin H or G -CH2-Ph
19 p-Hydroxybenzylpenicillin Penicillin III or X -CH2-Ph-OH N-Heptylpenicillin 20 Penicillin IV or K -(CH2)6CH3 21 Phenoxymethylpenicillin Penicillin V -CH2-O-Ph 22 N-Amylpenicillin Dehydro-F-penicillin -(CH2)4CH3
1.5.3 Sulphonamides
The chemically useful antibacterial sulphonamides are derived from sulphonamide by substitution on the amide moiety such as sulphanilamide (23), sulphamethoxine (24) and sulphamethoxazole (25). Sulphonamides have some side effects such as headache, mental depression and vomiting.
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OMe OMe O
H2N SO2NH2 H2N S N N H N 23 H O
O O 24 N H2N S N
H O 25
1.5.4 Flouroquinolones
Flouroquinolones are synthetic antibiotics, which are in clinical practice. These include the broad spectrum drugs such as ciprofloxacin (26), ofloxacin (27), norfloxacin (28), perfloxacin (29) and enoxalin (30). They are used for complicated urinary tract infections in patients with gonorrhoea and bacterial prostatitis (Knowles, 1997). Flouroquinolones as a bacterial drug eradicate bacteria by interfering with DNA replication.
O O O O O O F F OH F OH OH N N N N N N HN N N 26 28 27 O O O O F F OH OH N N N N N HN HN 30
29
14
Flouroquinolones are generally well tolerated with most side effects being mild and serious side effects being rare. Some of the serious side effects common with these antibiotics include cardiac arryhythmias and tendon toxicity (Ball et al., 1999; Owens and Ambrose, 2005).
1.5.5 Quinolones
Quinolones such as nalidixic acid (31) inhibit the bacterial DNA gyrase or the topoisomerase IV enzyme, inhibiting DNA replication and transcription. Quinolones can enter cells easily via porins and therefore are often used to treat intracellular pathogens such as Legionella pneumophila and Mycoplasma pneumoniae. For many Gram-negative bacteria DNA gyrase is the target, whereas topoisomerase IV is the target for many Gram-positive bacteria (Ball et al.,
1999; Owens and Ambrose, 2005).
O COOH
Me N CH CH 31 2 3
1.6 Fungal infections
Fungi are classified in the kingdom fungi, which includes mushroom, yeast, slime moulds, rust and smut. The most common fungi responsible for systemic infections in humans are the species of Candida including C. albicans, C. tropicalis, C. guillermond and C. parapsilosis (Newton et al., 2002). With few exceptions, pathogenicity among fungi is not necessary for the maintenance or dissemination of the species. The ability of fungi to cause human disease appears to be an
15
accidental phenomenon and the mycoses are primarily related to the immunological status of the host and environmental exposure, rather than to the infecting organism (Cleveland et al., 2012).
Most fungi are unable to grow at 37oC. Thus, thermo- tolerance to survive at this temperature is essential for fungal growth within human body. Similarly, as most fungi are saprophytic, their enzymatic pathways function better at the redox potential of non-living substrates than at the lowered oxidation-reduction state of living tissue. However, many fungi prove to be able to surpass these two major physiologic barriers (Ellis, 1999).
Host defenses are of non-specific and specific in nature. The non-specific defenses include the antifungal activity of natural excretions, such as saliva and sweat; the protective effects of the endogenous normal micro- biota of the skin and mucous membranes in competing for space and nutrients, thus limiting the growth of potential pathogens, and the mechanical barrier of the mucous membranes preventing entry of fungi (Cleveland et al., 2012). Additionally, the body has the highly efficient non-specific inflammatory system to combat fungal proliferation involving the action of neutrophils, mono-nuclear phagocytes and other granulocytes. The specific or acquired immunity defending from fungal growth in tissue consist basically of the cell-mediated immunity regulated by T-lymphocytes. The role of specific antibodies or humoral immunity regulated by B-lymphocytes is not so clear (Ellis, 1999). The spectrum of fungal infections is different according to the major deficit in host defenses.
16
1.7 Antifungal drugs
The last two decades have witnessed a dramatic rise in the incidence of life threatening systemic fungal infections. The challenge has been to develop effective strategies for the treatment of candidiasis and other fungal diseases, considering the increase in opportunistic fungal infections in human immunodeficiency virus-positive patients and in others who are immune compromised due to cancer chemotherapy and the indiscriminate use of antibiotics (Myers, 2006).
Majority of the clinically used antifungals have various drawbacks in terms of toxicity, efficacy and cost, and their frequent use has led to the emergence of resistant strains. Additionally, in recent years public pressure to reduce the use of synthetic fungicides in agriculture has increased.
Concerns have been raised about both environmental impact and the potential health risk related to the use of these compounds in the past few decades. A worldwide increase in the incidence of fungal infections has been observed as well as a rise in the resistance of some species of fungus to different fungicides used in medical practice (Graybill, 1989; Vanden, 1994).
1.7.1 Flucytosine
Flucytosine (32) is a synthetic antifungal agent, which is active only to a few organisms mainly those caused by yeast (Candida). When administered on its own, resistance is likely to develop; hence it is usually combined with amphotericin to severe infections such as Cryptococcal meningitis. Other antifungal drugs include diazole (33) and Griseofulvin (34) (Myers, 2006).
17
NH N O 2 O F Cl O N N Cl O O N H O O Cl 32 Cl 34 Cl 33
1.7.2 Amphotericin
Amphotericin (35) is a macrolide antibiotic of complex structure and many carbon atoms. It acts by binding on the cell membranes hence interfering with permeability and transport functions.
When administered orally amphotericin is poorly absorbed though it is the only route into the gastrointestinal tract. The commonest and most serious side effects of amphotericin are renal toxicity, potassium loss, fever and chills. Some degree of reduction of renal function occurs in more than 80% of patients using the drug during the time of use (Schoffski et al., 1998).
OH OH OH O
O OH OH OH OH OH
O O
35 OH OH NH2
18
1.8 Statement of the problem
Infectious diseases are the world's leading cause of premature deaths, killing almost 50,000 people every day; especially bacterial infections which has remained a large public health issue and approximately 50-75% of hospital deaths are reported to be due to secondary infections
(Mokaddas et al., 1998). Another important factor is that drug resistance to human pathogenical bacteria has been increasing not only in the developing countries but throughout the world due to indiscriminate use of antibiotics. The drug resistance bacterial and fungal pathogens have further complicated the treatment of infectious diseases in immune compromised AIDS and cancer patients. In the present scenario due to emergence of multiple drug resistance to human pathogenic bacteria and fungi, especially the antibiotic penicillins, cephalosporins and chloromphenical types involve the enzymatic inactivation of the antibiotic by hydrolysis or by the formation of an active derivative (Myers, 2006). This has opened a new vista for the search of new antimicrobial substance.
Search for newer drugs from plant has been increasing day by day due to the emergence of new diseases and alarming side-effects of synthetic drugs. Several plant species have been used by ethnic groups for the treatment of various diseases like dysentery, skin diseases, asthma, malaria, and a horde of other indications (Dahanukar et al., 2000; Perumal and Ignacimuthu, 2000).
Natural products of higher plants give a new source of antimicrobial agents. This study presents the antibacterial and antifungal screening of the stem bark of Bersama abyssinica.
19
1.9 Hypotheses
(i ) Bersama abyssinica contain constituents with antibacterial and antifungal
activities.
(ii) The constituents remain bioactive once isolated from the plant source.
1.10 Objectives of the study
1.10.1 General objective
The study was aimed at extraction, isolation and characterization of bioactive compounds with antibacterial and antifungal activities in Bersama abyssinica.
1.10.2 Specific objectives
(i) To evaluate antimicrobial properties of crude extracts of the stem bark of B.
abyssinica against Salmonella typhi, Shigella dysentriae, Vibrio cholorae,
diarrheagenic Escherichia coli, Candida albicans, Penicillium notatum, Klebsiella
pneumoniae, Pseudomonas aeruginosa and Bacillus subtilis.
(ii) To characterize the isolated compounds from the extracts using physical,
chemical, chromatographic and spectroscopic data.
(iii) To establish the bioactivity of the isolated compounds against Salmonella typhi,
Shigella dysentriae, Vibrio cholorae, diarrheagenic Escherichia coli, Candida
albicans, Penicillium notatum, Klebsiella pneumoniae, Pseudomonas aeruginosa
and Bacillus subtilis.
20
1.11 Justification and significance of the study
Most population in sub-Saharan Africa depends on traditional medicine for their primary healthcare (Kokwaro, 1996) and many of the drugs which are in use today were discovered through their ethno botanical route. This makes it necessary to have research on medicinal plants so as to obtain more potent pharmacological agents (WHO, 2000). Bersama abyssinica species are used by Kamba and Kipsigis communities in Kenya for traditional therapy of antimicrobial infections has not been fully investigated phytochemically for antibacterial and antifungal agents on the basis of literature search. This research study undertook phytochemical and biological investigations of the stem bark of Bersama abyssinica. This is because the stem bark is the most commonly exploited part in this species.
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CHAPTER TWO
LITERATURE REVIEW
2.1 The family Melianthaceae
The Melianthaceae family consists of two genera; Melianthus and Bersama. Melianthus consists of 8 species and a genus of shrubs endemic to southern Africa (Burkill, 1997). Melianthus major is a striking plant that has large, light blue-green pinnate leaves with toothed margins. Long stems support extended inflorescences of numerous reddish flowers above the leaves. Melianthus is well adapted to accommodate birds that rest on the strong stems and collect pollen as they drink nectar from within the flowers. In its native South Africa, Melianthus major is found in both the winter and summer rainfall areas of the country where it favours wetter soils. The genus
Bersama comprises of four species namely; B. swinnyi, B. yangambiensis, B. engleriana and B. abyssinica (Burkill, 1997). Trees and shrubs belonging to the genus Bersama are spread throughout tropical and sub-tropical Africa, and various parts of the plant such as the leaves, bark, roots and seeds have been used for medicinal purposes (Burkill, 1997).
2.1.1 Bersama yangambiensis Toussaint
Bersama yangambiensis is a tree that occurs in tropical forest of Africa. It is used to treat a range of diseases for example stomach infections and cancer related illnesses (Heywood, 1993).
Phytochemical investigations on this species led to the isolation of triterpenoid saponins, flavonoids and xanthones. The triterpenoids were found to exhibit antimicrobial activity for
Vibrio cholerae and Salmonella typhi. Bufadienolide glycosides (36 and 37) and bufadienolide
(38) were isolated which had cardiotonic and antiturmor properties (Mikkelsen and Seberg,
2001).
22
OH CH3 H C 3 O O R 36 :R=TWO ALDOHEXOSE 37 :R= ONE ALDOHEXOSE 38 :R=H
2.1.2 Bersama engleriana Gurke
Bersama engleriana Gurke is a tree that occurs in forests and forest margins of tropical and subtropical Africa. This species is used to treat a range of diseases by various communities in
Africa. Phytochemical studies revealed the presence of five 3-O-glucuronide triterpene saponins isolated from the stem bark of B. engleriana along with two known saponins, and one major C- glycoside xanthones; mangiferin (39). The structures of the saponins were established mainly by means of spectroscopic methods (one- and two-dimensional NMR spectroscopy as well as FAB-,
HRESI-mass spectrometry) as 3-O-[β-d-glucopyranosyl-(1 → 2)-β-d-glucuronopyranosyl]-28-O-
[β-d-gluco-pyranosyl]-betulinacid (40); (3-O-[β-d-glucopyranosyl-(1 → 2)-[β-d-galactopyranos- yl-(1 → 3)]-β-d-glucuronopyranosyl]-oleanolic acid (41); 3-O-[β-d-glucopyranosyl-(1 → 3)-β-d- glucurono-pyranosyl]-28-O-[β-d-xylopyranosyl-(1 → 6)-β-d-glucopyranosyl]-oleanolic acid (42)
; 3-O-[β-d-galactopyranosyl-(1 → 3)-β-d-glucuronopyranosyl]-28-O-[β-d-glucopyranosyl-(1 →
4)-β-d-gluco -pyranosyl]-oleanolic acid (43) and 3-O-[β-d-glucopyranosyl-(1 → 3)-β-d- galactopyranosyl-(1 → 3)-β-d-glucuronopyranosyl]-28-O-[β-d-xylopyranosyl-(1 → 6)-β-d-gluc- opyranosyl]-oleanolic acid (44) (Tapondjou et al., 2006).
