Isolation and Structural Elucidation of Antibacterial Triterpeniods Compounds from Stem Bark of Psorospermum Febrifugum (Spach Var

ISOLATION AND STRUCTURAL ELUCIDATION OF ANTIBACTERIAL TRITERPENIODS COMPOUNDS FROM STEM BARK OF PSOROSPERMUM FEBRIFUGUM (SPACH VAR. FEBRIFUGUM)

BY JENNIFER NAMBOOZE BSc Industrial Chemistry (Hons) , Mak 2015/HD13/1655U

A DISSERTATION SUBMITTED TO THE DIRECTORATE OF RESEARCH AND GRADUATE TRAINING IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF MASTER OF SCIENCE (CHEMISTRY) OF MAKERERE UNIVERSITY

OCTOBER, 2019

DEDICATION

With honor, I dedicate this thesis to my parents, Mr. and Mrs. Ssetimba, my brother Arnold Phillip Kasumba, my sisters, Dorothy Namata and Marion Kugonza, my friends Mariana Namugwe, Barbara Nayiga, Cissy Nanteza, Dianah Nnakayima, Khadija Nanjala and my employer, Mr. Bitew Marcos.

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ACKNOWLEDGMENT

A number of people deserve thanks for their support towards the successful completion of this work. It is therefore my greatest pleasure to express my gratitude to them all in this acknowledgement.

First of all, I would like to convey my deepest gratitude and appreciation to my supervisors: Prof. Robert Byamukama and Dr Jane Namukobe for their generous guidance, encouragement and support from the start until the end of my study.

My sincere appreciation goes to Dr. Susanna Bonnet of the University of Free State for availing me with NMR and MS instruments, as well as solvents and guidance during my short research stay in South Africa. I am very grateful to Ms. Madina Adia for her help, support and guidance at the beginning of this course of study.

My warm gratitude goes to the members of the natural products research group: Mr Peter Ssekandi, Mr. Peter Kavuma for the valuable support, guidance and encouragement that they offered me throughout this study.

I thank my dear mother, Mrs Grace Nakayiza, for not only being a strong pillar in my life but also for being the most comfortable shoulder to lean on.

TABLE OF CONTENTS

DECLARATION...... i DEDICATION...... iii ACKNOWLEDGMENT ...... iv ABBREVIATIONS ...... xi ABSTRACT ...... xii CHAPTER ONE: INTRODUCTION ...... 1 1.1Background ...... 1 1.2 Statement of the problem ...... 3 1.3 Objective ...... 3 1.4 Justification of the study ...... 4 CHAPTER TWO: LITERATURE REVIEW ...... 5 2.1 Plants as sources of medicine ...... 5 2.1.1 Plants in Uganda used to treat bacterial infections ...... 7 2.2 Psorospermum species ...... 8 2.2.1 Medicinal uses of the genus Psorospermum ...... 8 2.2.2 Pharmacological Investigation of members of Psorospermum genus ...... 8 2.2.3 Some of the compounds that have been isolated from the genus Psorospermum ...... 10 2.3 Psorospermum febrifugum ...... 10 2.3.1 Medicinal uses of Psorospermum febrifugum ...... 11 2.3.2 Bioactive compounds isolated from Psorospermum febrifugum ...... 12 2.3 A review of some analytical methods used in natural products research ...... 13 2.3. 1 Extraction...... 14 2.3.2 Chromatographic Methods ...... 15 2.4 Spectroscopic Techniques ...... 20 2.4.1. Nuclear Magnetic Resonance Spectroscopy (NMR) ...... 20 2.4.1.1. One Dimensional NMR ...... 20

2.4.1.2. Two dimensional NMR ...... 21 2.4.2 Mass spectrometry (MS) ...... 23 CHAPTER THREE: MATERIALS AND METHODS ...... 24 3.2 Preparation and Partitioning of the Crude Extract ...... 24 3.3 Antibacterial Activity Tests ...... 24 3.4 Phytochemical Analysis ...... 27 3.4.1 Test for alkaloids (Dragendorff‟sreageant) ...... 27 3.4.1 Test for anthroquinones (Borntrager‟s test) ...... 27 3.4.2 Test for flavoniods (sodium hydroxide test) ...... 27 3.4.3 Test for steroids (Liebermann-Burchardt test) ...... 27 3.4.5 Test for terpeniods (Salkowski test) ...... 27 3.4.7 Test for saponins (foam test) ...... 28 3.4.8 Test for phenols (ferric chloride) ...... 28 3.4.9 Test of Carbohydrates ...... 28 3.4.10 Test for glycoside ( Kellar-Kiliani) ...... 28 3.5 Isolation and Purification of Antibacterial Compounds from the Ethyl acetate Crude extract...... 28 3.5.1 Thin layer chromatography ...... 28 3.5.2 Column chromatography ...... 29 3.5.3 Structure determination of the isolated pure compounds ...... 30 3.4. 3.1 Nuclear magnetic resonance (NMR) spectroscopy ...... 30 3.5.3.2 Mass Spectrometry (MS) ...... 31 3.6 Melting Point Determination of the Isolated Pure Compounds ...... 31 CHAPTER FOUR: RESULTS AND DISCUSSION ...... 32 4.1 In -Vitro Activity of the Extracts Against Selected Bacteria ...... 32 Phytochemical screening ...... 35 4.3 Characterization of the compounds from ethyl acetate extract ...... 37 4.3.1 Compound 1 ...... 37 4.3.1.1 1H NMR spectrum of compound 1 ...... 37 4.3.1.2 13C NMR spectrum of compound 1 ...... 38 4.3.1.3 1H-1H COSY of compound 1 ...... 40

4.3.1.4 HSQC for compound 1 ...... 41 4.3.1.6 Mass spectrum for compound 1...... 43 4.3.2 Compound 2 ...... 46 4.3.2.1 1H NMR spectrum of compound ...... 46 4.3.2.2 13C NMR spectrum of compound 2 ...... 47 4.3.2.3 1H-1H COSY of compound 2...... 48 4.3.2.4 HSQC for compound 2 ...... 49 4.3.2.5 HMBC for compound 2 ...... 50 4.3.2.6 Mass spectrum of compound 2 ...... 51 4.3.3 Compound 3 ...... 54 4.3.3.1 1H NMR spectrum of compound 3 ...... 54 4.3.3.2 13C NMR spectrum of compound 3 ...... 55 4.3.3.3 1H-1H COSY of compound 3...... 56 4.3.3.4 HSQC for compound 3 ...... 57 4.3.3.5 HMBC for compound 3 ...... 58 4.3.3.6 Mass spectrum of compound 3 ...... 59 4.4 Melting point determination for compound 1, compound 2 and compound 3 ...... 62 4.5 Antibacterial activity of compound 1, 2 and 3 ...... 62 CHAPTER FIVE: CONCLUSION AND RECOMMENDATION ...... 65 5.1 CONCLUSION ...... 65 5.2. RECOMMENDATION ...... 65 CHAPTER SIX: APPENDICES ...... 66 REFERENCES ...... 70

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LIST OF TABLES Table 4. 1: Zone of inhibition of plant extracts against Pseudomonas aeruginosa ,Escherichia coli, Staphylococcus aureus and Streptococcus pyogenes…………………………………… ...... 32 Table 4.2: Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the crude extract ...... 34 Table 4.3 Phytochemical/ metabolites found in the ethyl acetate crude extract for Psorospermum febrifugum...... 35 Table 4.4 : 1H and 13C NMR spectral data together with the literature values for 1H and 13C NMR spectral data for compound 1 in MeOH ...... 45 Table 4.5: 1H and 13CNMR spectral data together with the literature values for 1H and 13C NMR spectral data for compound 2 in acetone ...... 53 Table 4.6 : 1H and 13C NMR spectral data together with literature values for 1H and 13C NMR spectral data for compound 3 in MeOH ...... 61 Table 4.7: Melting point for compound 1, compound 2 and compound ...... 62 Table 4.8: Zone of inhibition of compound 1, 2 and 3 against Pseudomonas aeruginosa and Staphylococcus aureus ...... 63 Table 4.9 : Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the compound 1, compound 2 and compound 3 ...... 63

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LIST OF FIGURES

Figure 2.1: The leaves of Psorospermum febrifugum ...... 11 Figure 2.2: The bark of Psorospemum febrifugum ...... 11 Figure 2.3 : Structure of 2-isopentenylemodine from the berries (Botta et al, 1983) ...... 12 Figure 2.4: The structure of xanthonoligniod cadensin from the root bark (Abou-Shoer et al., 1989) ...... 13 Figure 3.1 : Summary of ...... 12 Figure 4.1.: plates showing zone of inhibition for the ethyl acetate extract against the four tested bacteria strains...... 33 Figure 4.2: plates showing the minimum inhibitory Concentration (MIC) tests for the ethyl acetate ...... 34 Figure 4.3: plates showing the minimum bactericidal Concentration (MBC) for the ethyl acetate extract ...... 35 1 Figure 4.4: H NMR spectrum in MeOD at 600 MHZ of compound 1 ...... 38 13 Figure 4.5: C NMR spectrum in MeOD at 150 MHZ of compound 1 ...... 39 13 Figure 4.6: C NMR-APT spectrum in MeOD at 150 MHZ of compound 1 ...... 39 1 1 Figure 4.7: H- H COSY in MeOD at 600 MHZ in MeOD of compound 1……………...... 38

Figure 4.8: HSQC in MeOD at 600 MHZ for compound 1 ...... 39

Figure 4.9: HMBC in MeOD at 600 MHZ for compound 1 ...... 42 Figure 4.10: EI-MS for compound 1 ...... 43 Figure 4.11: The structure of compound 1 (betulinic acid) ...... 44 1 Figure 4.12: H NMR spectrum in CD3COCD3 at 400 MHZ of compound 2 ...... 46 13 Figure 4.13: C NMR spectrum in CD3COCD3 at 400 MHZ of compound 2 ...... 47 1 1 Figure 4.14: H- HCOSY in CD3COCD3 at 400 MHZ of compound 2 ...... 48

Figure 4.15: HSQC in CD3COCD3 at 400 MHZ of compound 2 ...... 49

Figure 4.16 : HMBC in CD3COCD3 at 400 MHZ of compound 2 ...... 48 Figure 4.17: EI-MS of compound 2 ...... 49 Figure 4.18: The structure of compound 2 (oleanolic acid) ...... 50 1 Figure 4.19: H NMR spectrum in MeOD at 600 MHZ of compound 3 ...... 54

13 Figure 4.20: C NMR spectrum in MeOD at 600 MHZ of compound 3 ...... 55

Figure 4.21: COSY in MeOD at 600 MHZ of compound 3 ...... 56

Figure 4.22: HSQC in MeOD at 600 MHZ for compound 3 ...... 57

Figure 4.23: HMBC in MeOD at 600 MHZ for compound 3 ...... 58 Figure 4.24: EI-MS of compound 3 ...... 59 Figure 4.25: The structure of compound 3 (oleanolic acetate acid) ...... 58 Figure 6.1: TLC plates showing the different forms of solvent system for the ethylacetate extract ...... 66 Figure 6.2: Packing of the column using silica gel as the stationary phase ...... 66 Figure 6.3: TLC plates showing a pure sample for compound 1 and compund 2 repectively .... 67 Figure 6.4: TLC plates showing a pure sample for compound 3 ...... 67 Figure 6.5: Plates showing the zone of inhibition for compound 1,2 and 3 against S. aureus .... 68 Figure 6.6: Plates showing the zone of inhibition for compund 1,2 and 3 against P. aeruginosa...... 68 Figure 6.7: Plates showing MIC for compound 1 against S. aureusa and P.aeruginosa ...... 68 Figure 6.8: Plates showing MIC for compound 2 against S. aureus and P.aeruginosa ...... 69 Figure 6.9: Plates showing MIC for compound 3 against S. aureusa and P.aeruginosa ...... 69

ABBREVIATIONS

NMR: Nuclear Magnetic Resonance Spectroscopy 13C NMR: C-13 Nuclear Magnetic Resonance Spectroscopy 1H NMR: Proton Nuclear Magnetic Resonance Spectroscopy 1 D: One Dimensional 2 D: Two Dimensional COSY: Correlation spectroscopy DEPT: Distortionless Enhancement by Polarization Transfer HMBC: Heteronuclear Multiple Bond Correlation HSQC: Heteronuclear Single Quantum Coherence Hz: Hertz MIC: Minimum Inhibitory Concentration MBC Minimum Bactericidal Concentration NOESY: Nuclear Overhauser Effect Spectroscopy TLC: Thin Layer Chromatography