23
CO
O HO O HOOC O O HO HO HO OH Glc.A O HO Glc.2
OH O 40 OH HO HO Glc.1
COOR2
R1O
R2 R1 H O 41 Glc- (1 2)[Gal-(1 3)]-GlcA- HO OH 42 Glc-(1 3)-GlcA- Xyl-(1 6)-Glc- 43 Gal-(1 3)-GlcA- Glc-(1 4)-Glc- HO O OH 44 Glc-(1 3)-Gal-(1 3)GlcA- Xyl-(1 6)-Glc- OH OH O
HO HO 39
2.1.3 Bersama swinnyi Phil
Bersama swinnyi Phil is a tree that occurs in forests, on forest margins and sandstone outcrops of
eastern South Africa. It is one of the most widely used medicinal plants by the Zulu of KwaZulu-
Natal and is becoming rare outside nature reserves. The stem bark is used traditionally by the
Zulu to treat barrenness, impotence, menstrual pain, leprosy and as a protective charm
(Hutchings et al., 1996).
24
Phytochemical investigations on this species led to isolation of Swinniol (3β, 23-dihydroxylup-
20(29)-en-28-al) (45), a triterpenoid, along with lupeol (46) and betunal (47) from the chloroform extract of the bark of Bersama swinnyi (Thabo et al., 1998). Oleanolic acid (48) was isolated from the leaves (Monkhe et al., 1998).
CHO H H H O HO H HO CH2OH HO H H 45 46 47
OH
COOH
HO 48
2.1.4 Bersama abyssinica Fresen
Bersama abyssinica Fresen is a species of medium-sized evergreen tree in the Melianthaceae family. The leaves are pinnately divided with a strongly winged rachis (hence the common name
Winged Bersama (Heywood, 1993). The inflorescence is a spike (figure 1.2). This species is distributed across sub-Saharan Africa (Mikkelsen and Serberg, 2001).
25
Figure 2.1: Flowering branch of Bersama abyssinica (Londian forest, Feb. 2011)
2.1.4.1 Biological and pharmacological activities of Bersama abyssinica
All parts of Bersama abyssinica are poisonous when ingested and have been implicated in killing human and livestock. For internal use, the dosage is therefore critical (Burkill, 1997). Bark, leaf and root decoctions are widely taken as purgative to treat a range of stomach disorders such as abdomen pains, colic, diarrhea, cholera, intestinal worms, amoebiasis and dysentery. Rabies, syphilis, gonorrhea and malaria are also treated by these decoctions. A stem bark decoction is drunk to cure cancer and rheumatism (Makonnen and Hagos, 1993).
26
As an aphrodisiac, powdered stem bark or leaves is added to beer. A stem bark poultice is applied on the back, a leaf decoction is drunk to cure lumbago, stem bark and leaves decoction is used to treat diabetes mellitus. Leaf decoctions are also taken to treat feverish pains, loss of appetite, delibility, jaundice diarrhea and leprosy. Extracts of growing shoots are used for external treatment of burns, ulcer and to clean wounds. To treat convulsions and snake bites, leaves are pounded and mixed with water and the mixture is drunk and applied the body (Edeoga et al., 2005). The root bark infusion is drunk, leaf sap is applied as eye drops or leaf powder is sniffed to treat migraine, headache and colds. A root decoction is used to treat hemorrhoids and epilepsy (Taniguchi and Kubo, 1993). Shoots and leaves are pounded and used to control stalk borers in maize. An aqueous stem bark crude extracts showed activity against Candida albicans
(Makonnen and Hagos, 1993). Bersama abyssinica, species was reported to possess anthelmintic, antitumour (Burkill, 1997) purgative, pesticidal and cardiogenic activities.
Antispasmodic use of the plant is one of the claimed traditional applications (Asres et al., 2001).
Cardiac glycosides and unsaturated sterols were identified in tests in Ethiopia on stem bark and root bark. Leaf extracts have cardiogenic, spasmolytic and hypoglycaemic activities. Crude stem bark extracts slow down growth of Bacillus cereus, Staphylococcus aureus, Shigella flexineri and Shigella dysentriae, a root bark extract slows down that of Bacillus subtilis (Zekeya et al.,
2014). An aqueous stem bark extract showed antispasmodic effects on isolated guinea-pig ileum.
A methanolic leaf extract had an inhibitory effect on HIV-1 replication (Makonnen and Hagos,
1993; Ngemenya et al., 2005). Much of the work on the species B. abyssinica have been stimulated by the reported three hellebrigenin acetates isolated from the bark of that species with anti-tumour activities . Since that time several other acetate and orthoacetate derivatives have
27
been reported. Bersama abyssinica was reported to possess anthelmintic, antitumour, purgative
(Ngemenya et al., 2005) and cardiogenic activities.
2.2 Biosynthetic pathway of anthraquinone
Biosynthetic pathway leading to anthraquinones; Rings A and B of AQs are derived from chorismic (or isochorismic) acid and α-ketoglutarate via o-succinylbenzoic acid (Leistner, 1985).
The origin of ring C is either via the MVA or the MEP pathway (El-Gamal et al., 1995).
28
Shikimate pathway MVA pathway MEP pathway
COOH CHO Glucose CHO + COOH O SCoA O + OP pyruvate OP AcetylCoA OP HO glycealdeh-yde OH TCA +TPP PEP O CO2 E-4-P HO
shikimic acid SCoA COOH O 3-hydroxyl-3-methyglutaryl CoAl OP chorismic acid OH 1-deoxy-D- xylulose 5-P
COOH HOOC O HO HO OH OH OP O COOH + COOH COOH MVA OH OH MEP O-ketoglutaric acid Isochorismic acid
OH +TPP CO2, PEP COOH
+ COOH O COOH 1,4-Dihydroxy OPP -2-naphtoic acid OPP
IPP DMAPP O O-succinylbenzoic acid
O OH O OH IPP +
O O 2,3,4-trimethyl-8-hydroxy-9,10-anthraquinone
Scheme 2.1: Biosynthesis of anthraquinone (Leistner, 1985; El-Gamal et al., 1995; Dewick,
2004).
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2.3 Biosynthesis of lupeol
Lupeol is formed when two molecules of farnesyl pyrophosphate (FPP) are joined tail to tail to form squalene. Squalene undergoes multiple cyclizations because of its six double bonds.
Cyclization starts with the formation of an incipient carbon cation at the tertiary carbon of the end double bond of squalene. The cation undergoes many transformations mostly by shifting hydride ions and methyl groups before stabilizing through expulsion of a proton (Dewick, 2004).
Squalene O2 NADPH
Enzymatic + cyclisation protosteryl cation
HO NADPH O squalene epoxide Wagner-Meerwein 1, 2 alkyl shift
+
+
H
H H HO HO baccharenyl cation H H Lupenyl cation -H+
HO lupeol
Scheme 2.2: Biosynthesis of lupeol from squalene (Dewick, 2004).
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2.4 Biosynthetic pathway of steroids
The natural steroids are derived by a series of chemical transformations from two parent triterpenes; lanosterol and cycloartenol. The biosynthesis of all natural steroids is believed to proceed from acetic acid to lanosterol or cycloartenol through mevalonic acid and squalene. It is generally recognized that all animal steroids originate from lanosterol, whilst cycloartenol is the precursor of plant steroids. The biosynthesis of sitosterol and stigmasterol is summarized in the scheme below.
O2 Squalene NADPH
Enzymatic cyclisation cycloartenol
HO O
NADPH squalene epoxide
HO HO Sitosterol Stigmasterol
Scheme 2.3: Biosynthesis of stigmasterol and sitosterol from squalene (Swartz, 2006)
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2.5 Biosynthetic pathway of oleanolic acid
The first committed step in triterpenoid biosynthesis is the cyclization of 2,3-oxidosqualene. This reaction is catalyzed by specific oxidosqualene cyclases (OSCs), including β-amyrin synthase
(bAS), and has been functionally characterized in several plants (Dewick, 2004). Subsequent modifications that impart functional properties and diversify the basic triterpenoid backbone include the addition of functional groups including carboxyl. In this way, β-amyrin 28-oxidase catalyzes three sequential oxidation reactions at C-28 of the oleanane backbone.
Squalene O2 SQE NADPH
Enzymatic cyclisation beta Amyrin bAS HO
+ bAS O squalene epoxide CPR NADPH
COOH
HO oleanolic acid
Scheme 2.4: Biosynthesis of oleanolic acid from squalene (Dewick, 2004)
32
CHAPTER THREE
MATERIALS AND METHODS
3.1 General procedures
Hexane, dichloromethane (DCM), ethyl acetate (EtOAc) and methanol (MeOH) used in this study were of laboratory grade (general purpose), hence they were freshly distilled before use.
Dimethylsulphoxide (DMSO), sulphuric acid, acetic acid and p-anisaldehyde were of analytical grade. All these chemicals were purchased from Avon Chem., Nairobi, Kenya. The glassware used was soaked in chromic acid overnight, rinsed with tap water and finally dried in an electric oven at 1100C for 1 hour.
3.2 Plant material and preparation
The stem bark of Bersama abyssinica was collected under supervision of a taxonomist, from
Kericho County, Londian forest, between the towns of Kericho and Nakuru. Reference materials for Bersama abyssinica were placed in Kenyatta University Herbarium, voucher specimen number AO/025/15/04/2007. The plant materials were dried in air under the shade and then, they were ground to a fine texture using the grinding mill (Christy and Norris Ltd: Chelmsford,
England). The powdered plant part was weighed using a top-loading analytical balance (Denver instrument, Golorado, USA) and extracted sequentially using n-hexane, dichlomethane, ethyl acetate and methanol.
33
3.3 Chromatographic techniques
3.3.1 Column chromatography
Column chromatography was carried out using glass columns of internal diameter 3.0cm and of length of 0.8m. Silica gel (Merck, 70-230 mesh/0.63-0.2mm) was used and slurry packing method was employed. Vacuum liquid chromatography was also used with silica gel 60G
(0.040-0.060mm Merck, Germany). Further purification was done using a filter gel sephadex
LH-20. Packing was by slurry method.
3.3.2 Spray reagents
p-Anisaldehyde spray used was made by mixing p-anisaldehyde (0.5ml), acetic acid (10ml), chilled methanol (90 ml), and concentrated sulphuric acid (5ml) (Krishnaswany, 2003). This was used for detection of flavanoids and terpenoids. Sulphuric acid 5% on methanol was made by slowly adding 5ml concentrated sulphuric acid into distilled (95ml methanol (Krishnaswany,
2003). Larger quantities of the reagent were obtained by increasing the ratios accordingly. This was for detection of terpenoids.
3.3.3 Detection of terpenoids
Analytical pre-coated TLC plates on aluminium sheets were used throughout the present study for establishment of optimum solvent systems, separations, complexity of the extracts and purity of isolated compounds. Spots on the chromatograms were detected under UV light at 254 and
365nm by p-anisaldehyde and ammonia. Terpenoids would give blue fluorescent in UV 365nm and turn purple when plates are sprayed with anisaldehyde and then heated at 110oC for about 10 minutes. However, most terpenoids did not fluoresce in UV.
34
3.4 Bioactivity screening of the plant extracts
Antimicrobial activity of the plant species involved was undertaken in two phases: screening of crude extracts to detect the presence or absence of activity. This was done against a number of bacteria and fungi strains as described by Radovanovic et al. (2009). Screening of isolated pure compounds to determine their activity was done against the antimicrobial strains that gave positive response in the primary screening.
3.4.1 Anti-bacterial activity test
This was carried out in vitro using disc diffusion method. Small, sterile discs of filter papers (5 mm) were used as carriers of antibiotics and/ (or) crude extracts solutions to be tested. The test organisms (Gram-positive and Gram-negative) used in the study are listed in Table 3.1. These were obtained from Botany Department, Kenyatta University, Nairobi. The test bacteria were prepared by culturing the required bacterium in nutrient broth medium from stock cultures and later were transferred onto the nutrient agar in the Petri dishes (Clinical and Laboratory Standard
Institute, 2013). Table 3.2 gives a list of the standard antibiotics used as reference drugs.
Table 3.1: Bacteria strains used in the bioassay
Name (code) ATCC number Type Staphylococcus aureus (Sa) 25923 Gram-positive Bacillus subtilis (Bs) 202638 Gram-positive Escherichia coli (Ec) Local isolate Gram-negative Pseudomonas aeruginosa (Pa) 10622 Gram-negative Vibrio cholera (Vc) 14035 Gram-negative Klebsiella pneumoniae (Kp) 93883 Gram-negative
35
Table 3.2: Standard antibiotics used as reference drugs Standard drug name Abbreviations Weight μg/discs
Amplicillin AMP 25
Tetracycline TET 25
Kanamycin KAN 30
μg/discs: amount of the drug in the disc
3.4.1.1 Preparation of the nutrient agar media and growing of bacterial cultures
Nutrient agar 28g (Oxoid Ltd. Basingstoke, England) was dissolved in distilled water to make one litre of the solution and placed in an autoclave at 121 - 1240C and 15psi pressure for 30 minutes. Portions of sterilized nutrient agar medium (15ml) were dispensed into 90mm diameter pre-sterilized Petri-dishes to yield a uniform depth of 40mm under septic conditions in a laminar flow. The Petri dishes were covered and allowed to cool at room temperature undisturbed until the culture media hardened. They were then incubated at 37 - 390C for 24 hours in an inverted position to test their sterility. Using a sterile wire loop under septic conditions, bacteria cultures from stock cultures were scooped and spread on the nutrient agar surface and incubated aerobically at 37 - 390C for 24 hours (Radovanović et al., 2009).
Nutrient broth powder 13g, (Oxoid Ltd. Basingstoke England) was dissolved in distilled water to make one litre of solution. Portions of the solution (25ml) were dispensed into bijou bottles and steam sterilized in an autoclave instrument at 121 - 1240C and 15 psi pressure for 20 min. on cooling, one loopful of the bacterial strain from the 24 hours culture was added into the sterile nutrient broth medium and incubated at 37 - 390C for 24 hours in a rotator shaker (Radovanović
36
et al., 2009). The 24 hours broth bacteria culture (0.1ml) was pipette into the nutrient agar media in the Petri dishes and spread evenly suing a sterilized L-shaped glass rod under sterile conditions.
3.4.1.2 Introduction of the crude plant extract into the inoculated Petri dishes
Antimicrobial efficacies were tested using the filter paper disc diffusion method (Buwa and Van
Staden, 2006). A solution of each extract was prepared by dissolving 50mg per ml of the crude extract in DMSO and 10μl of the solution were dispensed on 6mm sterile absorbent filter paper discs (500µg/disc). Some other discs were dipped in DMSO alone for use as a negative control.
All discs were removed and dried in the oven at 500C for about an hour to expel the solvent. The dry impregnated filter paper discs containing 500µg/disc of the extract were then firmly placed on the inoculated Petri dishes using sterile forceps under sterile conditions. They were then pressed down with a slight pressure to ensure complete contact of the disc with the inoculated agar surface and incubated at 370C aerobically in an inverted position. The zones of inhibition (if any) were measured after 24 and 48hours in triplicates. Common antibiotics (amplicillin, tetracycline and kenamycin) were used as standard for comparing with the plant extracts by noting their activity against the bacterial strains used (Chhabra and Uiso, 1991; McChesney et al., 1991).
3.5 Antifungal activity test
Agar-well plate diffusion method was used. Two fungal species, Candida albicans and
Penicillium notatum that affect human beings were obtained from the Botany Department,
Kenyatta University, Nairobi. Commercial potato dextrose agar powder 39g (Himedia
37
laboratories, Pvt. Ltd., Bombay) was dissolved in distilled water to make a litre of the solution followed by steam sterilization in an autoclave at 1200C and 15psi pressure for 20min. on cooling to 500C, portions of this solution (15ml) were dispensed into sterile Petri dishes under sterile conditions and left to solidify. This provided the medium for growing the fungal spores.
3.5.1 Preparation of the drugs
Each crude extract (1mg) was weighed and dissolved in DMSO (50μL) and the solution made to
1ml using methanol. This gave a stock solution of 1,000 ppm used for initial test. Pure culture for the concerned fungus was made on the PDA surface in the Petri dishes from stock culture and incubated to 300C for seven days to produce a good crop of spores.
The fungal inoculums was prepared by harvesting the spores from the crop of spores with a bent spore-harvesting needle in a sterile tube containing sterile distilled water (Radovanovic et al.,
2009). The suspension (0.5ml) was pipetted onto the PDA medium in the Petri dishes. The plate was then tilted several times to spread the inoculums and left still for about 10 min. Using a sterile cork borer (6mm) 14 agar wells were cut out in the inoculated PDA medium. The drug
(0.1ml) of 25µg/ml was pipetted into each of these wells in triplicates. The Petri dishes were then covered, sealed and kept aerobically at 300C for 72-96 hours. At the end of the incubation period the diameter of zone of inhibition produced around the agar wells (if any) were measured with a transparent measuring scale for three consecutive days. Sterile distilled water and the solvent mixture in the ratios used to prepare the drugs being screened were used as controls
38
3.6 Extraction procedure for the stem bark of Bersama abyssinica
Based on the results obtained from preliminary screening the amount of extract obtained was isolated and characterized as shown in the Scheme 3.1 that follows.
Stem bark 4kg (4000g)
n-Hexane 2 days x2
Hexane extract (4g) Residue
DCM 2 days x2
DCM extract Residue (16.5g) Ethyl acetate 2 days x2
Ethyl acetate Residue extract (63 .7 g)
Methanol 2 days x2
Methanol extract Residue (82.1g)
Scheme 3.1: Sequential extraction of Bersama abyssinica Fresen
39
3.7 Instrumentation
3.7.1 Melting point
Uncorrected melting points were recorded with open capillary tubes using Gallenkamp melting point apparatus (Sanyo, West Sussex, UK).
3.7.2 Ultraviolet (UV)
Visualization of spots on a developed plate was done using long and short wave lengths (365 and
254nm, respectively) on an ENF-240 C/F UV lamp (spectronics Co., Westburg).
3.7.3 Nuclear magnetic resonance (NMR) spectroscopy
1H (1D, 2D, NOESY and 13C spectra were recorded using Varian Gemini 200 and 600 MHz (1H
13 NMR) and 150 MHz ( C NMR) machine using CD2Cl2 as a solvent. This was done in Germany.
In addition, 1H NMR, 13C NMR and 2D NMR were recorded on Bruker Advance at 400MHz for
1 13 HNMR and C NMR at 100 MHz using CDCl3 and DMSO-d6 as solvents. These analyses were done in South Africa at Kwa Zulu Natal University. 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 a 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 CDCl3, in a siglet sample tube and mixed thoroughly. The solution was transferred into NMR tube and spectrum recorded.
40
3.7.4 Gas chromatography linked with MS
The component separation of the crude extracts of MeOH and DCM were determined using GC system (GC-800 series) with fused silica capillary column (15m length, 0.25mm internal diameter and 0.25µm film thickness). Static phase methylsilicone (SE-30) directly coupled to quadrupole M/S (Hewlett Packard 5973). Electron Impact Ionization was carried out with ionization energy of 70eV. Inert gas helium was used as a carrier gas at constant flow rate of 1 ml/min. Mass transfer line and injector temperatures were set at 200 and 2500C, respectively.
The oven temperature was programmed starting from 60 to 1500C. Then held isothermally for 15 min and finally raised to 2500C at a rate of 150C/min. The instrument was scanned at a mass range of 60 to 400 a.m.u. The crude samples were diluted with appropriate solvent (1/100, v/v) and filtered. The particle-free diluted crude extracts were taken in a syringe and injected into injector with split mode. The split ratio was of 1:120. The organic chemical compounds were identified and characterized in various crude extracts was based on GC retention time. The mass spectra were computer matched with those of standards available in existing computer library
(Mainlab, Replib and Tutorial data of GC –MS system). This analysis was done at the Kenya
Bureau of Standard laboratories in Kenya. This analysis was done for preliminary studies.
3.8 Isolation of compounds from plant extracts
A combination of chromatographic techniques: VLC, CC, and TLC were used. The different solvent extracts of the stem bark were separately fractioned by VLC on Kieselgel silica gel 60G
(0.040-0.060) mm, Merck, Germany. The solvent system was varied gradually by increasing the more polar solvent starting with n-hexane. DCM was eventually added until 100% DCM. This was further polarized using MeOH up to 20% MeOH in DCM.
41
Further purification was achieved by use of column chromatography, on silica gel 60 (0.63-
0.2mm/70-230mesh. Mesh ASTM, Merck, Germany) and eluted with a slow gradient of solvent system. Packing of both types of column was done using slurry method with silica gel suspended in the least polar solvent (n-hexane). 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 and ground into a fine powdery form and air dried to remove the solvent. This powder was then applied at the top of the column and finally covered by a small amount of the CC silica gel to minimize disturbance of the sample when eluting solvent is applied.
Analytical pre-coated plastic (PolyGram R silica G/UV 254) and aluminum sheets (Alugram R sil G/UV 254, MACHERY- Nagel GmbH and Co., Germany). The plates were used throughout the purification process. These were mainly for establishment of optimum solvent systems for separations, complexity of extracts and purity of isolated compounds. Sports on the chromatogram were detected under UV light at a λ 254 and 366nm for UV active compounds and visualized upon development by separately spraying p-anisaldehyde as well as 5% sulphuric acid in methanol and heating for 10 minutes at 1100C in an oven. Fractions that showed homogeneity were combined and concentrated together to give pure compounds or semi-pure compounds for further purification. Sephadex columns were used during these purifications. The
Sephadex (LH-20) columns were run using 1:1 (DCM: methanol). The specific chromatographic techniques used are shown in the Schemes 3.2 and 3.3.
42
Column chromatography was used to fractionate 16.5g of the Bersama abyssinica DCM extract.
The fractions were combined depending on the spots they gave on a TLC. The fraction obtained with DCM: Hexane (20:80) was further fractionated using a column chromatography using
DCM: hexane (70:30), this gave the first and second fraction which were combined and fractionated in a column of smaller diameter. This gave fraction three of the small column which was finally recrystallized in methanol to yield 10mg of compound (TN3) and further recrystallization of the fraction in acetone yielded 16mg of compound TN5. The fraction obtained with DCM: hexane (70:30) was fractionated using column chromatography and fractions 70-100 showed two spots on a TLC. The compounds in this fraction were separated through preparative thin layer chromatography to yield 16mg of compound (TN6) and 10mg of compound (TN4). DCM extract obtained from the fraction of DCM: hexane (40:60) was further separated in small column. The TLC analysis showed three spots on the fraction that was further subjected to sephadex column and resulted 10mg of the compound (TN1) was obtained.
3.9 Fractionation of the extract
The DCM extract was fractionated in the column starting with 100% hexane and increasing the percentage of DCM at the rate of 5% till 20% DCM: hexane. Then the percentage increased to
10% uniformly until 100% DCM. This extract resulted to five pure compounds as shown in the
Scheme 3.2 below.
43
DCM extract 16.5g
40% DCM: Hex. 70% DCM: Hex. 20% DCM: Hex.
Fraction 3 and PTLC Fraction 6 and 7 Fraction 1 and 2 (1- 4 (59-70, 2g) (70-100, 1.2g) 50, 2g)
Small column PTLC Small column (50cm height and 50mm diameter) Fraction 3 and 4 (59 - 70, 2g) 70%DCM: Hex. Compound TN4 1:1 MeOH: DCM Fraction 3 Sephadex column (30-70, 1.3g)
Compound TN1
Recrystallization
Compound TN6 By MeOH
By acetone
Compound TN5 Compound TN3
Scheme 3.2: Chromatographic separation of DCM crude extract of the stem bark of Bersama abyssinica.
44
The methanol extract was gummy and yielded 82.1g which was further re-extracted by 200ml of
DCM three times and 200ml of ethyl acetate three times separately. The DCM and ethyl acetate extracts were spotted on TLC plates and the spots that developed were similar. This necessitated combination of the two extracts from methanol which yielded 8g and separated by column chromatography. The fraction obtained at 40:60 (DCM: hexane) were combined and separated through PTLC which yielded 8mg of compound (TN2) as shown in the Scheme 3.3 below.