ABSTRACT

In this study, Psorospermum febrifugum stem bark was investigated for the antibacterial compounds present in its stem bark. The stem bark of Psorospermum febrifugum was collected in Mpigi District, Misindye hill in Buwungu village- Buwama sub country in Uganda. Extraction of the plant material was done using ethyl acetate and methanol in a ratio of 1:1. The crude extract was separated using a separating funnel into hexane extract, ethyl acetate extract and methanol extract. The crude extracts (hexane, ethyl acetate and methanol) were screened for antibacterial activity against P. aeruginosa, E. coli, S. aureus and S. pyogenes using the modified agar diffusion method. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were done on the most active antibacterial crude extract using broth diffusion method. Phytochemical screening was done on the active antibacteraial (ethyl acetate) extract. Isolation and purification of the compounds on the most active antibacterial (ethyl acetate) extract was done using a combination of column chromatography and thin layer chromatography. The pure compounds were identified using 1D and 2D 1H and 13C NMR techniques as well as Mass spectrometry (MS). The results of the bioactivity tests carried out in this study indicate that Psorospermum febrifugum has potential antibacterial activity. The ethyl acetate extract showed the highest zone of inhibition of (19.1± 0.14) mm and (18.3±0.07) mm against S. aureus and S. pyogenes respectively. The hexane extract showed no zone of inhibition against any tested bacterial strain and the methanol extract had moderate antibacterial activity. Phytochemical screening of ethyl acetate extract confirmed the presence of terpeniods, phenols, reducing sugar, carbohydrates and tannins. Three compounds, Betulinic acid (1), Oleanolic acid (2) and Oleanolic acetate acid (3) were identified and characterized from the ethyl acetate extract. A promising antibacterial activity was exhibited by all the three compounds. Compound 2 exhibited the highest zone of inhibition of (14.2± 0.07) mm against both bacterial strains. Compound 3 exhibited the lowest zone of inhibition of (6.5± 0.2) mm against S.aureus and (8.1±0.14) mm against P.aeruginosa and compound 1 showed a moderate zone of inihibition. This justifies the use of this plant in traditional medicine and indicates a promising potential for the development of medicinal agents from Psorospermum febrifugum stem bark.

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CHAPTER ONE: INTRODUCTION

1.1Background

Investigations on plants with medicinal properties and identification of the chemical components responsible for their activities have justified the ancient traditional healing wisdom and have proven the enduring healing potential of many plant medicines (Babu et al., 2009). Wild plants have always been a major source of primary health care and other necessities of daily life for local communities throughout the world, an indication that medicinal plants can provide the best alternative sources to obtain a variety of drugs (Idamokoro et al., 2013).

Despite the extensive use of antibiotics and vaccination programmes, infectious diseases continue to be a leading cause of morbidity and mortality worldwide (WHO, 2014). The worldwide burden of microbial infections has increased in recent years with 3.3 million cases of protozoan, viral and infections reported annually ( WHO, 2013; WHO, 2014). By the year 2012, several approaches including chemotherapy, ethno-medicine, vector control and vaccination have been reinforced to control different microbial infections (WHO, 2002 ; WHO, 2012). However, the increasing prevalence of multidrug resistant bacteria, the recent appearance of strains with reduced susceptibility to antibiotics, the side effects associated with antibiotics, the high costs of antimicrobial drugs and the re-emergence of diseases like tuberculosis are the key factors that obstruct resonant management of bacterial infections in many developing countries including Uganda (Kisangau et al, 2007). The increased prevalence of HIV/AIDS virus has also in the recent past augmented the magnitude of many bacterial and fungal opportunistic infections with frequent episodes resulting from the immune suppression of the affected persons (Jiofack et al., 2010).

Medicinal plants are possible sources of antimicrobial agents (Sati and Joshi, 2011). The discovery of modern drugs such as copsin, eugenol, etc from medicinal plants signifies the enormous potential that still exists for the production of many more novel pharmaceuticals (Geyid et al., 2005). For this reason, the ethno pharmacology of medicinal plants has fascinated increasing attention in new drugs research and development (Jazari et al., 2011). Globally, millions of people rely on traditional remedies due to the high prices of most pharmaceutical drugs, the limited availability of the drugs, lack of access to medical clinics and hospitals by the

1 rural population of developing countries, the various side-effects and toxicities of modern synthetic drugs (WHO, 2014). This further gives good reasons for the search for alternative products from plants used in traditional medicine.

The World Health Organization, (2000) defines traditional medicine as “the diverse health practices, approaches, knowledge and beliefs incorporating plant, animal and/or mineral based medicines, spiritual therapies, manual techniques and exercises applied singularly or in combination to maintain well-being, as well as to treat, diagnose or prevent illness”. Traditional medicine utilizes biological resources and the indigenous knowledge of plant groups conveyed verbally through generations. This is closely linked to the conservation of biodiversity and the related intellectual property rights of indigenous people (Timmermans, 2003). It is however necessary to validate the information through an organized infrastructure for it to be used as an effective therapeutic means, either in conjunction with existing therapies or as a tool in novel drug discovery. Although it is these traditional medicines that provide the link between medicine and natural products, it was not until the 19th century that active compounds were isolated and principles of medicinal plants identified (Phillipson, 2001). The isolation of morphine from opium started the chemistry of natural products (Patwardhan et al, 2005).

In Uganda, up to 80% of the population uses traditional medicine due to the cultural acceptability of healers and local pharmacopeias, the relatively low cost of traditional medicine and difficult access to modern health facilities (Hamill et al., 2000; UNAS, 2015; CDCP, 2015). This makes Uganda to be well known for its rich ethno botanical prosperity, particularly regarding medicinal plants which are traditionally used in the treatment of diseases and could be an excellent source for finding of new and biodegradable drugs ( Tabuti et al., 2003 ; Basha et al., 2010). Because there is an extensive spread of many infectious disease in Uganda, they are a number of medicinal plant species prescribed traditionally against infectious diseases ( Tabuti et al., 2003; Namukobe et al., 2011).

In Uganda, Psorospermum febrifugum is known locally as, akanzironziro (Luganda). The bark of Psorospermum febrifugum is harvested locally and its decoction is used for the treatment of various skin problems including leprosy, the parasitic disease craw-craw, scabies, rashes eczema and insect bites (Rukangira et al., 2001; Tabuti et al., 2003; Lamorde et al., 2010). However

Psorospermum febrifugum stem bark have not been investigated for its efficacy and chemical constituent. Therefore, this research isolated, characterized and identified the antibacterial compounds from the stem bark of Psorospermum febrifugum which are used to traditionally treat bacterial infection.

1.2 Statement of the problem

Psorospermum febrifugum stem bark is traditionally used in treatment of several skin infections such as scabies, leprosy, wounds and insect bites (Lamorde et al., 2010 ). However, the compounds that could be responsible for the physiological action of this traditional remedy are not known. This has not only hindered the standardization and formulation of this herb drug, but also limits its recognition; acceptance and utilization. Therefore, this study was aimed at isolating, characterization and identification of the bioactive compounds in the stem bark of Psorosperumum febrifugum that could be responsible for some of its medicinal properties.

1.3 Objective

1.3.1 General Objective

To characterize the compounds in the stem bark extract of Psorosperumum febrifugum which are responsible for its antibacterial properties.

1.3.2 Specific Objective

The general objective will be achieved through the following specific objective:

1. To determine the antibacterial activities of crude extracts from the stem bark of Psorosperumum febrifugum.

2. To isolate the phytochemical constituents of the active antibacterial crude extract from stem bark of Psorosperumum febrifugum and elucidate the chemical structures of the isolated compounds. 3. To determine the antibacterial activities of isolated compounds from the stem bark of Psorosperumum febrifugum.

1.4 Justification of the study

The resistance of Staphylococcus aureus to the commonly synthetic antimicrobials has called for more research in drug discovery in order to discover new chemotherapeutic compounds which will enhance effective treatment. Psorospermum febrifugum herbal medicine has been used to treat bacterial infections but difficult to standardize. The identified bioactive compounds can be used as starting material for production of drugs and may as well be used as markers for the standardization of herbal formulations of Psorosperumum febrifugum. The bioactivity results can be used to validate the use of this plant hence providing preliminary scientific justification for the traditional medicinal uses of this ethno remedy, an important step towards its acceptance and development as alternative therapeutic agent. Therefore this research may establish the antibacterial activity, identify compounds in plant material and this can help in formulation of products based on the information from the findings.

CHAPTER TWO: LITERATURE REVIEW

2.1 Plants as sources of medicine

Several investigations and modern methods have confirmed folkloric accounts of the use of higher plant preparations for the treatment of infections. The natural products have reasonable potency, are comparatively easy to synthesize and their synthetic analogues can possess enhanced therapeutic potential (Aburjai et al., 2001). In Traditional Chinese Medicine (TCM) for example, many medicinal herbs have been used for hundreds of years to treat respiratory complaints such as bronchial inflammation, pneumonia, expectoration and cough, and have shown less or no side effects as compared to synthetic drugs (Shang et al., 2010).

Plant metabolites include compounds that aid in the growth and development of plants; however some of them are not required for the plant to survive (Dos Santos et al., 2015). Each plant family, genus, and species produces a characteristic mix of these chemicals, and they can sometimes be used as taxonomic parameters in classifying plants. Humans use some of these compounds as medicines, flavoring agents, or recreational drugs such as the alkaloids, nicotine, cocaine, and the terpene cannabinol (Owolabi et al., 2013).

Each plant is unique in its nature because of its biodiversity and certain medicinal value it has. Medicinal values of the different plant species is due to the presence of unique type of chemical compounds. These chemical compounds vary in different plant species. Each plant species contains different concentration of the chemical compound inside of plant body. This is because of the plant types, their age, plant health and also regulated by the effect of the environmental variables (Patel et al., 2015).

The search for medicines, which undoubtedly began in prehistorical times, has led to compounds such as copsin, engueol, penicillin, morphine, atropine, tubocurarine, quinine, anticholinestrace and digoxin which have been isolated from plants (Khan et al., 2006).

Copsin is an antimicrobial polypeptide secreted from the inky cap mushroom (Poumale et al., 2008). Copsin is a novel fungal antimicrobial peptide that binds in a unique manner to the cell wall precursor lipid II (Poumale et al., 2008). It was reported to be potent in the petri dish against Gram positive bacteria which have a cell wall, including Enterococcus faecium and Listeria monocytogenes.

Eugenol is a naturally occurring phenolic molecule found in several plants such as cinnamon, clove, and bay leaves (Venskutonis et al., 2009). It has been used as a topical antiseptic as a counter-irritant and in dental preparations with zinc oxide for root canal sealing and pain control. Eugenol has been found to have anti-inflammatory, antibacterial, neuroprotective, antipyretic, antioxidant, antifungal and analgesic properties (Venskutonis et al., 2009).

Penicillin is naturally produced from pencillium molds (Zubair et al., 2009). Penicillin is a group of antibiotics which include penicillin G (intravenous use), penicillin V (use by mouth), procaine penicillin, and benzathine penicillin (intramuscular use).Penicillin antibiotics were among the first medications to be effective against many bacterial infections caused by staphylococci and streptococci (Zubair et al., 2009).

Morpine was first isolated from poppy straw of the Opium poppy (Martı et al., 2002). Morphine is a pain medication of the opiate variety which is found naturally in a number of plants and animals. It acts directly on the central nervous system (CNS) to decrease the feeling of pain. It can be taken for both acute pain and chronic pain. It is frequently used for pain from myocardial infarction and during labor. It can be given by mouth, by injection into a muscle, by injecting under the skin, intravenously, into the space around the spinal cord, or rectally (Butler et al., 2004).

Digoxin was first isolated from the foxglove plant, Digitalis lanata (Toyang et al., 2013) Digoxin, sold under the brand name Lanoxin among others, is a medication used to treat various heart conditions. Most frequently it is used for atrial fibrillation, atrial flutter, and heart failure (Toyang & Verpoorte, 2013). Digoxin is taken by mouth or by injection into a vein.