Methanolic extract 82.1 g
DCM (200 Ml ×3)
Filtration
Residue DCM extract
40% DCM in hexane
VLC
Compound TN2 Fraction 10 (56-59) PTLC
Scheme 3.3: Chromatographic separation of the methanol crude extract of stem bark of Bersama abyssinica
45
3.10 Physical and spectroscopic data
3.10.1 Physical and spectral data of compound TN1 (Appendix 1a and 1b)
White needle like crystals from DCM extract with uncorrected melting point of 182-1840C; Rf of
1 0.6 (1:1) Hexane: DCM. H NMR (CD2Cl2, (ppm), 600 MHz) δ 10.36 (1H, s), 11.95 (1H, s),
12.50 (1H, s), 12.56 (s), (1H, s), δ 6.41 (1H, s) and 6.52 (1H, s), δ 3.89 (3H, s), δ 1.99 (3H, s),
13 2.42 (3H, s) and 2.61 (3H, s). C NMR (CD2Cl2, (ppm), 150 MHz) δ 194 (C-9), 194 (C-10),
172.5 (C-3), 169.9 (C-8a), 169.2 (C-7a), 167.9 (C-14), 162.9 (C-7), 162.9 (C-11), 153 (C-6),
152.3 (C-5), 140.2 (C-4), 116.9 (C-8), 116.2 (C-1), 112.9 (C-13), 110.5 (C-12), 108.7 (C-2),
103.3 (C-9a), 103.3 (C-10a), 52.6 (C-17), 29.9 (C-16), 25.6 (C-15), 23.9 (C-18).
3.10.2 Physical and spectral data of compound TN2 (Appendix 2a and 2b)
White amorphous powder from MeOH with uncorrected melting point of 284-2860C; Rf of 0.65
(8:2) DCM: MeOH); 1H NMR (DMSO-d6, (ppm), 400 MHz) δ 5.36 (1H, s), δ 3.70 (m), δ 4.2
(d), J = 1.30 Hz (3H), δ 0.98 (3H, s), δ 0.90 (9H, s), δ 0.80 (3H, s), δ1.5 (m), δ 2.9 (d), δ 3.0
13 (m). C NMR (DMSO-d6, (ppm), 100 MHz). δ 12.2 (C-29), 12.3 (C-18), 19.1 (C-26), 19.4 (C-
19), 19.6 (C-21), 20.2 (C-27), 21.1 (C-11), 23.1 (C-28) 24.3 (C-15), 26.0 (C-23), 28.3 (C-16)
29.2 (C-25), 31.2 (C-7), 31.9 (C-2), 32.0 (C-22), 33.9 (C-8), 36.0 (C-10), 36.7 (C-20), 37.3 (C-
1), 38.8 (C-12), 42.3 (C-13), 45.7 (C-4), 45.7 (C-24), 50.1 (C-9), 56.0 (C-17), 56.7 (C-14), 62.0
(C-6’), 70.6 (C-3), 77.2 (C-4’), 77.4 (C-2’), 74.0 (C-5’), 77.3 (C-3’), 101.3 (C-1’), 121.7 (C-6),
141.0 (C-5).
46
3.10.3 Physical and spectral data of compound TN3 (Appendix 5a and 5b)
White feather like compound from DCM with uncorrected melting point of 168-1700C; Rf of
1 0.66 (100% DCM); H NMR (CDCl3, (ppm), 400 MHz) δ 5.15 (1H), δ 5.09 (1H, d, J = 8.5 Hz),
13 δ 5.33 (1H, d, J = 3.2 Hz), 3.51 (1H, m), δ 1.01, 0.91, 0.85, 0.81, 0.78, 0.66; C NMR (CDCl3,
(ppm), 100 MHz). δ 12.0 (C-18), 12.2 (C-29), 19.0 (C-26), 19.4 (C-19), 21.0 (C-21), 21.1 (C-
11), 21.2 (C-27), 24.4 (C-15), 25.4 (C-28), 28.9 (C-16), 31.7 (C-2), 31.7 (C-8), 31.9 (C-7), 31.9
(C-25), 36.5 (C-10), 37.3 (C-1), 39.7 (C-12), 40.4 (C-20), 42.2 (C-4), 42.3 (C-13), 50.2 (C-9),
51.2 (C-24), 56.0 (C-17), 56.7 (C-14), 71.8 (C-3), 121.7 (C-6), 129.3 (C-23), 138.3 (C-22),
140.7 (C-5).
3.10.4 Physical and spectral data of compound TN4 (Appendix 3a and 3b)
White crystalline solid from DCM with uncorrected melting point of 128-1300C; Rf of 0.56
1 (100% DCM) H NMR (CDC13, (ppm), (400 MHz) δ 5.33 (1H, d, J = 7.2 Hz), δ 3.53 (m), δ 1.03
13 (3H, s), δ 0.94 (3H, d, J = 8.4Hz), δ 0.86 (9H, m), δ 0.70 (3H, s); C NMR (CDC13, (ppm), 100
MHz). δ 11.8 (C-29), 12.0 (C-18), 18.7 (C-26), 19.0 (C-19), 19.4 (C-21), 19.7 (C-27), 21.1 (C-
11), 23.1 (C-28), 24.3 (C-15), 26.1 (C-23), 28.2 (C-16), 29.2 (C-25), 31.7 (C-7), 31.9 (C-2), 31.9
(C-22), 34.0 (C-8), 36.1 (C-10), 36.5 (C-20), 37.3 (C-1), 39.8 (C-12), 42.3 (C-4), 42.3 (C-13),
45.8 (C-24), 50.1 (C-9), 56.1 (C-17), 56.8 (C-14), 71.8 (C-3), 121.7 (C-6), 140.8 (C-5).
47
3.10.5 Physical and spectral data of compound TN5 (Appendix 4a and 4b)
White needle like crystals from DCM with uncorrected melting point of 126-1280C; Rf of 0.57
1 (1:1 n-hexane: DCM); H NMR (CDCl3, (ppm), 400 MHz) δ (4.67, 4.58, 3.16, 2.30, 1.66, 1.01,
13 0.95, 0.92, 0.81, 0.77 and 0.74); C NMR (CDCl3, (ppm), 100MHz). δ 14.6 (C-27), 15.4 (C-24),
16.0 (C-26), 16.1 (C-25), 18.0 (C-28), 18.3 (C-6), 19.3 (C-29), 20.9 (C-11), 25.1 (C-12), 27.4 (C-
2), 27.5 (C-15), 28.0 (C-23), 29.8 (C-21), 34.3 (C-7), 35.6 (C-16), 37.2 (C-10), 38.1 (C-13), 38.7
(C-1), 38.9 (C-4), 40.0 (C-22), 40.8 (C-8), 42.8 (C-14), 43.1 (C-17), 48.0 (C-19), 48.3 (C-18),
50.4 (C-9) 55.3 (C-5), 79.0 (C-3), 109.3 (C-30), 150.9 (C-20).
3.10.6 Physical and spectral data of compound TN6 (Appendix (6a and 6b)
White amorphous powder from DCM with uncorrected melting point of 271-2730C; Rf of 0.22
1 in DCM: Hexane, 4:1. H-NMR (CDCl3, (ppm), 400 MHz) showed seven methyl protons at, δ
0.75, 0.77, 0.90, 0.91, 0.92, 0.98, 1.13 (7 x 3H, s, CH3), 2.52 (l H, dd, J = 13.3, 4.4 Hz, H-18),
13 3.21 (l H, dd, J = 8.8, 6.6 Hz, H-3) and 5.49 (l H, t J 4.00 Hz, H-12). C NMR (CDCl3, (ppm),
100 MHz) δ15.6 (C-25), 16.6 (C-24), 17.5 (C-26), 18.8 (C-6), 23.7 (C-11), 23.8 (C-16), 24.0 (C-
30), 26.2 (C-27), 28.2 (C-2) 28.4 (C-15), 28.8 (C-23), 31.1 (C-20), 33.2 (C-22), 33.3 (C-7), 33.4
(C-29), 34.3 (C-21), 37.4 (C-10), 39.0 (C-1), 39.4 (C-4), 39.8 (C-8), 42.0 (C-18), 42.2 (C-14),
46.5 (C-19), 46.7 (C-17), 48.2 (C-9), 55.9 (C-5), 79.2 (C-3), 122.6 (C-12), 144.8 (C-13), 183.8
(C-28).
48
CHAPTER FOUR
RESULTS AND DISCUSSIONS
4.1 Crude extracts fraction yields
Dry powdered stem bark of B. abyssinica weighing 4.0kg were extracted sequentially in four solvents in order of increasing polarity starting with n-hexane, DCM, ethyl acetate and MeOH.
The amount of the extract obtained was recorded and tabulated as in table 4.1 below.
Table 4.1: Yield of extracts from 4kg of the stem bark of B. abyssinica
Extract Hexane DCM EtOAc MeOH
Yield (gms) 4.00 16.50 63.71 82.11
Percentage (%) yield 0.09 0.41 1.59 2.05
Hexane extract had the lowest percentage and was oily in nature. The methanol extract had the highest percentage yield of 2.05% than the rest. This is because methanol is more polar compared to the other solvents used hence extracts both polar and non-polar compounds. These extracts were subjected to antibacterial and antifungal tests before fractionation commenced.
4.2 Bioassay tests
4.2.1 Antibacterial assay for the extracts
The crude extract were subjected to bioassay tests using P. aeruginosa, B. subtilis, S. aureus, E. coli, V. cholerae and K. pneumoniae in agar diffusion assay method. The 500µg/disc were loaded on sterile filter paper diameter 6mm and incubated at 370C for 24hrs. The inhibition zones were measured in mm as described by Chhabra and Usio (1991) and the result tabulated in Table
4.2.
49
Table 4.2: Antibacterial activities of the crude extract and the standard drugs (Inhibition zones in mm)
Extract B.subtilis E.coli K.pneumoniae P.aerginosa S.aureus V.cholerae Hexane 6.00±0.00a 6.00±0.00a 6.00±0.00a 6.00±0.00a 6.00±0.00a 6.00±0.00a DCM 8.83±0.76b 6.00±0.00a 7.77±0.40b 6.00±0.00a 8.00±0.00b 6.00±0.00a EtOAc 10.00±0.00c 7.83±0.29b 7.90±0.17b 7.00±0.00b 9.87±0.23c 7.00±0.00b MeOH 10.53±0.50c 9.67±0.58c 15.67±0.29c 15.93±0.12c 11.00±0.00d 9.67±0.29c Tetracycline 20.83±0.29d 18.17±0.76d 21.83±0.29e 20.33±0.58e 22.00±0.00f 22.67±0.29f Amplicillin 23.00±0.00e 21.00±0.00e 21.00±0.00d 20.00±0.00e 23.00±0.00g 22.00±0.00e Kanamycin 22.00±0.00f 20.00±0.00f 23.00±0.00f 18.00±0.00d 21.00±0.00e 21.00±0.00d P-Value <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
Mean values followed by the same small letter within the same column do not differ significantly from one another (One-way Anova, SNK test,α=0.05)
The MeOH extract had activity against all test bacteria. K. pneumoniae and P. aeruginosa
recorded highest inhibition zones of approximately 16 mm each. The extract had activity that
was significantly different from the other extracts except in B. subtilis where the activity is not
significantly different from EtOAc. The EtOAc extract had significantly different activity in
comparison to DCM in some test strains except in K. pneumoniae where the activity is
significantly not different. It had mild activity against E. coli, P. aeruginosa, V. cholerae and K.
pneumoniae. This is in comparison with the standards as shown in table 4.2. DCM had mild
activity against three test bacteria. This was done in comparison with the inhibition zones of the
standard reference as shown in Table 4.2. The values that are close to the values of the standard
used show high activity and those that are half show moderate activity while those that are far
below shows mild activity. Hexane did not have any activity since all the inhibition zones had
6mm which is the same value that was the diameter of the disc.
50
4.2.2 Antifungal assays for the extracts
The crude extract was subjected into antifungal assays using Candida albicans and Penicillium notatum in agar diffusion method. The zones of inhibition were measured in mm as described by
Chhabra and Usio (1991) and the result recorded in table 4.3.
Table 4.3: Antifungal activities of the crude extract and standard drug (Inhibition zones in mm)
Extract Candida albicans Penicillium notatum Hexane 6.00±0.00a 6.00±0.00a DCM 20.00±0.00c 11.00±0.00b EtOAc 21.83±0.29d 12.00±0.00c
MeOH 18.90±0.46b 11.93±0.12c e d Amphotericin 28.00±0.00 32.00±0.00 p-value <0.001 <0.001
The crude extracts of DCM, EtOAc and MeOH had moderate activity against Candida albicans and mild activity against Penicillium notatum .These activities were compared to the activities of the standard drugs as shown in table 4.3 above. Each of the extract was significantly different from the other in their activity. For example, DCM and EtOAc show different activities. The hexane extract had no activity because its zones of inhibition are similar to those of the disc that was used (6mm).The values that are half to those of the standard are shown to have moderate activity while those whose values are far below are shown to have mild activity.