Quinine was first isolated from the bark of Cinchona officinalis (Nguta et al., 2010). Quinine is a medication used to treat malaria. Malaria is a life-threatening disease caused by Plasmodium parasites that are transmitted to people through the bites of infected female Anopheles mosquitoes. There are 5 parasite species that cause malaria in humans, and 2 of these species – P. falciparum and P. vivax – pose the greatest threat (Jaiswal et al., 2016)

2.1.1 Structures of some compounds isolated from medicinal plants described above

2.1.1 Plants in Uganda used to treat bacterial infections

In Uganda, many plants have been used to treat bacterial infections. Plants like Mangifera indica l (common name: mango) has been used as gargle to treat mouth infections in children and the infusion of its leaves singly or combined with leaves of Citrus sinensis are used in treating diarrhea, dysentery, gastrointestinal tract disorders, typhoid fever, sore throat and scurvy (Doughari et al., 2008). Aloe vera and other aloe species (common name: Barbados aloe) are used in treating of mouth ulcers, reducing dental plagues, speeding up wound healing and can help inhibit the growth of certain bacteria that can cause infections in humans since it contains polyphenols (Gruca et al., 2015)

The fresh leaves decoction of Eucalyptus grandis, Lantana trifolia, Momordica foetida, Spathodea nilotica, Zingiber officinale roscoe, Thevetia peruviana, Plectranthus barbatus Andrews and Ocimum rothii Bak are used to treat cough (Namukobe et al., 2011). The leaves of Spathodea nilotica seem, Allium cepa and Vigna unguiculata are squeezed and the juice is applied on the ear infection or dropped in the eyes (Namukobe et al., 2011).

2.2 Psorospermum species

2.2.1 Medicinal uses of the genus Psorospermum

A good number of the species have been used for centuries in the ethno medical traditions of indigenous African populations as febrifugal, antidote against poisons, purgative, stomach and as a remedy for the treatment of leprosy, skin diseases (like dermatitis, scabies and eczemas) and subcutaneous wounds (Meepagala et al., 2005). For example, Psorospermum androsaemifolium, the extracts from this plant are used as therapeutic remedies for spiders or scorpions bites and also for healing stomach disease (Mathur et al., 2011). Also Psorospermum corymbiferum, the roots and barks infusion is drunk for treatment of dermal infections such as leprosy, scabies, eczema, and herpes (Kouam et al., 2010) and Psorospermum guineense Hochr, the bark decoction is taken to cure skin disease like scabies, cold sores, eczema, leprosy and psoriasis, syphilis and neuralgia (Djoukeng et al., 2005).

2.2.2 Pharmacological Investigation of members of Psorospermum genus

(i) Antimalarial Activity Bioassay-guided fractionation of the n-hexane extract of the stem bark of Psorospermum glaberrimum showed that this crude extract exerted a good anti-plasmodial activity against P. falciparum W2 strain, with IC50 of 0.87 l g/mL, among the compounds, resulting from the purification of the extract, 3-geranyloxy emodinanthrone and acetylvismione D were seen to exert a good level of anti-plasmodial activity providing IC50 values of 1.68 and 0.12 l M respectively (Ndjakou et al., 2008). The extracts of different polarity from the leaves of P.senegalense were studied as form of anti plasmodial remedy against Plasmodium falciparum.

It was found that the dichloromethane extract exerted a valuable activity (IC50 = 10.03 g/mL) (Epifano, Fiorito, & Genovese, 2013)

(ii) Antimicrobial activity A bianthrone named Adamabianthrone isolated from the bark extract of Psorospermum adamauense was tested against five Gram-positive bacteria (Citrobacter freundii, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Salmonella typhi) and three Gram negative bacteria (Bacillus cereus, Staphylococcus aureus, and Staphylococcus faecalis). Also Adamabianthrone was tested against two fungi (Candida albicans and Microsporum audounii). The lowest MIC value was 27.34 mg/mL against S. faecalis, while other values ranged from 54.68 to 218.74 mg/mL (Tsaffack et al., 2009). Adamabianthrone did not show activity against C. albicans (Tsaffack et al., 2009). The antimicrobial activity of the root extract of Psorospermum corymbiferum was tested and it was found to be active against a wide panel of microorganisms, including bacteria and fungi like B. subtilis, S. typhi, S. aureus, P. aeruginosa, Proteus mirabilis, Bacillus cereus, and C. albicans (Zubair et al., 2009). (iii) Anti cancer activity Kupchan et al., (1980) provided the first evidence of the anti-leukemic properties of Psorospermin isolated from the root of P. febrifugum on P-388 cell line. Also 3- geranyloxyemodin anthrone isolated from the same plant was proven to have anti-leukemic effect on P-388 cell line, although with much less efficacy when compared to Psorospermin (Amonkar et al., 1981). Leet et al., (2008) performed a preliminary screening of the in vitro anti-cancer activity of the dichloromethane and methanol extracts from the wood stems and roots of Psorospermum molluscum Hochr and found that both exerted a potent effect in a panel of mammalian cancer cells with IC50 values which ranged between 0.2 to 4.0 g/ mL.

(iv) Anti-leishmanial activity Extracts of the leaves and root bark of P. guineense obtained after maceration with dichloromethane, methanol and water were tested as anti-leishmanial agents. The result from this study indicated that only the dichloromethane extract of root bark was active against both the extracellular and the intracellular form of leishmania major with percentages of survival of 3.5 and 14.0 % at a dose of 35 g/mL respectively (Ahua et al., 2007).

(v) Anti-inflammatory activity An essential oil obtained by stem distillation from the leaves and roots of P. tenuifolium was assayed for its anti-inflammatory activity using the tetradecanoylphorbol-13-acetate induced ear

9 oedema in mice. At concentration values of 5.0 and 2.5 mg/mL, a significant anti-inflammatory effect with percentages of oedema reduction of 92.3 and 76.9 % respectively was recorded (Zubair et al., 2009).

2.2.3 Some of the compounds that have been isolated from the genus Psorospermum

Anthraquinones, xanthones trepeniods and visminoes are commonly described compounds in the genus psorspermum. Phytochemicals isolated from psorospermum spp. consist of steroids, tannins, terpenes, simple and prenylated anthraquinones, alkaloids, flavonoids, long chain alcohols and hydrocarbons, and simple and prenylated xanthones (Epifano et al., 2013).

Phytochemical studies have been carried out on several psorospermum species which has led to the isolation and characterization of several bioactive compounds like the bark of Psorospermum adamauense which revealed the presence of four anthraquinones, namely emodin , madagascin, 3-geranyloxyemodin and 2-geranylemodine , one anthrone, 3-geranyloxyemodin anthrone. and two bianthrones, bianthrone and adamabianthrone (Tsaffack et al., 2009). The leaves of Psorospermum androsaemifolium which revealed five compounds namely quercetin 8, acanthophorin B,3A-(2A,4Adihydroxyphenoxy) acanthophorin B , 6-hexahydroxy-a-(a-L- rhamnopyranosyl) dihydrochalcone, vismiaquinone , and a- and b-amyrine (Poumale et al., 2008). The roots of Psorospermum aurantiacum which revealed several metabolites including the anthranoids ferruginin B , vismin , vismione D, haronginanthrone, kenganthranol B, kenganthraquinone, psorantin , and kenganthranol E , the xanthones 1,7-dihydroxyxanthone, the sesqui-, di-, and triterpenes paradisiol , friedelan-3-one , friedelan-3-ol , and finally betulinic acid (Kouam et al. 2010). The root bark of Psorospermum corymbiferum which revealed three anthranoids, acetylvismione F, bianthrone A, and 1,8-dihydroxyanthraquinone, and one steroid, diosgenin 34 (Zubair et al., 2011).

2.3 Psorospermum febrifugum

Psorospermum febrifugum belongs to the psorospermum Baker (tribe Vismieae, family Guttiferae, sub family Hypericoideae) which comprises 55 species, most of which are shrubs or small trees typically growing in the tropical regions of South America, Africa, and Madagascar

(Epifano et al., 2013). Psorospermum febrifugum is a shrub or small tree about 4-6 m tall with bumpy stem that grows in open woodland over a wide range of altitude. The inconspicuous flowers are fragrant, creamy-white, densely covered in hairs with 8 mm in diameter. The fruit is a small berry about 6 mm in diameter and bright red when mature. The leaves of Psorospermum febrifugum are opposite, at right angles, very variably pubescent, always much lighter underneath. The bark of Psorospermum febrifugum is fissured; slash dark brown, rough and raggedly scaling (Lamorde et al., 2010).

Figure 2.1: The leaves of Psorospermum febrifugum

Figure 2.2: The stem bark of Psorospemum febrifugum 2.3.1 Medicinal uses of Psorospermum febrifugum

Psorospermum febrifugum is one of the many plants used in folklore medicine to treat a variety of ailments. In Uganda, Psorospermum febrifugum is known locally as, akanzironziro (Luganda).

The bark of Psorospermum febrifugum is harvested locally and its decoction is used for the treatment of various skin problems including leprosy, the parasitic disease craw-craw, scabies, eczema and insect bites (Rukangira et al., 2001; Tabuti et al., 2003; Lamorde et al., 2010). Its bark has also been reported to have antioxidant, anti-acne and anti-lipase activity (Elufioye et al, 2016)

2.3.2 Bioactive compounds isolated from Psorospermum febrifugum

Investigations on the berries of Psorospermum febrifugum provided several anthraquinones, namely 3-geranyloxyemodine3, 2-isopentenylemodine (figure 2.33.1) and vismiones like ferruginin B (Botta et al, 1983). In subsequent years several other studies about the isolation and structural characterization of novel secondary metabolites from the root bark of Psorospermum febrifugum have been reported in the literature. These isolates included the xanthones (Habib et al., 1987), the xanthonolignoids cadensin D (figure 2.3.3.2 ) , isocadensin D and diosgenin (figure 2.3.3.3 ) (Abou-Shoer et al., 1989). The bioactive compounds from the root bark of Psorospermum febrifugum have been isolated and structure elucidated, however bioactive compounds from the stem bark and the leaves of the same plant has not been isolated and characterized. This study enabled us to isolate compounds from the stem bark of Psorospermum febrifugum.

2.3.2.1 Structures of some compounds isolated from Psorospermum febrifugum

2-isopentenylemodine isolated from the berries of Psorospermum febrifugum (Botta et al, 1983)

Figure 2.3: Structure of 2-isopentenylemodine isolated from the berries of Psorospermum febrifugum (Botta et al, 1983)

Diosgenin isolated from the root bark of Psorospermum febrifugum (Abou-Shoer et al., 1989).

Figure 2.4: Structure of diosgenin isolated from the root bark of Psorospermum febrifugum (Abou-Shoer et al., 1989).

Xanthonoligniod cadensin isolated from the root bark of Psorospermum febrifugum (Abou-Shoer et al., 1989).

Figure 2.5: The structure of xanthonoligniod cadensin isolated from the root bark of Psorospermum febrifugum (Abou-Shoer et al., 1989).

2.3 A review of some analytical methods used in natural products research

The qualitative and quantitative studies of bioactive compounds from plant materials mostly rely on the selection of proper methods. In this section, some of the commonly used methods in natural products research are discussed

2.3. 1 Extraction

Extraction is the crucial first step in the analysis of medicinal plants, because it is necessary to extract the desired chemical components from the plant materials for further separation and characterization. Extraction methods are sometimes referred to as “sample preparation techniques”. It is true that the development of modern chromatographic and spectrometric techniques make bioactive compound analysis easier than before but the success still depends on the extraction methods, input parameters and the exact nature of plant parts.

The basic operation includes steps such as, pre-washing, drying of plant materials or freeze drying, grinding to obtain a homogenous sample and often improving the kinetics of analytic extraction and also increasing the contact of sample surface with the solvent system. Proper actions must be taken to assure that potential active constituents are not lost, distorted or destroyed during the preparation of the extract from plant samples. If the plant was selected on the basis of traditional uses, then it is advisable to prepare the extract using a procedure close to the method described by the traditional healer in order to mimic as closely as possible the traditional „herbal‟ drug (Fabricant and Farnsworth, 2001).

The selection of solvent systems largely depends on the specific nature of the bioactive compound being targeted. Different solvent systems are available to extract the bioactive compound from natural products. The solvent to be used during extraction is chosen depending on the polarity of the compounds in the plant for examples if the compounds are non-polar, a non polar solvent like hexane would be used to extract it because there are stronger forces of attraction between a non polar solvent and non-polar compound and vice versa for polar compounds. Occasionally, for better extraction efficiency, mixtures of solvents can be used (Venskutonis et al., 2009). The extraction of hydrophilic compounds uses polar solvents such as methanol, ethanol or ethyl-acetate. For extraction of more lipophilic compounds, dichloromethane or a mixture of dichloromethane/methanol in ratio of 1:1 are used. In some instances, extraction with hexane is used to remove chlorophyll (Cosa et al., 2006).

The most common factors affecting extraction processes are the matrix properties of the plant part, the solvents used, and temperature and extraction time (Ngaha et al., 2017). It is only

14 possible to conduct further separation, identification, and characterization of bioactive compounds if the extraction process has been appropriately done. Bioactive compounds from plant materials can be extracted by various classical extraction techniques. Most of these techniques are based on the extracting power of different solvents used and the application of heat and/or mixing. The commonly used classical techniques are: soxhlet extraction, maceration, infusion, decoction and hydro distillation to obtain a crude extract which is then concentrated using a rotary evaporator (Gradé et al., 2009).

2.3.2 Chromatographic Methods

Chromatography is the method of choice in handling the problem of isolation of a compound of interest from a complex natural mixture. Therefore, the chromatographic methods used during the present work are briefly described.

2.3.2.1 Column chromatography

In column chromatography, the stationary phase (a solid adsorbent) is placed in a vertical glass column and the mobile phase (a liquid) is added to the top and flows down through the column (by either gravity or external pressure). Column chromatography is generally used as a purification technique to isolate desired compounds from a mixture (Kenkel, 2003).

The crude extract to be purified by column chromatography is applied at the top of the column. The liquid solvent (the eluent) is passed through the column by gravity or by the application of air pressure. Equilibrium is established between the solute adsorbed on the adsorbent and the eluting solvent flowing down through the column. Because the different components in the mixture have different interactions with the stationary and mobile phases, they will be carried along with the mobile phase to varying degrees and a separation will be achieved. The individual components, or elutants, are collected as the solvent drips from the bottom of the column (Harvey, 2000).

Silica gel (SiO2), alumina (Al2O3), sephadex, zirconium oxide, Florisil, and ion-exchanger. are the adsorbents commonly used for column chromatography. These adsorbents are sold in different mesh sizes, indicated by a number on the bottle label. The polarity of the solvent which is passed through the column affects the relative rates at which compounds move through the column (Harvey, 2000). Polar solvents can compete more effectively with the polar molecules of

15 a mixture for the polar sites on the adsorbent surface and will also solvate the polar constituents better. Consequently, a highly polar solvent will move even highly polar molecules rapidly through the column. If a solvent is too polar, movement becomes too rapid, and little or no separation of the components of a mixture will result. If a solvent is non polar, non polar compounds will elute from the column. Proper choice of an eluting solvent is thus crucial for the successful application of column chromatography as a separation technique. Often a series of increasingly polar solvent systems are used to elute a column. A non-polar solvent is first used to elute the less-polar compounds. Once the less-polar compounds are off the column, a more-polar solvent is added to the column to elute the more-polar compounds (Kenkel, 2003). Gel permeation chromatography (Size Exclusion Chromatography) is based on the principle that molecules move through a column of gel that has pores of clearly defined sizes. The larger molecules cannot enter the pores, and therefore, they move faster through the column and elute first. Slightly smaller molecules can enter some pores, and so take longer to elute, while small molecules can be delayed further. The advantage of the technique is its simplicity, isocratic, and large molecules rapidly elute. However, the matrices or gels of this kind are expensive and sensitive to contamination; consequently they are mainly used in applications where alternative separation techniques are not available, and samples are fairly clean. The commonly used gel in natural products lab is sephadex used to separate chlorophyll from compounds of interest, where usually chlorophyll elutes first as well as polyvinyl alcohol (PVA50) (König & Hochmuth, 2015).

2.3.2.2 Thin Layer Chromatography

TLC is a simple, quick, and inexpensive procedure that gives the researcher a quick answer as to how many components are in a mixture. TLC is also used to support the identity of a compound in a mixture when the retention factor / retardation factor, Rf of a compound is compared with the retention factor, Rf of a known compound. Additional tests involve the spraying of phytochemical screening reagents, which cause color changes according to the phytochemicals existing in a plants extract; or by viewing the plate under the UV light (Shahverdi et al., 2007). TLC has also been used for confirmation of purity and identity of isolated compounds i.e. used to analyze the fractions obtained from column chromatography to assess whether the fraction

16 contains more than one component and if fractions can be combined without affecting their purity (Tradit et al., 2011). The separation by TLC depends on the relative affinity of compounds towards stationary and mobile phase. The compounds under the influence of mobile phase (driven by capillary action), travel over the surface of the stationary phase. During this movement, the compounds with higher affinity to the stationary phase travel slowly while those with less affinity to the stationary phase travel faster. Thus separation of components in the mixture is achieved. Once separation occurs, the individual components are visualized as spots on the plate after staining with vanillin hydrochloric acid reagent spray (Harvey, 2000).

2.3.2.3 High Performance Liquid Chromatography

High performance liquid chromatography (HPLC) is a versatile, robust, and widely used technique for the isolation of natural products (Cannell, 1998). HPLC can separate a mixture of compounds and it is used in phytochemical and analytical chemistry to identify, quantify and purify the individual components of the mixture (Ngaha Njila et al., 2017). Currently, this technique is gaining popularity among various analytical techniques as the main choice for fingerprinting study for the quality control of herbal plants (Fan et al., 2006). In order to improve resolution, HPLC columns are packed with small sized particles with a narrow size distribution (Ye et al., 2007). Flow rate and column dimensions can be adjusted to minimize band broadening (Lia et al., 2004). The required pressures are supplied by pumps that could withstand the involved chemicals. There are two types of phase i.e. normal phase columns where non polar solvents such hexane are used as the mobile phase and polar surface such as silica is used as the stationary phase. The other phase is reverse phase (RP) columns where polar solvents such as water, methanol, acetonitrile etc are used as the mobile phase and a non polar surface as the stationary phase.

Natural products are frequently isolated following the evaluation of a relatively crude extract in a biological assay in order to fully characterize the active entity. The biologically active entity is often present only as a minor component in the extract and the resolving power of HPLC is ideally suited to the rapid processing of such multicomponent samples on both an analytical and preparative scale. Many bench top HPLC instruments now are modular in design and comprise a solvent delivery pump, a sample introduction device such as an auto-sampler or manual injection

17 valve, an analytical column, a guard column, detector and a recorder or a printer. Chemical separations can be accomplished using HPLC by utilizing the fact that certain compounds have different migration rates given a particular column and mobile phase. The extent or degree of separation is mostly determined by the choice of stationary phase and mobile phase. Generally the identification and separation of phytochemicals can be accomplished using isocratic system (using single unchanging mobile phase system). Gradient elution in which the proportion of organic solvent to water is altered with time and may be used if more than one sample component is being studied and differ from each other significantly in retention under the conditions employed. Purification of the compound of interest using HPLC is the process of separating or extracting the target compound from other (possibly structurally related) compounds or contaminants. Each compound should have a characteristic peak under certain chromatographic conditions. Depending on what needs to be separated and how closely related the samples are, the chromatographer may choose the conditions, such as the proper mobile phase, flow rate, suitable detectors and columns to get an optimum separation. Identification of compounds by HPLC is a crucial part of any HPLC assay. In order to identify any compound by HPLC, a detector must first be selected. Once the detector is selected and is set to optimal detection settings, a separation assay must be developed. The parameters of this assay should be such that a clean peak of the known sample is observed from the chromatograph. The identifying peak should have a reasonable retention time and should be well separated from extraneous peaks at the detection levels which the assay will be performed. UV detectors are popular among all the detectors because they offer high sensitivity (Lia et al., 2004; Tsao, R. and Deng, 2004) and also because majority of naturally occurring compounds encountered have some UV absorbance at low wavelengths (190-210 nm) (Cannell, 1998). The high sensitivity of UV detection is bonus if a compound of interest is only present in small amounts within the sample. Besides UV, other detection methods are also being employed to detect phytochemicals among which is the diode array detector (DAD) coupled with mass spectrometer (MS) (Tsao and Deng, 2004). Liquid chromatography coupled with mass spectrometry (LC/MS) is also a powerful technique for the analysis of complex botanical extracts. It provides abundant information for structural elucidation of the compounds when tandem mass spectrometry (MS) is applied. Therefore, the combination of HPLC and MS facilitates rapid and accurate identification

18 of chemical compounds in medicinal herbs, especially when a pure standard is unavailable (Ye et al., 2007).

The processing of a crude source material to provide a sample suitable for HPLC analysis as well as the choice of solvent for sample reconstitution can have a significant bearing on the overall success of natural product isolation. The usage of guard columns is necessary in the analysis of crude extract. Many natural product materials contain significant level of strongly binding components, such as chlorophyll and other endogenous materials that may in the long term compromise the performance of analytical columns. Therefore, the guard columns will significantly increase the lifespan of the analytical columns.

2.3.2.4 Gas Chromatography (GC) GC is most useful for the analysis of trace amounts of organically extractable, volatile compounds and highly volatile compounds. In GC the mobile phase is gaseous. The mixture to be analyzed is vaporized into the column. The stationary phase in the column can be solid or liquid. In GC, the carrier gas conveys the sample in a vapor state through a narrow column made from usually fused silica tubes (0.1 to 0.3mm ID) that have refined stationary phase films (0.1 to 5μm) bound to the surface and cross linked to increase thermal stability. The column is installed in an oven that has temperature control, and the column can be slowly heated up to 350-450 °C starting from ambient temperature to provide separation of a wide range of compounds. The carrier gas is usually hydrogen or helium under pressure, and the eluting compounds can be detected in several ways, including flames (flame ionization detector), by changes in properties of the carrier (thermal conductivity detector), or by mass spectrometry.

The availability of "universal" detectors such as the FID and MS, makes GC the appropriate tool in the investigation of essential oils. However, GC is restricted to molecules (or derivatives) that are sufficiently stable and volatile to pass through the GC system and remain intact at the operating temperatures (König and Hochmuth, 2015; Niessen, 2001; Rödel and Wölm, 1982).

2.3.2.5 Chromatographic solvent "polarity"

There are four major kinds of intermolecular interactions between sample and solvent molecules in liquid chromatography .i.e. dispersion, dipole, hydrogen-bonding, and dielectric. Dispersion

19 interactions are the attractions between each pair of adjacent molecules, and are stronger for sample and solvent molecules with large refractive indices. Strong dipole interactions occur when both sample and solvent have permanent dipole moments that are aligned together. Strong hydrogen-bonding interactions occur between proton donors and proton acceptors. Dielectric interactions favor the dissolution of ionic molecules in polar solvents. The total interaction of the solvent and sample is the sum of the four interactions. The total interaction for a sample or solvent molecule in all four ways is known as the "polarity" of the molecule. Polar solvents dissolve polar molecules and, for normal phase partition chromatography, solvent strength increases with solvent polarity, whereas solvent strength decreases with increasing polarity in reverse-phase systems (Ernst, Bodenhausen & Wokaun, 1987).

2.4 Spectroscopic Techniques

2.4.1. Nuclear Magnetic Resonance Spectroscopy (NMR)

Spectroscopy is the study of the interaction of electromagnetic radiation (EMR) with matter. NMR spectroscopy is the study of interaction of radio frequency (RF) of the EMR with unpaired nuclear spins in an external magnetic field to extract structural information about a given sample. NMR spectroscopy is routinely used by chemists to study chemical structure of simple molecules using simple one dimensional technique (1D-NMR). Two-dimensional techniques (2D-NMR) are used to determine the structure of more complicated molecules. The organic chemist is principally concerned with the study of carbon compounds. As a consequence, he/she is interested in 1D and 2D NMR involving protons (1H) and carbons (13C).

2.4.1.1. One Dimensional NMR

2.4.1.1.1. 1D-Proton NMR (1H-NMR)

Proton NMR is a plot of signals arising from absorption of radio frequency during an NMR experiment by the different protons in a compound under study as a function of frequency (chemical shift). The area under the plots provides information about the number of protons present in the molecule, the position of the signals (the chemical shift) reveals information regarding the chemical and electronic environment of the protons, and the splitting pattern provides information about the number of neighboring (vicinal or geminal) protons (Sanders & Hunter, 1993; Abraham, Fisher, & Loftus, 1988; Derome, 1987).

2.4.1.1.2. 1D-Carbon NMR (13C-NMR)

Similar to proton NMR, carbon NMR is a plot of signals arising from the different carbons as a function of chemical shift. The signals in 13C-NMR experiments normally appear as singlets because of the decoupling of the attached protons. Different techniques of recording of the 1D carbon NMR has been developed so that it is possible to differentiate between the various types of carbons such as the primary, secondary, tertiary and quaternary from the 1D 13CNMR plot. The range of the chemical shift values differs between the 1H (normally 0-10) and 13C NMR (normally 0-230) that arises from the two nuclei having different numbers of electrons around their corresponding nuclei as well as different electronic configurations (Al-Musayeib et al., 2000).