4.3 Structural elucidation of isolated compounds
4.3.1 Structure of compound TN1
Compound TN1 appeared as white needle like crystals after drying. It was isolated from the
DCM extract, with an uncorrected melting point range of 182-1840C. It was obtained from a
51
second smaller column after combining closely related fractions from the main column and eluted by 50:50 DCM: Hexane from a DCM fraction of the crude extract of the main column. On spraying with anisaldehyde on a TLC plate, it turned orange. It was UV active on short wave
(254nm) indicating conjugation or aromaticity in the compound (Newman and Cragg, 2007). The
1 H-NMR CD2Cl2 data revealed presence of four chelated hydroxyl groups δ 10.36 (s), 11.95 (s),
12.50 (s) and 12.56 (s), two aromatic protons δ 6.41 (s), 6.52 (s), one methoxy group protons δ
3.89 3H, (s) and three aromatic methyl groups δ 1.99 3H, (s), 2.46 3H (s), 2.61 3H, (s). The chelation of the four hydroxyl groups is a characteristic of an anthraquinone type of skeleton
(Williams and Lemke, 2002). In NOESY the aromatic protons δ 6.33 and 6.46 were correlated to
2.6 and 2.46 hydrogen atoms, respectively. The methoxy hydrogen atoms δ 3.89 were correlated to 2.46 hydrogen atoms of the methyl group.
13 A total of 20 signals were noted in C NMR CD2Cl2 data. The signal at δ 52.6 indicated the presence of a methoxy group. The signal at 194.0 is of conjugated carbonyl groups which are two in number because of them being magnetically equivalent. The peaks δ 23.9, 25.6 and 29.9 are for aromatic methyl carbon atoms. This is evident from HMBC in which the aromatic protons correlate with the methyl carbon at long range. The peaks at δ 108.7, 110.5 and 140.2 are for the aromatic carbon atoms into which aromatic methyl groups are attached. The aromatic carbon attached to the methoxy group has a peak at δ 172.5.
The four aromatic carbon atoms with hydroxyl groups have peaks at (δ 116.9 162.9 and 167.6).
The peak at 162.9 represents two carbon atoms that are magnetically equivalent. This can be seen from the long range correlation of the aromatic protons and the aromatic carbon atoms
52
bearing the hydroxyl groups. The aromatic carbon atoms with hydrogen atoms have peaks at
δ112.9 and 116.3. This is evident from the HSQC correlations in which the two aromatic protons are attached to aromatic carbon atoms through one bond. The quaternary carbon atoms have peaks at (δ 103.3, 152.3, 153.0, 169.2 and 169.9). The peak at δ103.3 also represents two carbon atoms that are magnetically equivalent. The peak at δ 9.3 is an impurity as most of the aromatic methyl groups fall in the region above 20ppm (Kim et al., 2004). This compound is being reported for the first time in this plant and was given the name bersamanone (2,4,12-trimethyl-3- methoxy-7,8,11,14- tetrahydroxy-9,10 anthraquinone).
Table 4.4: NMR spectral data for compound TN1
Carbon no. 1H NMR 13C NMR HMBC
1 6.46,s 116.3 C18, C2 2 - 108.7 3 - 172.5 4 - 140.2 5 - 152.3 6 - 153.0 7 12.44, s 116.9 C8a 7a - 169.2 8 12.42, s 162.9 C8a 8a - 169.9 9 - 194.0 - 9a - 103.3 - 10 - 194.0 - 10a - 103.3 -
11 11.98, s 162.9 C12, C18 12 - 110.5 - 13 6.33, s 112.9 C14, C15, C10a,C12
14 10.26, s 167.6 C14 15 2.61, s 25.6 - 16 1.99, s 29.9 - 17 3.89, s 52.6 C3
18 2.46, s 23.9 C17
53
OH O OH H 18 8 1 H 14 9 CH3 6 8a 13 9a 2 CH 12 10a 7a 5 3 3 4 17 H C 11 10 7 O 3 15 Compound TN1 OH O OH CH3 16
6.46 OH H O OH H 6.33 H 2.46 167.9 162.9 H 194 116.3 C 23.9 112.9 103.3 169.9 153 108.7 H 110.5 103.3 152.3 H 169.2 172.5 H 25.6 194 162.9 116.9 140.2 O C H 3.89 C H 29.9 52.6 2.6 OH O HO C H 1.98 H H H H HMBC ( ) correlations
6.46 OH H O OH H 6.33 H 2.46 H 116.3 C 23.9 H
H 25.6 O C 52.6 H 3.89 C H 29.9 2.6 O OH C OH H H H H 1.98 H H
NOESY( ) correlations
54
4.3.2 Structure of compound TN2
This compound was isolated from the MeOH extract as white amorphous powder with a melting point range of 284 - 286oC. On TLC the compound had an Rf of 0.65 (8:2) DCM: MeOH). The compound was not UV active and when the plate was sprayed with anisaldehyde the spot turned purple suggesting that the compound was a terpenoid (Dey and Harborne, 1991; Prestch et al.,
2000).
1H NMR spectrum displayed three regions; aliphatic, hydroxylated and allylic region on the spectrum and strongly suggested a triterpenoid structure. A signal at δ 5.35 (1H, s) suggested a hydrogen attached to a double bond carbon atom next to a quaternary carbon atom. A doublet at
δ 4.2 suggests hydrogen on the anomeric carbon. A multiplet centred at δ 3.7 was a characteristic of a proton germinal to a 3-hydroxyl group at C-3 in terpenoids. Signals representing the methyl groups were observed at δ 1.18 (3H, s), 0.98 (3H, s), 0.93 (3H, s), 0.92
(3H, s), 0.84 (3H, s) 0.67 (3H, s) which is characteristic of a modified triterpenoid (Dey and
Harborne, 1991). 1H NMR spectra data of this compound compared closely with that of β– sitosterol. However, there appeared six additional peaks between δ 3.0 and 4.2, characteristic of hydroxylation suggesting existence of a sugar moiety.
13C NMR spectrum was in close agreement to that of β-sitosterol except for the six peaks at δ
101, 77.4, 77.3, 77.2, 74.0 and 62.0 associated with a sugar moiety. The signals associated to the double bond carbon atoms appeared at δ 141 and 121.7 for the aglycone part. The assignment of the peak at δ 70.6 was to C-3 due to hydroxylation. The methyl groups were represented by the signals δ 12.2, 12.3, 19.1, 19.4, 19.6 and 20.2 characteristic of triterpenoid (Dey and Harborne,
55
1991). The compound was finally assigned a structure of β–sitosterol glucoside. The anomeric carbon resonance was observed at 101.2 in the 13C NMR spectrum and indicated the presence of a monosaccharide. The C-3 resonance showed correlations to the anomeric proton doublet at 4.2 in HMBC spectrum. In the HMBC spectrum, the sugar moiety was placed at C-3.
All the glycoside resonances were assigned making use of HSQC and HMBC spectra as well as literature values (Dey and Harborne, 1991; Mimaki et al., 2004). The NMR data indicated that the sugar could be a glucopyranose or a galactopyranose. These two sugars differ only in the stereochemistry of the H-4' proton. The resonance of importance (H-4') in this case correlates with the H-3' proton resonance. A NOESY correlation between the H-2' resonance and the H-
3'/H-4' resonance gave evidence that the sugar is a glucopyranose. A NOESY correlation would not be possible between the H-2' resonance and the H-3' resonance in gluco- or galactopyranose.
Thus, NOESY correlation can only exist between the resonances of H-2' and H-4' of glucose
(Agrawal, 1992). The NOESY correlations giving evidence of a 3-0-glucopyranose.
Finally, the 13C NMR data of compound TN2 was compared to the literature 13C-NMR data. A
13 literature search showed that the C NMR data was run in CDCl3. Compound TN2 was dissolved in DMSO-d6, the chemical shift values of compound TN2 were, in all cases, in conformity with the reported values for sitosterol glucopyranose (Agrawal, 1992; Mahato and
Kundu, 1994). The melting point and values of compound TN2 were also identical to those reported for sitosterol 3-0-glucopyranose (2840C) (Gohar et al., 2000). The literature data of the aglycone of sitosterol 3-O-glucopyranose which was run in a suitable solvent (CDCl3) was compared to the aglycone of compound TN2 (Mahato and Kundu, 1994).
56
29 21 22 28 19 12 20 24 23 27 4' OH 17 18 11 13 25 HO 1 16 5' O 14 26 3' 2 9 HO 2' 10 8 15 1' 3 5 7 Compound TN2 OH O 4 6 H
Table 4.5: 13C NMR data of the aglycone of compound TN2 and aglycone of sitosterol (Mahato and Kundu, 1994; Mimaki et al., 2004)
Carbon Compound 3-O-β-Sitosterol Carbon Compound 3-O-β-sitosterol No. TN2 No. TN2 1 37.3 37.0 17 56.0 55.9
2 31.9 29.5 18 12.3 11.7 3 78.9 79.9 19 19.4 19.2 4 38.5 38.7 20 36.7 34.0 5 141.0 140.2 21 19.6 18.6 6 121.7 121.9 22 31.9 34.0 7 31.2 31.7 23 26.0 26.0 8 33.9 31.7 24 45.7 45.7 9 50.1 50.0 25 29.2 29.2 10 36.7 36.6 26 19.1 19.6 11 21.1 21.0 27 20.2 18.9 12 38.8 39.6 28 23.1 23.0 13 42.3 42.2 29 12.2 11.8 14 56.7 56.6 15 24.3 24.2 16 28.2 28.1
NMR data obtained in DMSO-d6, 100MHz
57
Table 4.6: NMR data for aglycone [Non-sugar part (sitosterol)] of compound TN2
Carbon 1H NMR 13CNMR DEPT HMQC NOESY
1 0.91,1.22 37.3 CH2 9
2 1.42,1.86 31.9 CH2 - 19 3 3.66 m 78.9 CH 1 , 4’ 4
4 2.37, 2.10 38.5 CH2 - 6, 4 5 - 141.0 19 - 6 5.35 br, s 121.7 CH 4’ -
7 1.82 31.2 CH2 - - 8 1.40 33.9 CH - 18, 19 9 0.83 50.1 CH 19 - 10 - 36.7 - -
11 1.41 21.1 CH2 - -
12 1.17 38.8 CH2 18 - 13 - 42.3 18 14 1.25 56.7 CH 9, 18 9, 17 15 1.04 24.3 CH - -
16 1.79 28.2 CH2 - - 17 0.92 56.0 CH 18 21
18 0.67 s 12.3 CH3 - 8, 20
19 0.84 s 19.4 CH3 9 8 20 1.25 36.7 CH 21 18
21 0.83 19.6 CH3 - -
22 1.73 31.9 CH2 21 -
23 1.18 26.0 CH2 - - 24 0.83 45.7 CH 26, 27, 28, 29 - 25 1.51 29.2 CH 26, 27 -
26 0.70 s 19.1 CH3 27 -
27 0.70 s 20.2 CH3 26 -
28 0.70 s 23.1 CH2 26, 27 -
29 0.83s 12.2 CH2 - -
NMR data obtained in DMSO-d6, 100MHz
58
Table 4.7: NMR data for the sugar moiety (Dey and Harborne, 1991; Mimaki et al., 2004)
Carbon 1H NMR 13C NMR HMBC NOESY
1’ 4.20 d (7.8) 101.3 3,2’ 5’
2’ 3.10 77.4 - 4’
3’ 3.32 77.3 2’, 4’ 5’
4’ 3.32 77.2 3’ 2’
5’ 3.03 m 74.0 6’ 1’
6’ 3.17 61.6 4’ -
1 13 NMR data obtained in DMSO-d6, H NMR at 400MHz and C NMR at 100MHz
OH H 4' HO 6' CH2 H O 3' HO 2' H O OH H 1'
H
Fig 4.1 NOESY correlations of the sugar moiety
59
4.3.3 Structure of compound TN3
This compound was isolated from DCM as a white feather like compound with an uncorrected melting point range of 168-1700C. On spraying with anisaldehyde, the spot turned purple suggesting that it is a terpenoid (Dey and Harborne, 1991). The 1H NMR spectrum (Appendix
5b) displayed several peaks between δ 1.01 and 0.66 characteristic of tritepenoid.