2.4.1.2. Two dimensional NMR

Currently, the common 2D-NMR experiments that appear in research papers concerned with structural elucidation of natural products include the homonuclear 1H, 1H-COSY as well as NOESY and the heteronuclear 1H, 13C-HMQC as well as HMBC.

2.4.1.2.1 2D Correlated Spectroscopy (1H-1H-COSY)

Correlation spectroscopy is one of the most useful experiments. It is a plot that shows coupling among neighboring protons involving coupling constant, J 2, J 3 as well as J 4. It provides information on the connectivity of the different groups within the molecule (Kessler, Gehrke, & Griesinger, 2014;Ernst, Bodenhausen, & Wokaun, 1987).

2.4.1.2.2. 2D Nuclear Overhauser Enhancement Spectroscopy (NOESY)

Nuclear Overhauser Enhancement Spectroscopy is a homonuclear correlation via dipolar coupling; dipolar coupling may be due to NOE or chemical exchange. It is one of the most useful techniques as it allows to correlate nuclei through space (distance smaller than 5Å) and enables the assignment of relative configuration of substituents at chiral centers (Kessler et al., 2014; Ernst, Bodenhausen, & Wokaun, 1987).

2.4.1.2.3 Heteronuclear Single Quantum Coherence Spectroscopy (HSQC)

Heteronuclear single quantum coherence spectroscopy (HSQC) is used to correlate the chemical shift of protons (displayed on the F2 axis) to the 13C chemical shift (on the “indirect,” F1 axis) of 1 13 their directly attached carbons via the JCH coupling. Taking the natural abundance of C into account, roughly every 100th molecule responds in the HSQC experiment. A particularly useful, so-called phase-sensitive or multiplicity-edited HSQC variant enables making a distinction between carbons bearing an even (CH2) or odd number (CH or CH3) of hydrogens. The advantage of HSQC as compared to the 1D carbon sequences is twofold: firstly, it is a proton- detected experiment, consequently it is more sensitive and less time-consuming to acquire; secondly, it is richer in information since it simultaneously allows the list of directly bound 1H- 13C pairs to be assembled (König & Hochmuth, 2015).

2.4.1.2.4 Heteronuclear Multiple Quantum Correlation (HMQC)

The Heteronuclear Multiple Quantum Correlation experiment provides correlation between protons and their attached heteronuclei through the heteronuclear scalar coupling. This sequence is very sensitive as it is based on proton detection instead of the detection of the least sensitive low gamma heteronucleus carbon. The basic idea behind this experiment is related to the echo difference technique which is used to eliminate proton signals not coupled to the carbon. From this experiment important information regarding the number and chemical shifts of methyl, methylene and methine groups can be extracted (König & Hochmuth, 2015).

2.4.1.2.5 Heteronuclear Multiple Bond Correlation (HMBC)

The HMBC (Heteronuclear Multiple Bond Correlation) experiment gives correlations between carbons and protons that are separated by two, three, and, sometimes in conjugated systems, four bonds. Direct one-bond correlations are suppressed. This gives connectivity information much like a proton-proton COSY. The intensity of cross peaks depends on the coupling constant, which for three-bond couplings follows the Karplus relationship. For dihedral angles near 90 degrees, the coupling is near zero. Thus, the absence of a cross peak doesn't confirm that carbon- proton pairs are many bonds apart. Because of the wide range (0-14 Hz) of possible carbon- proton couplings, one often does two experiments. One optimized for 5 Hz couplings and the second optimized for 10 Hz. This gives the optimum signal-to-noise. Alternatively, a comprise

22 value of 7-8 Hz can be used. There are also "accordion" versions that attempt to sample the full range of couplings (König & Hochmuth, 2015).

2.4.2 Mass spectrometry (MS)

Mass spectrometry allows the determination of the molecular mass and the molecular formula of a compound, as well as certain structural features. A small sample of the compound is vaporized and then ionized as a result of an electron being removed from each molecule, producing a molecular ion (a radical cation). Many of the molecular ions break apart into cations, radicals, neutral molecules or other radical cations (McMurry, 2000). The bonds most likely to break are the weakest ones and those that result in the formation of the most stable products. These fragments of the molecules are detected individually on the basis of their mass-to-charge ratios (Bruice, 2000). The details of exactly how these positively charged fragments are separated and detected differ according to the specific design of the mass analyzer portion of the instrument. In any case, the information acquired and displayed by the data system (the so-called mass spectrum) allows the analyst to reconstruct the original molecule and thereby identify it. Besides the significant applicability to molecular compound identification, mass spectrometry also finds application in elemental analysis, such as to determine what isotopes of an element might be present in a sample (Kenkel, 2003).

CHAPTER THREE: MATERIALS AND METHODS

3.1 Plant Sample Collection

The stem bark of Psorosperumum febrifugum was collected from Mpigi district, Misindye hill in Buwungu village- Buwama sub country, Uganda in August 2017 and was identified by a taxonomist from Makerere University Herbarium under the management of Department of Plant Science, Microbiology & Biotechnology, College of Natural Sciences, Makerere University.

3.2 Preparation and Partitioning of the Crude Extract

The bark was chopped into small pieces and air dried at room temperature for 21 days. The dry bark was ground into fine powder using an electric grinder. The powdered plant material (1 Kg) was extracted three times with 5 litres of a mixture ethyl acetate and methanol in a ratio of 1:1 at room temperature for 48 hours. The extracts were filtered through cotton wool and concentrated with a rotary evaporator at 40 0C to dryness. The crude extract was partitioned into hexane extract, ethyl acetate and methanol using a separating funnel. The extracts were again concentrated with a rotary evaporator at 40 0C to dryness and were transferred to sample bottles which were placed in a dessicator containing anhydrous sodium sulphate to remove any traces of water that could have been present. The ethyl acetate extract yielded 40.06 g of a brick red powder upon concentration. The methanol extract yielded 36.49 g of a dark brown coloured powder while the hexane extract yielded 24.26 g of a brown powder. The dried extracts were later kept in tightly stoppered bottles in a refrigerator for further analysis (Zygmunt and Namiesnik, 2003; Tradit et al., 2011).

3.3 Antibacterial Activity Tests

Plant extracts were tested against four American Type Culture Collection (ATCC) bacterial strains: Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922) which are gram negative, Staphylococcus aureus (ATCC 25923) and Streptococcus pyogenes (ATCC 700294) which are gram positive. These organisms were obtained from the Department of Pharmacology, College of Health Sciences, Makerere University. These four bacteria strain were chosen because they causes many common bacterial infections, including cholecystitis, bacteremia, cholangitis, urinary tract infection (UTI), urinary tract infections, respiratory system

24 infections, soft tissue infections, bacteremia, bone and joint infections, skin infections, such as pimples, impetigo, boils, cellulitis, folliculitis, carbuncles, scalded skin syndrome, and abscesses, pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome, bacteremia, and sepsis. Also activities of the isolated compounds were tested against two bacterial strains namely: Pseudomonas aeruginosa (ATCC 27853) and Staphylococcus aureus (ATCC 25923). Testing of the plant extracts for antibacterial activity was done by the modified agar disc diffusion method (Baris et al., 2006). Testing of the plant extracts and the isolated compounds was done at department of Bio-molecular Resources and Bio-lab Sciences, College of Veterinary Medicine, Animal Resources and Biosecurity at Makerere University.

3.3.1. Modified Agar Disc Diffusion Assay for Bacterial Screening

The agar disc diffusion assay was carried out as described by (Baris et al) 2006 with minor modifications. Thus, a stock concentration of 1000 µg/ml of each crude extract was prepared. Briefly, 1000 µg (1.0.mg) of each crude extract was weighed and dissolved into 1 ml of dimethyl sulfoxide (DMSO). 1000 µg/ml of ciprofloxacin was prepared as the a positive control, 1000 µg (1.0 mg) of ciprofloxacin was weighed and dissolved into 1 ml of distilled water. 99% dimethyl sulfoxide (DMSO) was used as a negative control. The test microorganism was aseptically inoculated (approx. 1.0 x 108 colony forming units/ml) on sterile Mueller Hinton agar by surface spreading to make a uniform microbial inoculum. Using sterile glass cork borers (6 mm in diameter), three wells were carefully made on the agar plate without distorting the media; one well contained the test extract/, the second well contained ciprofloxacin which was used as a positive control. The third well contained dimethyl sulfoxide (DMSO) which was used as a negative control. 50 µl of the stock concentration of each extract and the controls were carefully dispensed into the respective wells and the plates left on the bench for 60 minutes to allow the system stabilize as the inoculated microorganisms get acclimatized to the new environment. The culture plates were then incubated at 37 0C for 24 hours. Using a metric ruler, the diameter of the zone of inhibition was measured for hexane, ethyl acetate and methanol extracts. This experiment was run in duplicates. This same method was used to determine the zone of inhibition for the three isolated compounds and the stock concentration that was used was 0.1µg/ml.

3.3.2. Determination of Minimum Inhibitory Concentration (MIC) using Broth dilution method

The broth dilution method adopted was used to determine the MICs of the ethyl acetate extract and the isolated compounds from the stem bark of Psorospermum febrifugum. Here, a set of four test tubes was arranged on a test tube rack for an experiment of one extract. A stock concentration of 1000 µg/ml of each crude extract was prepared. Briefly, 1000µg (0.001g) of each crude extract was weighed and dissolved into 1 ml of dimethyl sulfoxide (DMSO). 1.0 ml of Mueller Hinton broth (Merk, GERMANY) was dispensed into each of these tubes. 1.0 ml of the stock concentration of the extract was then added to the first tube, thereby diluting the contents by 1/2 (500 µg/ml). 1.0 ml of the extract mixture from this tube was then transferred to the next tube hence causing a dilution of 1/4 (250 µg/ml). This process was carried on the next tube up to the last where the concentration was 1/16 (62.5 µg /ml). The excess 1.0 ml from the last tube was then discarded 18 hr old cultures of the standard laboratory organisms was used to make a suspension of concentration approximately 1.0 x 108 CFU/ml. This was adjusted using the 0.5 McFarland turbidity standard. 0.1 ml of this suspension was then added to each of the tubes with the diluted extract and thoroughly mixed on a vortex mixer. The tubes were then incubated at 37 ˚C for 24 hr and observed for growth in terms of turbidity. The tube with the least dilution that didn‟t show turbidity due to growth was considered as the MIC (Baris et al., 2006). This same method was used to determine the Minimum Inhibitory Concentration (MIC) for the three isolated compounds and the stock concentration that was used was 0.1µgml.

3.3.3. Determination of Minimum Bactericidal Concentration (MBC)

Following MIC determination using broth dilution method, after incubation of each of those tubes for a minimum of 24 hr, the contents in each of the tubes were inoculated on individual plates of Nutrient agar (bacteria). After inoculation, these plates were then incubated at 370C for 24 hrs after which they were observed for characteristic growth of each of the bacteria. The tube with the least dilution whose contents resulted in no growth was considered as the MBC. The purpose of this test was to determine the lowest concentration at which the extract would kill Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Streptococcus pyogens (Baris et al., 2006).

3.4 Phytochemical Analysis

3.4.1 Test for alkaloids (Dragendorff’sreageant) A drop of the ethyl acetate extract was spotted on a small piece of precoated TLC plate. The plate was sprayed with Dragendorff‟s reagent. Formation of a yellow spot on the plate indicated the presence of alkaloids. As reported by Kumar et al., 2007 .

3.4.1 Test for anthroquinones (Borntrager’s test) Dilute (10%) ammonia (1 ml) was added to the ethyl acetate extract (2 ml). Formation of a pink red colour in the ammoniacal lower layer indicated the presence of anthroquinones. As reported by Kumar et al., 2007 .

3.4.2 Test for flavoniods (sodium hydroxide test) The ethyl acetate extract (2 ml) was treated with sodium hydroxide, followed by addition of dilute hydrochloric acid. Formation of a yellow solution with NaOH that turns colourless on addition of HCL indicated the presence of flavoniods. As reported by Kumar et al., 2007.

3.4.3 Test for steroids (Liebermann-Burchardt test) The ethyl acetate extract (2 ml) was treated with (10%) ferric chloride solution (1 ml) followed by adding 2 drops of concentrated sulphuric acid. Formation of a dark pink or red colour indicated the presence of steroids. As reported by Mawa et al., 2016.

3.4.5 Test for terpeniods (Salkowski test) The ethyl acetate extract (5ml) was added to chloroform (2 ml) and concentrated sulphuric acid (3 ml). Formation of reddish brown color of interface indicated the presence of terpeniods. As reported by Poumale et al., 2008.

3.4.6 Test for tannin (Braemer’s test)

(10%) alcoholic ferric chloride (1 ml) was added to the ethyl acetate extract (2 ml). Formation of a dark blue or greenish grey coloration of solution indicated the presence of tannins. As reported by Leopold et al., 2014.

3.4.7 Test for saponins (foam test) The ethyl acetate extract (2 ml) was added to water (6 ml) in a test tube. The mixture was shaken vigorously and observed for the formation of persistent foam that confirmed the presence of saponins. As reported by Mawa et al., 2016.

3.4.8 Test for phenols (ferric chloride) The ethyl acetate extract (2 ml) was treated with (5%) ferric aqueous (1 ml). Formation of deep blue or black colour indicated the presence of phenols. As reported by Mallikharijuuna et al, 2007.

3.4.9 Test of Carbohydrates

3.4.9.1 Fehling’s test

Both Fehling A (5 ml) and Fehling B (5 ml) were mixed together and the ethyl acetate extract (2 ml) was added to the mixture and gently boiled. A brick red precipitate that appeared at the bottom of the test-tube indicated the presence of reducing sugars ( Djoukeng et al., 2005).

3.4.9.2 Benedict’test

The ethyl acetate extract (2 ml) was added to of Benedict‟s reagent (2 ml) and boiled; a reddish brown precipitate formed which indicated the presence of carbohydrates ( Farooq et al., 2016)

3.4.10 Test for glycoside ( Kellar-Kiliani)

The ethyl acetate extract (2 ml) was added to glacial acetic acid (1 ml), ferric chloride (1 ml) and concentrated sulphuric acid (1 ml). Green blue coloration of solution indicated the presence of glycoside ( Farooq et al., 2016).

3.5 Isolation and Purification of Antibacterial Compounds from the Ethyl acetate Crude extract.

3.5.1 Thin layer chromatography

The solvent systems used to elute in column chromatography (CC) were determined using thin layer chromatography (TLC). Several solvent systems were tested and two of these were chosen as the mobile phase for eluting the column because they were separating the compounds in the ethyl acetate extract well compared to other solvent systems. The chosen solvent systems were;

28 hexane and dichloromethane in a ratio of 4:6 and hexane, ethyl acetate and methanol in a ratio of 7:2:1 respectively. Briefly, two spots of the ethyl acetate extract were carefully applied onto two thin layer chromatographic plate (coated with silica) and left to dry. After about five minutes, one plate was dipped in hexane: ethyl acetate and methanol in a ratio of 7:2:1 respectively and another plate was dipped in hexane: dichloromethane in a ratio of 4:6 respectively using tweezers. This allowed the compounds in the spots to move upwards by capillary attraction. The plates were then removed from the solvent systems when the solvent systems reached the eluent front and left to dry. The positions of different compounds were observed by fluorescence under UV-light followed by staining with vanillin hydrochloric acid reagent spray and then heated for 5 minutes (Mbaveng et al., 2008; Shahverdi et al., 2007). Also TLC was used to identify if a fraction is pure where by a fraction with one compound on the plate was considered to be pure.

3.5.2 Column chromatography

The ethyl acetate extract was subjected to silica gel (30-150 mesh) column chromatography. Briefly, silica gel (150 g) was mixed with hexane: dichloromethane in a ratio of 4:6 to form a homogenous suspension/slurry and stirred using a glass-stirring rod to remove bubbles. The silica gel slurry was then poured into a glass column. The sample to load on the column was prepared by dissolving 5 g of the extract in 40 ml of ethyl acetate. To the solution, 10 g of silica was added and mixed by stirring with a glass rod. The mixture was allowed to dry at room temperature. The dried silica extract mixture was layered on the column layer bed. The column was first eluted with hexane: dichloromethane in a ratio of 4:6 as the mobile phase. After which another second solvent was introduced, the solvent system became hexane, ethyl acetate and methanol in a ratio of 7:2:1 respectively. For each solvent system, two liters were used and 2 ml fractions collected in test tubes. TLC was used to combine the concentrated fraction from column chromatography where by fraction with similar retardation factor (Rf) were combined together. The collected fractions were concentrated to dryness using a rotary evaporator at 40 0C. (Mbaveng et al., 2008 ; Tradit et al., 2011). 24 fractions were collected from the column chromatography. Three fractions that were relatively pure were further fractionated using a small silica gel column with hexane: ethyl acetate and methanol (7:2:1) as the eluent system to obtain the pure compounds. Compound 1 (33.1 mg) which was an anamorphous white powder and soluble in methanol was obtained from fraction 7 (53.4 mg). Compound 2 (34.1 mg) which was

white powder and soluble in acetone was obtained from fraction 12 (51.4 mg). Compound 3 (80.1 mg) which was white powder and soluble in methanol was obtained from fraction 19 (133.4 mg).

Summary of fractionation and isolation results

Ethyl acetate (5 g)

24 fraction were collected from the column using a solvent system of HEX: DCM in a ratio of 4:6 and HEX: ETOH: MEOH in a ratio if 7:2:1

Fraction 12 Fraction 19 Fraction 7 (51.4 mg) (133.7 mg) (53.8 mg)

Compound 1 (33.1 mg) Compound 2 (34.9 mg) Compound 3 (80.1 mg) an anamorphous white white powder and white powder and powder and soluble in soluble in acetone soluble in methanol methanol

Figure 3.1: Summary of fractionation and isolation results

3.5.3 Structure determination of the isolated pure compounds

The structures of the isolated compounds were determined using spectroscopic methods like Mass spectrometry (MS) and Nuclear magnetic resonance (NMR) spectroscopy.

3.4. 3.1 Nuclear magnetic resonance (NMR) spectroscopy

For NMR, both one dimensional (1D) and two dimensional (2D) proton (1H) and carbon (13C) NMR experiments such as 1H-1H COSY, DEPT, APT, NOESY, HMBC and HSQC were

30 employed. Compound 1 and compound 3, the 600 MHZ NMR was used while for compound 2; the 400 MHZ NMR was used. Each pure sample was first stored in a vacuum oven for 4 hours at 400C in order to remove any solvent that might be in the sample. Each pure sample, the pure compound (5 mg) was dissolved in a suitable deuteriated solvent (1 ml) and then was filtered through a pasteur pipette equipped with a glass wool plug that discharged into a 5 mm NMR tube which was labeled clearly with a concentric label. The purpose of the filtration was to remove any undissolved sample, particulates and dust from the solution, which could affect the resolution and the line shape of the NMR spectra. The 1D and 2D spectra were recorded on Bruker AV-600 and AMX- 400 instruments (McMurry, 2000). All the spectra were analyzed and the results were compared with published information in literature in order to elucidate the structures of the isolated compounds (McMurry, 2000).

3.5.3.2 Mass Spectrometry (MS)

For MS, each pure compound (0.5 mg) was put in a labeled sample vial and was diluted with an appropriate solvent of HPLC grade up to a concentration of one micro molar (1 μM). The solution in each case was filtered in order to remove any insoluble material which would otherwise block the sample introduction line. Each sample solution (2 ml) was introduced into the liquid chromatography mass spectrometer (LC-MS) for analysis. The Turbo spray MS spectra were obtained using, turbo spray ionization source (ESI), at electron energy of 70 V, at source temperature 190 °C. The ion energy at 1.7 V and at pressures < 1.7e – 4 Torr and 1.1e – 5 Torr penning was used for MS analysis (Harvey, 2000; Tradit et al., 2011).

3.6 Melting Point Determination of the Isolated Pure Compounds

The melting point of the isolated pure compounds was determined using 00590Q fisher john melting point apparatus; 220VAC. Briefly, 1 mg of each pure compound was placed between two coverslip. The coverslips were placed on the hot plate of the melting point apparatus and the magnifying glass was moved over the well. The apparatus was turned on by fipping the switch. A light illuminated the pure compound. The pure compound was watched through the magnifying glass and the temperature at which the pure compound started to melt up to when it stoped to melt was recorded (Olila et al., 2002).

CHAPTER FOUR: RESULTS AND DISCUSSION

4.1 In -Vitro Activity of the Extracts Against Selected Bacteria

The ethyl acetate extract was found to be sensitive to P. aeruginosa, E. coli, S. aureus, and S. pyogenes (figure 4.1). The ethyl acetate extract exhibited the highest zone of inhibition (19.1 ± 0.14) mm against S .aureus and (18.3 ± 0.07) mm against S. pyogens which are gram negative and the lowest zone of inhibition (14 mm) against P. aeurginosa. The methanol extract exhibited moderate zone of inhibition i.e. (15.56 ± 0.21) mm against S. pyogen,, (14.1 ± 0.14) mm against S.aureus and (10.67±0.24) mm against E. coli. The hexane extract had no inhibition of growth of any of the bacterial strains tested (Table 4.1).

Table 4.1: Zone of inhibition (mm) of plant extracts against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Streptococcus pyogenes

Zone of inhibition (mm) Bacteria Ethyl acetate Hexane Methanol Control extract extract extract Positive control Negative control (CIPRO) (DMSO) S. aureus 19.1 ± 0.14 0 14.1 ± 0.14 41.7 ±0.27 0 E.coli 15.45 ± 0.21 0 10.67 ± 0.24 40.3 ±0.07 0 S.pyogenes 18.3 ± 0.07 0 15.56 ± 0.21 41.67 ± 0.26 0 P. aeurginosa 14.1 ±0.07 0 0 40.5 ±0.24 0 The diameters corresponding to zone of inhibition were measured for the crude extract (in mm). The letters, Cipro = Ciproflaxin, DMSO = Dimethylsulfoxide

Both bacterial strains, (S. aureus, and S. pyogenes) were found to be susceptible to the ethyl acetate extract as the zone of inhibition diameters (19.1 ± 0.14 mm and 18.3 ± 0.21 mm for S. aureus, and S. pyogenes respectively, Table 4.1) were within the range for standard antibiotics such as ampicillin (inhibition diameter 16-22 mm), oxacillin (inhibition diameter 17-23 mm), doxycycline (inhibition diameter 18-24 mm), and tetracycline (inhibition diameter 18-25 mm), as reported by the Clinical and Laboratory Standards Institute – CLSI (2007). The antibacterial results were considered evidence of the presence of more active compounds in the ethyl acetate extract compared to the methanol and hexane extracts, the probable reason being that the compounds in P.febrifugum stem bark were more soluble in ethyl acetate as compared to methanol and hexane. Staphylococcus aureus is a pyogenic bacterium known to

32 play significant role in various skin diseases including superficial and deep follicular lesion (Linn, 2014). The prevalence of Staphylococcus aureus resistant strains to conventional antibiotic has increased to high levels in some hospital (Linn, 2014).

Cip = Ciproflaxin, DMSO = Dimethylsulfoxide B1-ethylacteate crude extract Figure 4.1: plates showing zones of inhibition for the ethyl acetate extract against the four tested bacteria strains.

Since the ethyl acetate crude extract showed the highest sensitivity to zone of inhibition, it was considered for further Tests. Its Minimum Inhibitory Concentration (MIC) against each of the bacterial strains was investigated as well as its Minimum Bactericidal Concentration (MBC).

Table 4.2: Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the crude extract

Microorganisms Ethyl acetate extract (B1) MIC in µg/ml MBC in µg/ml S. aureus 62.5 125 E.coli 500 500 S.pyagens 125 500 P. aeruginosa 250 500

The ethyl acetate crude extract showed a minimum inhibitory concentration (MIC) that ranged from 62.5 µg/ml to 500 µg/ml (Table 4.2 and figure 4.1.3).The MIC of the ethyl acetate crude extract was taken as 62.5 µg/ml against S. aureus. This showed that the ethyl acetate crude extract was more effective against S. aureus than the rest of the tested bacterial strain. The Minimum Bactericidal Concentration (MBC) of the ethyl acetate crude extract was taken as 125 µg/ml against S. aureus (table 4.2 and figure 4.1.4) showed that the extract has a stronger activity towards S. aureus than E. coli, S. pyragens and P. aeurginosa since it had the lowest MBC. It also showed the ethyl acetate crude extract was more active on gram positive bacteria than on gram negative bacteria. This was evidenced from very low MIC value against S. aureus (62.5 µg/ml) and S. pyragens ( 125 µg/ml) both of which are gram positive bacteria as compared to and P. aeurginosa (250 µg/ml) than E. coli (500 µg/ml) which are both gram negative bacteria.