A broad singlet at δ 5.34 suggested presence of a proton of double bond at a carbon atom next to a quarternary carbon atom. Two signals at δ 5.12 and 5.02 suggested the presence of a double bond on the side chain of triterpenoid skeleton. The multiplet at δ 3.51 represented a proton attached to a hydroxylated carbon atom. The diagnostic chemical shift values of the angular methyl protons for C-18 and C-19 appeared as singlets at δ 0.66 and 1.01, respectively. The other four methyl groups appeared at δ 0.78, 0.81, 0.85 and 0.91 (Dey and Harbone, 1991).
The 13C NMR spectrum (Appendix 5a) displayed twenty nine carbon atoms confirming that it is a modified triterpenoid. It showed signals at δ 140.7 and 121.7 confirming the presence of olefinic carbons with the more deshielded signal assignable to the quarternary carbon at the bridge. The signal at δ 138.7 and 129.3 represents olefinic carbon on the side chain. The peak at
δ 71.8 was assigned to C-3 due to the presence of the hydroxyl group common in this class of compounds. (Dey and Harbone, 1991; Muhit et al., 2010).
60
Table 4.8: 13C NMR data for compound TN3 and that reported for stigmasterol (Mahato and Kundu, 1994; Alam et al., 1996; Muhit et al., 2010)
Carbon Compound Stigmasterol Carbon Compound Stigmasterol No. TN3 No. TN3 1 37.3 37.3 17 56.0 56.1 2 31.7 31.7 18 12.0 12.1 3 71.8 71.8 19 19.0 19.4 4 42.2 42.3 20 40.4 40.5 5 140.7 140.8 21 21.0 20.9 6 121.7 121.7 22 138.7 138.3 7 31.9 31.9 23 129.3 129.4 8 31.7 31.9 24 51.2 51.3 9 50.2 50.2 25 31.9 31.9 10 36.5 36.6 26 19.0 19.0 11 21.1 21.1 27 19.8 21.2 12 39.7 39.7 28 26.1 25.4 13 42.3 42.3 29 12.2 12.0 14 56.7 56.9 15 24.3 24.4 16 28.3 28.6
NMR data obtained in CDCl3, 100 MHz
29 21 22 28 19 12 20 24 17 23 27 18 11 13 25 1 16 14 26 2 9 10 8 15 3 5 7 Compound TN3 HO 4 6
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4.3.4 Structure of compound TN4
This compound was obtained from DCM as a white crystalline solid with a melting point range of 128-1300C. 1H NMR spectrum displayed three regions namely aliphatic, hydroxylated and allylic on the spectrum signals and strongly suggested a triterpenoid skeleton. A signal at δ 5.33
(1H, d, J = 7.2 Hz) suggested the presence of a double bond at a quarternary carbon atom. A multiplet centered at δ 3.53, characteristic of proton germinal to a hydroxyl group at C-3 in terpenoids was also observed. Six signals representing the methyl groups were observed at δ 1.03
(3H, s), 1.00 (3H, d, J = 8.4 Hz), 0.96 (9 H, m) and 0.94 (3H, s) which is characteristic of a modified triterpenoid (Dey and Harborne 1991; Pretsch et al., 2000).
13C NMR spectrum showed signals involving the double bonds at δ 140.8 and 121.7 with the former representing a quaternary carbon atom. The signal at 71.8 represents the hydroxylated carbon atom C-3. The methyl groups were represented by the signal at δ 11.8, 12.0, 18.7, 19.4,
19.4 and 19.7. The overall spectrum of the compound closely compared to that of β – sitosterol, a sterol that has been reported in various plants (Dey and Harborne, 1991).
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Table 4.9: 13C NMR data of compound TN4 and that reported for β-sitosterol (Mahato and Kundu, 1994; Alam et al., 1996).
Carbon Compound TN4 β – sitosterol Carbon Compound TN4 β – sitosterol No. No. 1 37.3 37.2 16 28.2 28.2 2 31.7 31.9 17 56.1 56.0 3 71.8 72.0 18 12.0 11.8 4 42.3 42.2 19 19.0 19.2 5 140.8 140.7 20 36.1 36.1 6 121.7 121.6 21 19.4 18.9 7 31.7 31.8 22 31.9 32.1 8 33.9 33.8 23 26.1 25.7 9 50.1 50.1 24 45.8 45.8 10 36.5 36.4 25 29.2 29.1 11 21.1 21.4 26 18.7 18.7 12 39.8 39.7 27 19.7 19.7 13 42.3 42.5 28 23.1 23.0 14 56.8 56.7 29 11.8 11.9 15 24.3 24.2
NMR data obtained in CDCl3, 100MHz
29 21 22 28 19 12 20 24 17 23 27 18 11 13 25 1 16 14 26 2 9 10 8 15 3 5 7 Compound TN4 HO 4 6
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4.3.5 Structure of compound TN5
Compound TN5 was isolated from DCM extract as needle like crystals with a melting point range of 126-1280C. On analytical TLC, the compound had an Rf of 0.57 in n-Hexane: DCM
(1:1). When the plate was sprayed with anisaldehyde it turned purple indicating that it is a triterpenoid. The 1H NMR strongly suggested that the compound was a triterpenoid due to appearance of the signals. Downfield signals at δ 4.80 and 4.68 appearing as broad singlets indicated the presence of a terminal double bond. The signal at δ 3.35 indicated presence of hydroxyl group normally at position 3. Seven singlets each integrating to three protons at δ 0.99,
1.00, 1.13, 1.26, 1.27, 1.27, 1.29 and 1.82 strongly suggested pentacyclic triterpenoid (Sen et al.,
1995).
13C NMR spectra for compound TN5 showed 30 carbons. This further confirms a pentacyclic structure. Two downfield signals at δ 151.4 and 110.0 with the downfield signal being of a quaternary carbon. The signal at δ 79.3 represented the hydroxylated carbon at position 3. The seven methyl carbon atoms appeared at δ 39.0, 28.6, 18.3, 16.5, 16.3, 15.4 and 14.6. They are closely compared to that of lupeol a common pentacyclic triterpenoid reported from various plants (Sen et al., 1995; Satomi et al., 2002).
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Table 4.10: 13C NMR data of compound TN5 compared to lupeol (Satomi et al., 2002)
Carbon No. Compound Lupeol Carbon No. Compound Lupeol TN5 TN5 1 40.0 40.0 16 35.6 35.8
2 27.4 27.8 17 43.0 43.2
3 79.0 78.2 18 48.3 48.6
4 39.1 39.6 19 48.0 48.3
5 55.3 55.9 20 151.0 151.1
6 18.3 18.8 21 29.8 30.2
7 34.3 34.6 22 39.0 39.3
8 41.0 41.2 23 28.1 28.7
9 50.5 50.8 24 16.1 16.6
10 37.2 37.5 25 15.4 15.4
11 20.9 21.2 26 14.6 14.8
12 28.0 28.3 27 16.0 16.2
13 38.1 38.3 28 18.0 18.2
14 42.8 43.1 29 19.3 19.5
15 30.1 30.1 30 109.3 110.0
NMR data obtained in CDCl3, 100MHz
30
20 29 21 19 12 22 13 18 25 26 17 28 1 14 2 9 16 10 8 15 3 27 4 5 7 HO 6 24 23 Compound TN5
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4.3.6 Structure of compound TN6
This compound was obtained from DCM as a white amorphous powder and it showed a positive test to anisaldehyde reagent giving a purple colour which is characteristic of triterpenoid (Dey and Harborne, 1991). It had a melting point range of 271-2730 C. The compound TN6 displayed three functional groups: secondary hydroxyl, olefinic bond and carboxylic group. The 1H NMR
(400 MHz, CDCl3) spectrum (Appendix 6a) displayed seven peaks between δ 1.30 and 0.88 characteristic of triterpenoid signals and this closely compared to oleanolic acid (Sen et al.,
1995). These seven tertiary methyl protons were at the peaks δ 0.88, 0.94, 1.02, 1.03, 1.04, 1.24 and 1.30. Vinyl protons at δ 2.52 and 5.49, hydroxyl proton at δ 3.44 and 5.49 suggesting olea-
12-ene skeleton (Tan et al.,1999).
13 C NMR (150 MHz, CDCl3): Indicated 30 carbon signals. The signal corresponding to the carboxyl C-28 appeared at δ 183.8 the hydroxyl carbon at 79.2 was assigned to C-3, the double bond carbons at δ 122.6 and 144.8 in carbon 12 and 13, respectively. These chemical shifts at δ
183.8, 122.6 and 144.8 are characteristic peaks of oleanolic type of skeleton. The methyl carbon atoms have peaks at δ 15.6, 16.6, 17.2, 23.8, 26.2, 28.8 and 33.4. The spectral data were similar to the one reported for oleanolic acid (Tan et al., 1999; Kowalski, 2007).
66
Table 4.11: 1H NMR (400MHz) and 13C NMR (100MHz) data of compound TN6 compared to oleanolic acid (Seebacher et al., 2003; Kowalski, 2007)
Carbon No. 1H NMR 13C NMR of TN6 13C NMR Oleanolic acid 1 39.0 39.0 2 28.2 28.1 3 3.21 dd, 8.8/6.6 79.2 78.2 4 39.4 39.4 5 55.9 55.9 6 18.8 18.8 7 33.3 33.4 8 39.8 39.8 9 48.2 48.2 10 37.4 37.4 11 23.7 23.8 12 5.49 br s 122.6 122.4 13 144.8 144.8 14 42.2 42.2 15 28.4 28.4 16 23.8 23.8 17 46.7 46.7 18 2.52 d,11 42.0 42.1 19 46.5 46.6 20 31.1 31.0 21 34.3 34.2 22 33.2 33.2 23 28.8 28.8 24 1.24 16.6 16.5 25 1.02 15.6 15.6 26 0.88 17.5 17.5 27 1.04 26.2 26.2 28 1.30 183.8 180.0 29 0.94 33.4 33.4 30 1.02 24.0 23.8
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29 30 20 19 21 18 12 22 11 13 O 25 26 17 1 14 28 2 9 16 10 8 15 OH 3 27 4 5 7 HO 6 23 24 Compound TN6
4.4 GC-MS data for the crude extracts of MeOH and DCM (appendices 7a and 7b)
The stem bark extracts of DCM and methanol were analyzed by GC-MS instruments and several compounds were obtained (Appendices 7a and 7b). Most of the compounds from the two extracts were similar with a few that were different. The compounds were classified into their natural product classes. The terpenoids that were present include amyrin and myristoylolean in methanol and amyrin, lupenone and squalene in DCM extract. Sesquiterpenes and diterpenes were also present. Steroids were also present in the crude extracts. They include; sitosterol, stigmasterol, norandrostane, cholesterol and ergosterol. These steroids are present in both extracts.
Phenolic compounds embrace a wide range of plan substances which possess in common an aromatic ring bearing one or more hydroxyl substituents. Among the natural phenolic compounds, flavonoids form the largest group. Simple monocyclic phenols, phenylpropanoids and phenolic quinones (Dey and Harborne, 1991). In both extracts, a number phenolic compounds and flavonoids were identified as shown in Appendices 7a and 7b. In addition several organic acids and fatty acids were identified for example linoleic acid ethyl ester, octadecanoic acid, nonanoic acid and many others as shown in Appendices 7a and 7b.
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4.5 Antibacterial test assay for the isolated compounds
The isolated compounds were subjected to antibacterial assay against B. subtilis, S. aureus, P. aeruginosa, E. coli, V. cholerae and K. pneumoniae and their inhibition zones are as shown in table 4.12.
Table 4.12: Antibacterial activities of isolated compounds in the diffusion assay after 24hrs; 500µg/disc (diameter 6mm) and inhibition zones of the standards. P. K. Compound B .subtilis aeruginosa S. aureus V. cholerae pneumoniae E. coli Sitosterol (TN4) 8.67±0.23a 6.00±1.00a 8.00±1.00a 6.00±1.00a 10.87±0.23b 6.00±1.00a Stigmasterol(TN3) 7.90±0.17a 6.00±1.00a 8.00±2.00a 6.00±1.00a 6.00±1.00a 6.00±1.00a Lupeol (TN5) 11.00±1.00b 6.00±1.00a 14.00±1.00b 6.00±1.00a 6.00±1.00a 7.67±0.58a Sitosterol glucoside (TN2) 14.00±2.00c 11.70±0.36b 10.00±2.00b 10.00±2.00b 13.20±0.17b 15.00±2.00c Anthraquinone (TN1) 14.00±2.00c 8.50±0.87a 14.00±1.00b 6.00±1.00a 6.00±1.00a 9.83±0.29b Tetracycline 20.83±0.29d 20.33±0.58c 22.00±1.00c 22.67±0.29c 22.00±2.00c 18.17±0.76d Amplicillin 23.00±2.00d 20.00±2.00c 23.00±2.00c 22.00±2.00c 21.00±2.00c 21.00±2.00e Kanamycin 22.00±2.00d 18.00±2.00c 21.00±1.00c 21.00±2.00c 23.00±2.00c 20.67±1.15e P-Value <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
Mean values followed by the same small letter within the same column do not differ significantly from one another (One-way Anova, SNK test,α=0.05)
The compounds showed varied significant activities in all the test strains. Stigmasterol and sitosterol did not show significant difference in most of the test strains except in K. pneumoniae.
Lupeol showed significant different in B. subtilis and S. aureus while sitosterol glucoside and anthraquinone had no significant different in the two strains. In P. aeruginosa, V. cholerae and
E. coli the anthraquinone and sitosterol glucoside showed significant difference as shown in table
4.12 above. Those compounds whose mean value is 6mm exhibited no activity since the diameter of the disc was 6mm. The compounds that had their mean value half those of the standards had moderate activity while those whose values were more than half had high activity.
69
Sitosterol showed low activity of 8.6 and 8 mm against two Gram-positive bacteria; B. subtilis and S. aureus, respectively. The compound showed no activity against Gram-negative bacteria.
An inhibition zone of 8 mm was shown by stigmasterol against both B. subtilis and S. aureus.
Lupeol showed moderate activity against S. aureus with an inhibition zone of 14 mm. It also had an inhibition zone of 11 and 7.7 mm against B. subtilis and E. coli, respectively. Sitosterol glucoside had moderate activity against S. aureus of 14 and 13 mm for K. pneumoniae. An inhibition zone of 10 and 15 mm was observed in V. cholerae and E. coli, respectively.
Compared to sitosterol, the sitosterol glucoside had relatively higher activity which meant that the sugar moiety modified its activity. The anthraquinone had moderate activity against B. subtilis and S. aureus of 14 mm each. It also had an inhibition zone of 8 and 9 mm against P. aeruginosa and E. coli, respectively.
The five compounds exhibited moderate to mild activity for both Gram negative and Gram positive bacteria. The fact that the five compounds tested the six bacteria and showed some moderate activity indicated their potency. The usage of Bersama in traditional medicine by different ethnic communities could be justified by the observed antibacterial activity.
4.6 Antifungal activities for isolated compounds
The isolated compounds were subjected to antifungal assay against C. albicans and P. notatum.
The inhibition zones were measured in mm and the activities of the compounds are shown in table 4.13.
Table 4.13: Antifungal activities of isolated compounds the diffusion method assay after 24hr and the standard (inhibition zones in mm)
70
compound C. albicans P. notatum Sitosterol (TN4) 6.00±0.58a 6.00±0.58a Stigmasterol (TN3) 6.00±0.58a 7.83±0.17a Lupeol (TN4) 6.00±0.58a 9.00±1.15b Sitosterol glucoside (TN2) 10.00±1.15b 12.00±1.15b Anthraquinone (TN1) 14.13±0.07c 11.90±0.32b Amphotericin 28.00±1.15d 32.00±1.15c p-value <0.001 <0.001
Mean values followed by the same small letter within the same column do not differ significantly from one another (One-way Anova, SNK test,α=0.05)
Sitosterol had no activity against the fungi used. Lupeol and stigmasterol had activities against
P. notatum with inhibition zones of 9 and 7.8 mm respectively. Sitosterol glucoside showed moderate activity of 12 and 10 mm against P. notatum and C. albicans, respectively. The anthraquinone showed moderate activity of 14 and 12 mm against C. albicans and P. notatum, respectively. Stigmasterol, sitosterol and lupeol had no significant differences in their activities against C. albicans. However, lupeol had a significant difference in P. notatum as compared to sitosterol and stigmasterol. Sitosterol glucoside and anthraquinone showed significant differences in both strains of fungi.
71
CHAPTER FIVE
CONCLUSION AND RECOMMENDATIONS
5.1 Conclusion
The result showed that the percentage yields of the crude extracts increased from n-hexane to
DCM to EtOAc to MeOH. These result suggested that the stem bark of B. abyssinica is very rich in polar metabolites. The n-hexane extract did not show significant antibacterial and antifungal activity. DCM, EtOAc and MeOH had significant antibacterial and antifungal activities. The percentage by mass extracted was very low since the extracted material is a mixture that contain numerous compounds of almost equal polarity making isolation process complex.
The study shows that the stem barks contain steroids, terpenoids and anthraquinone among other compounds. Some of these compounds include; β-sitosterol, stigmasterol, β-sitosterol glucoside, lupeol, oleanolic acid and an anthraquinone. There structures have been elucidated using NMR data. Although stigmasterol, sitosterol and sitosterol glucoside share a common biosynthetic pathway, they displayed varied bioactivities hence need for structural relation activity.
The compounds isolated had varied activities against the test bacteria and fungi and this confirms the claim by traditional practioners for use of the plant as an antimicrobial. From the GC-MS data of the crude methanol and DCM had several compounds and some of the isolated compound for example sitosterol and stigmasterol were present in the crude.
72
5.2 Recommendations
5.2.1 Recommendations from the study
(i) The isolated compounds had varied bioactivities on the test bacteria hence the plant can
be used in treatment of the diseases caused by the respective pathogens once the cyto-
toxicity tests have been done.
(ii) The isolated compounds had varied activities against Candida albicans and Penicillium
notatum and hence the plant can be used to treat the diseases caused by these pathogens.
(iii) The crude extract for methanol possesses significant activity as seen in its ability to
inhibit the growth of most of the test bacteria and fungi. Since methanol is a polar solvent
it should be most preferred solvent in extraction.
(iv) Sitosterol glucoside had better activity than sitosterol this could be due to the presence of
the sugar.
5.2.2 Recommendations for further studies
(i) Sitosterol, stigmasterol, sitosterol glucoside and lupeol share a common biosynthetic
pathway. However, they display varied bioactivities. Hence need for structural activity
relation studies.
(ii) Presence of glycoside saponin which from other sources have been reported to have
moluscicidal activities especially against Schistomiasis should be investigated in B.
abyssinica species.
(iiii) From the previous pharmacological studies, the stem bark of aqueous extract has been
shown to be active against schizonts which cause malaria. More study should be carried
out to the compounds that are responsible and if they can be isolated and be stable.
73
(iv) Synergic effects among the crude extracts, isolated compounds, the fractions where the
compounds were obtained from and the conventional antibiotics should be carried out.
(v) TLC survey of the crude extracts showed many trace compounds which were not isolated
owing to the small quantity of the plant materials, time and organic solvents needed. The
role played by these trace compounds in overall biological activities need to be studied
further.
(vi) Phytochemical studies and antimicrobial activity of roots and leaves should be studied.
74
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APPENDICES Appendix 1a: 1H NMR spectrum compound TN1 (2,4,12-trimethyl-3-methoxy-7,8,11,14- tetrahydroxy-9,10 anthraquinone)
OH O OH H 18 8 1 H 14 9 CH3 6 8a 13 9a 2 CH 12 10a 7a 5 3 3 4 17 H C 11 10 7 O 3 15 Compound TN1 OH O OH CH3 16
81
Appendix 1b: 13C NMR spectrum of compound TN1 (2,4,12-trimethyl-3-methoxy-7,8,11,14- tetrahydroxy-9,10 anthraquinone)
OH O OH H 18 8 1 H 14 9 CH3 6 8a 13 9a 2 CH 12 10a 7a 5 3 3 4 17 H C 11 10 7 O 3 15 Compound TN1 OH O OH CH3 16
82
Appendix 1c: HMBC for compound TN1 (2,4,12-trimethyl-3-methoxy-7,8,11,14- tetrahydroxy-9,10 anthraquinone)
83
Appendix 1d: HSQC spectrum of compound TN1 (2,4,12-trimethyl-3-methoxy-7,8,11,14- tetrahydroxy-9,10 anthraquinone)
84
Appendix 1e: NOESY spectrum for compound TN1 (2,4,12-trimethyl-3-methoxy-7,8,11,14- tetrahydroxy-9,10 anthraquinone)
85
Appendix 2a: 1H-NMR spectrum for compound TN2 (sitosterol glucoside)
21 29 19 22 28 12 20 OH 11 17 24 4' 13 23 18 HO 16 26 5' 1 14 25 O 9 3' 2 27 HO 2' 10 8 15 1' 3 OH 5 7 O 4 6
86
Appendix 2b: 13C-NMR spectrum for compound TN2 (sitosterol glucoside)
21 29 19 22 28 12 20 OH 11 17 24 4' 13 23 18 HO 16 26 5' 1 14 25 O 9 3' 2 27 HO 2' 10 8 15 1' 3 OH 5 7 O 4 6