Figure 4.2: plates showing the minimum inhibitory Concentration (MIC) tests for the ethyl acetate

Figure 4.3: plates showing the minimum bactericidal Concentration (MBC) for the ethyl acetate extract

Figure 3: plates showing the minimum bactericidal Concentration (MBC) for the ethyl acetate extract This study showed the highest zone of inhibition was (19.1 ± 0.14) mm against S. aureus, the MIC of 62.5 µg/ml against S.aureus and MBC of 125 µg/ml aganist S.aureus which were similar to those studies done by Tchakam et al., 2012, Ghaima et al., 2013, Tamokou et al., 2013 and Kamali et al., 2015. This indicated that the ethyl acetate extract from the stem bark of Psorospermun febrifugum had antibacterial activity.

Phytochemical screening

The phytochemical screening of the Psorospermum febrifugum crude extract showed the presence of different primary and secondary metabolites like terpeniods, phenols, reducing sugar, carbohydrates and tannins. The results and observation are summarized in table 4.3

Table 4.3: Phytochemicals/ metabolites found in the ethyl acetate crude extract for Psorospermum febrifugum.

Bioactive group Observation Conclusion Alkaloids A green spot was observed _

Anthraquinone A brown precipitate was formed _

Flavoniods A brown solution was formed with NaOH which _ turned into a brown precipitate on addition of HCl Steriods A dark green solution was observed when 10%of _ ferric chloride was added. It formed bubbles , became hot and formed a brown precipitate on addition of sulphuric acid

Terpeniods Formed a reddish brown colour at the interface that +++ bubbles and becomes hot when sulphuric acid is added Tannin Formed a dark blue greenish solution +++ Saponins The foam formed but it lasted for a short time _ Phenols Formed a deep blue greenish colour ++ Reducing sugars Formed a greenish precipitate when fehling A and + fehlin B was added to the extract. A brick red precipitate appeared at the bottom during the boiling of the mixture Carbohydrates Formed a brownish precipitate on addition of + Benedict‟s solution and a reddish brown precipitate was formed during the heating of the mixture Glycosides A dark brown precipitate was formed _ (-)Absence, (+) presence, (++) moderate presence, (+++) highly presence

4.3 Characterization of the compounds from ethyl acetate extract Three compounds were isolated from the ethyl acetate extract of the stem bark of Psorospermum febrifugum by a combination of chromatographic techniques. The structures of these isolated compounds were identified by NMR spectroscopy and Mass spectrometry as Betulinic acid (1), oleanolic acid (2) and oleanolic acetate acid (3). This is the first time these compounds are being reported from the plant.

4.3.1 Compound 1

Compound 1 was soluble in deuteriated methanol (MeOD)

4.3.1.1 1H NMR spectrum of compound 1

The 1H NMR spectrum of compound 1 showed 48 proton signals. The 1H NMR spectrum of compound 1 (Fig 4.4) showed resonances for olefinic methylene protons at 4.72 ppm and 4.61 ppm, a oxymethine proton at 3.14 ppm, a methyl singlet at 1.71 and five methyl singlets at 0.97ppm, 0.55ppm, 0.96ppm, 0.87ppm and 1.02ppm. The signals that showed values between 0.72 ppm and 1.71 ppm are due to the presence of a triterpene (Sallau et al., 2016). The spatial arrangement of the olefinic methylene protons (H-29a and H-29b) in compound 1 was assigned basing on their coupling constants. H-29a (4.72 ppm) was observed as a doublet (J = 2.5 Hz), hence suggesting its coupling with H-30. It was therefore assigned the position that is trans to C- 30 (Fig. 4.2.1f). H-29b (4.61 ppm) was observed as a triple doublet (J = 2.7 Hz, 2.6 Hz, 1.3 Hz), suggesting its coupling with H-19 (J = 2.7 Hz), H-30 (J = 2.6 Hz) and H-18 (J = 1.3 Hz). Therefore, H-29b was assigned the position that is cis to C-30 (Fig. 4.2.1g).

Figure 4.4: 1H NMR spectrum in MeOD at 600 MHz of compound 1

4.3.1.2 13C NMR spectrum of compound 1

The 13C NMR data (Fig 4.5 and Fig 4.6) of compound 1 revealed 30 carbon signals (six methyl, ten methylene, six methine, five quaternary carbons, one carboxylic acid, and two olefinic carbons). The data was also in complete agreement with the existence of an isopropenyl group, in particular, the characteristic vinylic carbon atom resonances at 150.58 ppm and 109.74 ppm, corresponding to carbon atoms 20 and 29 respectively. This supported the olefinic methylene protons seen as singlets at 4.72 ppm and 4.61 ppm in the 1H NMR spectrum of compound 1. The signal at 78.25 ppm was characteristic for the oxymethine carbon at position 3. The down field part of the 13C NMR spectrum of compound 1 showed a signal at 178.72 ppm, which was attributed to the carboxylic carbon atom. The 1H-NMR, 13C-NMR and MS spectra were typical of a pentacyclic triterpenoid of the lupane skeleton as well as by comparison of their spectral data with previously reported value (Koma & Sani, 2014; Sai Prakash et al, 2012).

Figure 4.5: 13C NMR spectrum in MeOD at 150 MHz of compound 1

Figure 4.6: 13C NMR - APT spectrum in MeOD at 150 MHz of compound 1

4.3.1.3 1H-1H COSY of compound 1

From the 1H-1H COSY spectrum of compound 1 (Fig 4.7), a number of cross peaks showed coupling between different sets of protons including that between H-29a (4.72 ppm) and H-30 (1.71 ppm), H-2 (1.47 ppm) and H-3 (3.13 ppm) and H-18 (1.33 ppm) and H-19 (2.32 ppm. The methyl singlet at 1.71 ppm was found to be coupled to one of the two methylene protons (H- 29b, 4.61 ppm) in the 1H-1H COSY experiment, thus indicating the presence of an isopropenyl group as well as a lupane skeleton (Shai et al., 2008).

Figure 4.7: 1H-1H COSY in MeOD at 600 MHz of compound 1

4.3.1.4 HSQC for compound 1

With the help of the HSQC experiment (Fig 4.8), a correlation was observed between the olefinic methylene protons: H-29a (4.72 ppm), H-29b (4.61 ppm) and C-29 (110.0 ppm). From the same spectrum, all the 1H resonances were assigned to their corresponding 13C resonances as shown in figure 4.8.

Figure 4.8: HSQC in MeOD at 600 MHz for compound 1

4.3.1.5 HMBC for compound 1 The HMBC spectrum of compound 1 (Fig 4.9) showed correlations between H-29a (4.72 ppm) and the carbon atoms C-30 (19.7 ppm) and C-19 (39.9 ppm). Correlations between H-30 (1.71 ppm) and the carbon atoms C-19 (39.9 ppm), C-29 (110 ppm) and C-20 (152.5 ppm) were also observed. Many other correlations in the HMBC spectrum of compound 1 made the assignment of the quaternary carbon atoms possible (Table 4.4).

Figure 4.9: HMBC in MeOD at 600 MHz for compound 1

4.3.1.6 Mass spectrum for compound 1

The mass spectrum of compound 1 exhibited a molecular Ion [M]+ peak at m/z 455.7 observed 1 from EI-MS (figure 4.10);.its molecular formula was assigned to be C30H48O3, since the H NMR revealed 48 proton signals and the 13C NMR revealed 30 carbon signals and three oxygens which corresponds to its molecular ion. Fragments at m/z 254.3 and 283.4, characteristic of pentacyclic triterpenes were also present (Joshi et al., 2013) . The other prominent fragment ion + + peak was at m/z 437.5 [M−CH2] and 471.1 [M−O] which is characteristic of a pentacyclic triterpene with an isopropenyl group (Joshi et al., 2013).

Figure 4.10: EI-MS for compound 1

Generally, the 1H and 13C NMR chemical shifts (Table 4.4) of compound 1 were found to be in close agreement with the 1H and 13C NMR chemical shifts of the structure reported for Betulinic acid (Sholichin et al., 1980; Koma et al., 2014; Sallau et al., 2016). Compound 1 was therefore assigned as Betulinic acid (Fig 4.11).

Figure 4.11: The structure of compound 1 (betulinic acid)

Betulinic acid is reported to have antiviral, antiplasmodial, antibacterial (against E.coli and staphylococcus aureus), anthelmintic as well as antidepressant effects (Machado et al., 2013; Carmona et al., 2010; Shai, McGaw, Aderogba, Mdee, & Eloff, 2008). It is also reported to have exhibited moderate antibacterial activity against Staphylococcus aureus, E.coli, P.aeruginosa and partial inhibition of Bacillus subtilis (Koma & Sani, 2014). This supports the use of Psorospermum febrifugum for the treatment of skin infections in African folk medicine.

Table 4.4 : 1H and 13C NMR spectral data together with the literature values for 1H and 13C NMR spectral data for compound 1 in MeOD

Position 13C,ppm Type 1H, ppm Literature value for Literature value for using 150 using 600 13C,ppm using 150 M for 1H, ppm using MHz in MHz in Hz in MeOD (Sallau 600 MHz in MeOD MeOD MeOD et al., 2016) (Sallau et al., 2016) 1 37.5 CH2 1.54 (m) 38.0 1.52 2 27.1 CH2 1.47 (m) 26.7 1.50 3 78.3 CH 3.14 (dd) 79.4 3.15 4 55.4 C 55.6 5 57.0 CH 0.72(d) 56.2 0.78 6 19.2 CH2 1.44 (m) 18.1 1.48 7 33.4 CH2 1.45 34.2 1.42 8 40.4 C 40.6 9 50.4 CH 1.66 (dd) 50.8 1.60 10 35.5 C 37.9 11 20.2 CH2 1.89 (d) 20.8 1.89 12 24.2 CH2 1.56 (m) 25.4 1.56 13 39.9 CH 1.02 39.0 1.01 14 42.0 C 43.3 15 27.2 CH 1.08 29.7 1.10 16 29.0 CH2 1.07(dd) 30.1 1.04 17 56.2 C 56.9 18 48.7 CH 1.33(dd) 47.7 1.34 19 49.2 CH 2.32( m) 49.8 2.40 20 152.0 C 152.8 21 31.5 CH2 1.94 (m) 30.0 2.03 22 36.5 CH2 1.63 (m) 36.3 1.60 23 26.7 CH3 0.97 (s) 27.2 0.94 24 15.4 CH3 1.02 (s) 15.1 1.02 25 16.6 CH3 0.85 (s) 16.1 0.84 26 16.5 CH3 0.87 (s) 16.2 0.87 27 14.8 CH3 0.96 (s) 15.1 0.95 28 178.4 C 178.9 29a 109.7 CH2 4.72 108.1 4.72 29b 4.61 4.62

30 19.7 CH3 1.71(s) 18.3 1.69

4.3.2 Compound 2

Compound 2 was soluble in deuteriated acetone (CD3COCD3).

4.3.2.1 1H NMR spectrum of compound

The 1H NMR data of compound 2 (Fig 4.12) were found to be closely similar to the 1H NMR data of compound 1. Just like in compound 1, the 1H NMR spectrum of compound 2 showed signals for seven methyl singlets, eight methylene groups, two methine groups and one oxygenated methine groups at 3.17 ppm. The1H NMR revealed 48 protons signals. The same spectrum also showed resonances between 0.79 ppm and 1.91 ppm which may be due to the presence of triterpene skeleton. The 1H NMR spectrum of compound 2 however did not show the methyl proton signal at 1.71 ppm as was the case for compound 1 and also compound 2 showed only one olefinic methylene proton at 5.26 ppm yet compound 1 showed two olefinic methylene at 4.72 ppm and 4.61ppm.

1 Figure 4.12: H NMR spectrum in CD3COCD3 at 400MHZ of compound 2

4.3.2.2 13C NMR spectrum of compound 2

The 13C NMR spectrum of compound 2 ( figure 4.13) revealed 30 carbon signals, which were shown by the HSQC experiment to be seven methyl, eight methylene, five methine, six quaternary carbons, one carbonyl and two olefinic carbons. Similar to compound 1, the 13C NMR data of compound 2 was also in complete agreement with the existence of a carbonyl group due to the characteristic carbon atom resonance at 178.4 ppm. The signal at 78.2 ppm was characteristic for the oxymethine carbon at position 3. The two olefinic carbon resonances were observed at 122.1 ppm and 140.0 ppm due to the presence of a pair of sp2 hybridized carbons. The 1H-NMR, 13C-NMR and MS spectra were typical of a pentacyclic triterpenoid of the lupane skeleton as well as by comparison of their spectral data with previously reported value (Ghosh et al., 2014).