87
Appendix 2c: DEPT spectrum for compound TN2 (sitosterol glucoside)
88
Appendix 2d: HMBC spectrum for compound TN2 (sitosterol glucoside)
89
Appendix 2e: HSQC spectrum for compound TN2 (sitosterol glucoside)
90
Appendix 2f: NOESY spectrum for compound TN2 (sitosterol glucoside)
91
Appendix 2g: COSY spectrum for compound TN2 (sitosterol glucoside)
92
Appendix 3a: 1H-NMR spectrum for compound TN4 (sitosterol)
29
21 22 28 19 20 24 12 27 11 17 23 25 18 13 16 1 14 26 2 9 10 8 15 3 5 7 HO Compound TN4 4 6
93
Appendix 3b: 13C- NMR spectrum for compound TN4 (sitosterol)
29
21 22 28 19 20 24 12 27 11 17 23 25 18 13 16 1 14 26 2 9 10 8 15 3 5 7 HO Compound TN4 4 6
94
Appendix 4a: 1H-NMR spectrum for compound TN5 (Lupeol)
30 20 29 21 19 12 22 25 26 17 1 28 2 9 16 8 15 3 5 27 7 HO 6 23 24
95
Appendix 4b: 13C-NMR spectrum for compound TN5 (Lupeol)
30 20 29 21 19 12 22 25 26 17 1 28 2 9 16 8 15 3 5 27 7 HO 6 23 24
96
Appendix 5a: 13C-NMR of compound TN3 (stigmasterol)
29
21 22 28 19 20 24 12 11 17 23 27 18 13 25 16 1 14 26 2 9 10 8 15 3 5 7 HO 4 6
97
Appendix 5b:1H-NMR of compound TN3 (stigmasterol
29
21 22 28 19 20 24 12 11 17 23 27 18 13 25 16 1 14 26 2 9 10 8 15 3 5 7 HO 4 6
98
Appendix 6a: 1HNMR for compound TN6 (oleanolic acid)
29 30 20 19 21 18 12 22 11 13 O 25 26 17 1 14 28 2 9 16 10 8 15 OH 3 27 4 5 7 HO 6 23 24
99
Appendix 6b: 13C NMR for compound TN6 (oleanolic acid)
29 30 20 19 21 18 12 22 11 13 O 25 26 17 1 14 28 2 9 16 10 8 15 OH 3 27 4 5 7 HO 6 23 24
100
Appendix 7a: GC-MS data for crude methanol extract
No RT FORMULA NAME CLASS PROPOSED STRUCTURE
1 3.8 C4H8O 2,3 – epoxybutane H C Fatty acid 3 CH O 3
2 4.3 C8H8O3 Methyl salicylate Phenolic O CH3 O OH
3 5 C6H6O3 3 - methyl-2- furoic Organic acid acid O O OH
CH3
4 5.4 C8H12O4 5- (hydroxymethyl)- Phenolic 2- (dimethoxy CH3 O methyl)furan O CH3 HO O
5 5.7 C9H10O2 1-(2- hydroxyl -5- Phenolic methyl pheny) HO ethanone H3C
O CH3
6 6.7 C7H8O2 Orcinol Phenolic OH H3C
OH
7 6.9 C6H6O3 Triolbenzene Phenolic
HO OH OH
101
8 7.6 C8H8O3 3-hydoxy-methyl Phenolic ester benzoic acid O
O OH CH3
9 8.5 C8H8O4 2,5- dihydroxy – Phenolic acid OH methyl ester benzoic O acid O OH CH3
10 8.7 C7H6O3 4- hydroxyl- benzoic Phenolic acid OH acid HO
O
11 8.8 C8H8O3 2,4- dihydroxy -6- Phenolic CH methyl 3 benzaldehyde OH O HO
12 8.8 C10H12O3 4- methyl- 2,5 – dimethoxybenzaldeh Phenolic CH3 O -yde CH3 H3C O
O
13 9.1 C6H10O5 1, 6 - anhydro -; OH .beta. –D- gluco Sugar alcohol HO O HO pyranose O
14 10 C9H10O4 Methyl2,6- Phenolic HO dihydroxy -4- O CH3 methyl benzoate O
H3C OH
15 10.5 C6H10O5 1,6 - anhydro - Sugar alcohol O O .beta.- D - gluco OH HO furanose OH
102
16 10.5 C10H12O4 2,1 – dihydroxy -3, Phenolic acid OH CH3 6- dimethyl – ester O benzoic acid OH O H3C CH3
17 11.2 C11H14O4 Desaspidinol Flavonoids OH O CH3 H3C
HO O
18 11.8 C12H16O4 Aspidinol Phenolic OH O H3C O H3C HO
CH3
HO O 19 11.9 C9H10O5 4- hydroxyl-3,5 Phenolic acid
dimethoxy – benzoic H3C CH3 O O acid OH
20 12.1 C15H30O2 Pentadecanoc acid Organic acid O CH3 HO
21 13 C17H34O2 Methylester hexadecanoic acid Organic acid O H3C
O CH3
O 22 13.5 C16H32O2 n Hexadecanoic acid Organic acid HO CH3
23 13.8 C12H18O4 3-methyl-4-propyl -, polyacetyline CH3 O dimethyl ester ,(E,Z) H3C O -2,4-Hexadienedioc O CH3 H3C O acid
103
24 14.1 C6H6O2 5- methyl-2- Pheromone furancarboxaldehyd e O CH3 O
O 25 16 C18H32O2 9,12 Polyacetyline octadecadienoic acid OH (z,z) \
26 16.1 C17H36O n –Heptadecanol -1 Alcohol OH
27 16.1 C10H16 Tricyclo Cycloalkane H H (4.3.1.0(2,5) decane
28 16.4 C18H36O2 Octadecanoic acid Organic acid OH
O
29 21.5 C19H38O4 2- hydroxyl -1- Organic acid O
hydroxyl O methylethylester; OH Hexadecanoic acid
104
30 24 C19H34 E,Z – 1,3,12 – nonadecatriene polyacetyline
31 24.3 C19H32O2 Methyl ester (z,z,z) Organic acid H3C O – 9,12,15 – CH3 octadecatrienoic O acid H3C CH3 CH3 O 32 28.8 C H O Beta – Triterpenoid CH3 44 76 3 O CH3 H3C CH3 myristoylolean – 12- H3C en-16. Beta- ol H3C CH3
33 33.9 C29H48O Gamma – sitosterol Steroid
HO
34 32.7 C29H48O Stigmasterol Steroid
HO
35 35.3 C15H26 1,3 – dimethyl -5- n- Cycloalkane CH3 propyl adamantane
CH3 CH3
36 35.7 C30H50O Alpha- amyrin CH3 triterpene CH3 CH3
H3C CH3 H3C CH3 H3C CH3
37 37 C18H3O (5. alpha., Steroid 14.alpha.) d - H3C
norandrostane CH3
105
Appendix 7b: GC-MS data for the crude DCM extract
NO. RT FORMULA NAME CLASS PROPOSED STRUCTURE H C 1 4.1 C8H10O2 1,3 – Phenol 3 O dimethoxy -
Benzene H3C O
2 4.2 C10H20O Decanal Aldehyde
O
O 3 4.9 C9H18O2 Nonanoic Organic acid OH acid
4 5.4 C12H22 Dodecyne Acetylene
5 6.7 C15H24 octahydro – 7-1 H – CH3 H3C CH2 Cyclopentan [1,3] CH3 Cyclopropan[ 1,2] benzene
6 7.1 C15H24 Cryophyllene Sesquiterpene
CH2 CH3 CH3
H3C
7 7.7 C15H24 Alpha Sesquiterpene CH3
muurolene CH3 H3C CH3
106
8 7.9 C13H28 4-ethyl- Alkane undecane
9 7.9 C15H24 1,2,3,5,6,7,8, H2C
8a-octahydro H3C H3C CH3 – 1,8a- dimethyl – 7- (1-methyl ethenyl)-,[15- (1. Alpha, 7 alpha, 8a. alpha) ]- naphthalene
10 8.7 C12H24O2 Dodecanoic Organic acid OH acid O
11 8.8 C8H8O3 2,4 Phenol H3C didhydroxy -
6- methyl – OH O Benzaldehy- OH de
12 9.0 C13H28O n-tridecan -1 Alcohol -ol OH
13 9.0 C15H22 1,2,3,4,4a,5,8 ,9,12,12a-
decahydro - 1,4-methano benzocyclo- dene
107
14 9.1 C12H2O 1,5- diethenyl -2, 3- H3C CH3
dimethyl CH2
cyclohexane CH2
15 9.6 C15H24O Octahydro- CH3 3,8,8- H3C
trimethyl -6- CH3 metthylene H2C HO 1H-3a, 7- methano azulen-5-01
16 9.9 C9H10O4 Methyl 2,6 – Phenol
dihydroxy -4- HO methyl O H3C CH3 benzoate O OH
17 10.2 C15H26O Alpha- H3C OH Bisabolol CH3 H3C
18 10.2 C13H28 3-methyl -5- Alkane propylnona- ne
19 10.4 C11H14O3 2,6- Phenol dimethoxy - CH3 CH2 O 4- (2- HO O propenyl)- CH3 phenol
20 10.5 C H O 2,4-dihydro- 10 12 4 HO CH3 O 3,6-dimethyl- OH H3C O methyl ester H3C benzoic acid
108
21 10.8 C10H12O3 4-(16)3- Phenol HO Hydroxy-1- H3C OH propenyl)-2- O
methoxy phenol
22 11.0 C14H28O2 Tetradecano- Organic acid O ic acid HO
23 11.2 C H O 4-isopropyl – 9 14 2 O CH3 5, 5- dimethyl CH3
– 5H-furan-2- CH3 one H3C
24 11.3 C14H30O Tetradecanol Alcohol OH
25 11.4 C10H18O (R,S)- 5- Ketone ethyl-6- methyl-3E- hepten-2-one O
26 5,8- Sesquiterpen- dihydroxy- oid O 11.8 C15H22O3 4a-methyl- OH H3C 4,4a,4b,5,6,7, 8,8a,9,10- HO decahydro- 2(3H)-
phenanthren- one
27 11.8 C15H30O Pentadecanal Aldehyde O
109
28 11.9 C18H36O 6,10,14- Ketone CH3 CH3 CH3 CH3 trimethyl-2-
pentadecanon O e
29 12.1 C15H30O2 Pentadecano- Organic acid OH ic acid O
30 12.2 C24H40O2 Methylester - Organic acid 10;12- O
tricosadiynoic acid
31 6 beta- Sesquiterpene OH OH hydroxyl-17- 12.6 C H O O 19 30 4 oxo-4,5-se coandrostan- H3C H3C O 4-oic acid
32 Methyl ester Organic acid O
hexadecanoic O 13 C17H34O2 acid
33 13 C15H24O 4,6-di-tert- CH3 butyl-m- H3C H3C OH H3C cresol CH3
H3C CH3
34 13.2 C16H24O P –octylaceto Phenol phenone
H3C O
35 13.7 C16H32O2 n- Organic acid O Hexadecanoic OH acid
110
36 13.9 C22H44 1-Docosene Alkene
37 14.0 C11H14O4 3,4- Organic acid O Diethoxybenz O
oic acid OH
O
38 14.2 C15H20O2 Eudesma- Sesquiterpene CH3 CH2 5,11(13)- O dien-8,12- O olide H3C
39 14.5 C20H38O4 Decyl-2- Organic acid ethylhexyl O O
ester oxalic O O acid
40 14.9 C10H16 Tricycle[4.3.1 Cycloalkane H .0] (2,5)} H decane
41 15.2 C13H26O2 Formic acid, Organic acid O dodecylester O
42 15.4 C12H26 3,8 – Alkane dimethyl decane
111
43 15.7 C20H40O Phytol Diterpene
HO
44 16.2 C11H18 I-ethenyl -2- Cyclic hydroc- H2C hexenyl-, (1. arbon Alpha., 2.
Beta.(E) (.+1- .)- cyclopropane
O 45 16.4 C16H16O3 (4- Flavonoid O methoxyphen O yl) methylester Benzene
46 16.5 C18H36O2 Octadecanoic Organic acid acid OH O
47 16.8 C17H36O n- Alcohol heptadecanol- OH 1
48 18.4 C16H34 Hexadecane Alkane
49 19.2 C10H12O3 Alpha- Ester O methoxy-, O methylester,(. O
+1-,)- Benzene acetic
112
50 C21H40O2 4,8,12,16- Vitamin Tetramethyl O 19.3 O heptadecan-4- olide
51 19.8 C19H38 I-nonadecene Alkene
52 19.9 C20H38O4 2-ethyl hexyl Organic acid O
isohexylester O O adipic acid O
53 20.4 C15H30O Pentadecanal Aldehyde O
54 21.3 C22H46O Behenic Alcohol HO alcohol
55 21.4 C24H5O Tetracosane Alkane
HO 56 21.6 C19H38O4 2-Hydroxy-1- O O (hydroxyl- HO methyl) ethylester
57 22.2 C24H38O4 Di(2- Organic acid propylpentyl) O
ester phthalic O CH3 O acid CH3 O
113
58 22.8 C18H38O octadecanal Alcohol OH
59 22.8 C16H34 Hexadecane Alkane
60 23.9 C16H14O5 I-hydroxy- Naphthoqu- O O O 3,6- inone O dimethoxy-8-
methyl-9H- xanthen -9- one
61 24.2 C16H14O5 Ethylester Fatty acid O linoleic acid O
62 24.3 C15H24 4-methylene - Sesquiterp-ene 2,8,8- trimethyl-2- vinyl Bicyclo (5.2.0) nonane
63 25.1 C12H26 3,8-dimethyl- Alkane Decane
64 26.1 C30H5 Squalene Triterpenes
65 27.6 C17H14O5 5-hydroxy-7- Flavonoids HO O metthoxy-2-
(4methoxyph O O enyl) 4H-1- O benzopyram- 4-one
114
66 29.8 C21H41 Heneicosane Alkane
67 30.1 C27H26O Cholesterol Steroid HO
68 30.9 C28H46O (3. beta.) Steroid HO 22E, 24s) Ergosta-5,22 –dien -3-oI
69 32.1 C28H48O (3.beta.)- Steroid HO ergost-5-en- 3-oI.
HO 70 32.8 C29H48O Stigmasterol Steroid
71 33 C28H48O (3.alpha. 5, Steroid HO beta; 22E)
Ergost-22-en H3C H3C CH3 3-oI(fungi H3C CH3 steroid H3C
72 4,5,7- Flavonoids O O O trimethoxy-3- H3C 33.9 C19H18O6 H3C (4- O O CH3 H3C O methoxyphen yl)-2H-1- Benzopyran- 2-one
115
73 34.2 C29H50O Gamma- Steroid HO sitosterol CH3 CH3 CH3 H3C
74 34.8 C30H50O beta. Amyrin Triterpenes CH3 CH3
CH3 H3C H C CH 3 3 CH3
H3C OH
75 35.5 C20H30O2 (5. alpha.)- Diterpene Androst-6-
ene-17- H3C O carboxyllic H3C OH
76 36.5 C30H48O Lup-20(29)- Triterpenes O H3C H C en-3-one 3 H C 3 CH2 H3C
H3C CH3 CH3