13 Figure 4.13: C NMR spectrum in CD3COCD3 at 400MHZ of compound 2

4.3.2.3 1H-1H COSY of compound 2

Several cross peaks were observed from the 1H-1H COSY spectrum of compound 2 (Fig 4.14) including those between H-12 (5.26 ppm) and H-11 (1.91 ppm), H-3 (3.17 ppm) and H-2 (1.73 ppm), H-19 (1.74 ppm) and H-18 (2.91 ppm).

1 1 Figure 4.14: H- H COSY in CD3COCD3 at 400MHZ of compound 2

4.3.2.4 HSQC for compound 2

Correlation of the carbon resonances with the resonances of their directly attached protons (Table 4.5) was made possible by the HSQC experiment (figure 4.15). The proton at 5.26 ppm was found to be attached to one of the olefinic carbons, C-12 (122.1 ppm), the proton at 3.17ppm was found to be attached to oxygenated methine groups, C-3 (78.2 ppm) and the proton 2.91ppm at was found to be attached to methine carbon, C-18 (41.0 ppm).

Figure 4.15: HSQC in CD3COCD3 at 400MHZ of compound 2

4.3.2.5 HMBC for compound 2

From the HMBC spectrum of compound 2 (Fig 4.16), assignment of all carbon atoms was made possible (Table 4.5). The methine carbon, C-5 (55.4 ppm) was assigned basing on its HMBC correlation with C-3 (78.2 ppm), C-4 (38.6 ppm), C-7 (18.3 ppm), C-24 (27.8) and C-23 (15.4 ppm) while the olefinic carbon, C-13 (140.8 ppm) was assigned basing on its HMBC correlation with C-11 (23.0 ppm), C-18 (41.0ppm), C-19 (45.9 ppm) and C-27 (16.8 ppm).

Figure 4.16: HMBC in CD3COCD3 at 400MHZ of compound

4.3.2.6 Mass spectrum of compound 2

The mass spectrum of compound 2 exhibited a molecular ion peak [M]+ at m/z 455.7, 1 corresponding to a molecular formula C30H48O3 in line with the H NMR revealed 48 proton signals and the 13C NMR revealed 30 carbon signals and three oxygens. Fragment at m/z 269.8 is a characteristic of pentacyclic triterpenes was also present in the mass spectrum of compound 2 (Joshi et al., 2013) (Fig 4.17). The mass spectrometry data also suggested the presence of a carbonyl group (fragment at m/z 407.7). The fragment at 471.7 corresponding to [M-O]+ was also observed (Joshi et al., 2013). Compound 2 has the same molecular weight like compound 1 meaning they are isomer.

Figure 4.17: EI-MS of compound 2 Generally, the 1H and 13C NMR chemical shifts ( Table 4.5 ) of compound 2 were found to be in close agreement with the 1H and 13C NMR chemical shifts reported for Oleanolic acid isolated from chloroform extract of Borreria stachydea (Prod and Resour, 2013). Hence compound 2 was assigned as Oleanolic acid (Fig 4.18).

Figure 4.18: The structure of compound 2 ( oleanolic acid) According to Jesus et al, (2015), Oleanolic acid (3훽-hydroxyolean-12-en-28-oic acid) is a pentacyclic triterpenoid that possess several interesting pharmacological activities, such as anti inflammatory, antioxidant, anticancer, and hepatoprotective effects. The same report indicates that oleanolic acid acts as an effective antibacterial agent when tested against both gram positive and gram negative bacteria such as Bacillus cereus, Bacillus megaterium, Bacillus subtilis, Mycobacterium tuberculosis Staphylococcus aureus, Staphylococcus lutea, Salmonella paratyphi, Salmonella typhi, Shigella boydi, Shigella dysentriae, Vibrio mimicus, E.coli, Klebsiella pneumoniae and P.aeruginosa. Furthermore, according Bisoli et al, (2008), oleanolic acid inhibited the growth of a variety of pathogenic fungal species such as Sporothrix schenckii, Microsporum canis, Aspergillus fumigates and Candida albicans; which also supports the traditional use of Psorospermum febrifugum in the treatment of candidiasis and skin diseases.

Table 4.5: 1H and 13CNMR spectral data together with the literature values for 1H and 13C NMR spectral data for compound 2 in CD3COCD3 Position δC,ppm in Type δH, ppm in Literature value for Literature value for 13 1 CD3COCD3 CD3COCD3 C,ppm using for H, ppm using at 100MHZ at 400MHZ 400MHz in CDCl3 ( 400MHz in CDCl3 Prod and Resour, 2013) (Prod and Resour, 2013) 1 38.4 CH2 1.59 (m) 38.41 1.60 2 27.0 CH2 1.73 (m) 27.20 1.62 3 78.2 CH 3.17( d) 79.04 3.2 4 38.6 C 38.41 5 55.4 CH 0.79 (d) 55.22 0.70 6 18.3 CH2 1.69 (dd) 18.31 1.52 7 32.7 CH2 1.78 (m) 32.65 1.43 8 39..2 C 39.27 9 47.9 CH 1.57 (dd) 47.53 1.53 10 36.7 C 37.06 11 23.0 CH2 1.91 (m) 22.96 1.87 12 122.2 CH2 5.26(d) 122.66 5.23 13 144.0 C 143.60 14 41.9 C 41.63 15 27.0 CH2 1.78 (m) 27.69 1.70 16 22.0 CH2 2.03 m) 23.46 1.88 17 46.9 C 46.63 18 41.0 CH 2.91 (dd dd) 41.04 2.88 19 45.9 CH 1.74 (d) 45.89 1.75 20 30.5 C 30.68 21 33.3 CH2 1.23(m) 33.81 1.30 22 32.4 CH2 1.03 (m) 32.44 1.03 23 27.8 CH3 1.00(s) 28.11 0.97 24 15.8 CH3 0.84 (s) 15.15 0.78 25 14.9 CH3 0.96 ( s) 15.33 0.91 26 16.5 CH3 0.85 (s) 17.11 0.85 27 26.8 CH3 1.11 (s) 25.92 1.11 28 178.4 C 182.38 29 33.1 CH3 0.82(s) 33.07 0.88 30 22.9 CH3 0.97 (s) 23.58 0.92

4.3.3 Compound 3

Compound 3 was soluble in deuteriated methanol (MeOD).

4.3.3.1 1H NMR spectrum of compound 3

The 1H NMR data of compound 3 (Fig 4.19) were found to be closely similar to the 1H NMR data of compound 2. Just like in compound 2, the 1H NMR spectrum of compound 3 showed signals for one oxygenated methine groups at 4.47 ppm and an olefinic group at 5.26 ppm. The same spectrum also showed resonances between 0.79 ppm and 1.91ppm which may be due to the presence of triterpene skeleton. The 1H NMR spectrum of compound 3 however, showed the vinyl methyl proton signal at 2.03 ppm,eight methyl singlets and nine methylene which was not the case for compound 2. The 1H NMR data of compound 3 showed 50 proton signals.

Figure 4.19:1H NMR spectrum in MeOD at 600 MHz of compound 3

4.3.3.2 13C NMR spectrum of compound 3

The 13C NMR (figure 4.20) spectrum of compound 3 revealed 32 carbon signals, which were shown by the HSQC experiment to be eight methyl, nine methylene, five methine, six quaternary carbons, two carbonyl and two olefinic carbons. The signal at 80.7 ppm was characteristic for the oxymethine carbon at position 3. Similar to compound 2, the 13C NMR data of compound 3 showed two peaks at 125.1 ppm and 138.2 ppm represent the presence of a pair of sp2 hybridized carbon atoms assigned to C-12 and C-13. However 13C NMR for compound 3 showed the existence of two carbonyl group due to the characteristic carbon atom resonance at 171.5 ppm and 180.2 ppm at C-3c and C-28 respectively which was not the case for compound 2, it showed one carbonyl group.

Figure 4.20: 13C NMR spectrum in MeOD at 150 MHz of compound 3

4.3.3.3 1H-1H COSY of compound 3

The 1H-1H COSY spectrum (Fig 4.21) of compound 3 displayed cross peaks between H-12 (5.25 ppm) and H-11 (1.95 ppm), H-3a (4.47 ppm) and H-2 (1.65 ppm) as well as between H-18 (2.22 ppm) and H-19 (1.32 ppm) among others.

Figure 4.21: 1H-1H COSY in MeOD at 600 MHz of compound 3

4.3.3.4 HSQC for compound 3

From the HSQC spectrum of compound 3 (Fig 4.22), 1H resonances were assigned to their corresponding 13C resonances (Table 4.6). Similar to compounds 2, the HSQC spectrum of compound 3 showed a correlation between the proton at 5.24 ppm and one of the olefinic carbons, C-12 (125.9 ppm), the proton at 4.47 ppm and oxygenated methine groups, C-3 (80.7 ppm) and the proton 2.87 ppm and methine carbon, C-18 (41.7 ppm). The same spectrum showed six quaternary carbon atoms as was the case with compound 2, but a difference in the number of methylene groups and methine was observed. The HSQC spectrum of compound 3 showed nine methylene groups and eight methyl and not eight methylene and seven methyl as was the case for compound 2.

Figure 4.22: HSQC in MeOD at 600 MHz for compound 3

4.3.3.5 HMBC for compound 3

From the HMBC spectrum of compound 3 (Fig 4.23), assignment of all carbon atoms was made possible (Table 4.6). The methine carbon, C-5 (55.4 ppm) was assigned basing on its HMBC correlation with C-3a (80.7 ppm), C-4 (38.1 ppm), C-7 (17.9 ppm), C-6 (32.9) and C-24 (23 ppm) while the olefinic carbon, C-15( ppm) was assigned basing on its HMBC correlation with C-13 (138.4 ppm), C-14 (39.4 ppm) and C-16 (23.4 ppm).

Figure 4.23: HMBC in MeOD at 600 MHz for compound 3

4.3.3.6 Mass spectrum of compound 3

The mass spectrum of compound 3 exhibited a molecular ion peak [M] + at m/z 497.8, 1 corresponding to a molecular formula C32H50O4 in line with the H NMR revealed 50 proton signals and the 13C NMR revealed 32 carbon signals and four oxygens. Fragments at m/z 269.3 and 423.5, characteristic of pentacyclic triterpenes were also present in the mass spectrum of compound 3 (Joshi et al., 2013) (Fig 4.24). The mass spectrometry data also suggested the presence of a carbonyl group (fragment at m/z 301.5 and 543.8). The fragment at 557.8 corresponding to [M-CH2]+ was also observed (Joshi et al., 2013).

Figure 4.24: EI-MS of compound 3

Generally, the 1H and 13C NMR chemical shifts (Table 4.8) of compound 3 were found to be consistent with the 1H and 13C NMR chemical shifts and structure reported for oleanolic acetate acid isolated from Glossonema boveanum (Hwang et al., 2014). Hence compound 3 was assigned as Oleanolic acid acetate (Fig 4.25).

Figure 4.25: The structure of compound 3 (oleanolic acetate acid)

Oleanolic acetate acid is reported to have antimicrobial potential as it exhibited antifungal activity against C. albicans, Trichophyton mentagrophytes and Aspergillus niger (Sharma et al., 2016; Tamokou et al., 2013).

Table 4.6 : : 1H and 13C NMR spectral data together with literature values for 1H and 13C NMR spectral data for compound 3 in MeOD

Position δC,ppm Type δH, ppm in MeOD Literature value in MeOD at 600 MHz for 13C,ppm Literature value 1 at 150 using 150MHz in for H, ppm MHz MeOD at (Hwang using 600MHz in et al., 2014) MeOD (Hwang et al., 2014) 1 37.2 CH2 1.54 (m) 38.0 1.53 2 27.7 CH2 1.65(m) 27.7 1.62 3a 80.7 CH 4.47 (dd,11.3,3.9) 80.9 4.42

3b 21.3 CH3 2.03 (s) 22.8 2.06 3c 171.1 CHOO 171.1 4 38.1 C 39.0 5 55.4 CH 0.88(m) 55.4 0.89 6 17.9 CH2 1.55 (dd,14.5,4.9) 18.1 1.55 7 32.9 CH2 1.63 (m) 32.8 1.69 8 39.4 C 39.2 9 47.7 CH 1.44 (m) 48.0 1.43 10 37.4 C 37.7 11 23.2 CH2 1.95(dd, 9.1,3.1) 24.0 1.90 12 125.9 CH2 5.25 (d,3.7) 122.5 5.23 13 138.2 C 140.0 14 39.4 C 41.6 15 27.7 CH2 1.25 (dd,14.1,12.2) 28.0 1.25 16 23.9 CH2 1.64(dd,12.8,4.6,2.5) 22.8 1.63 17 50.8 C 50.6 18 41.9 CH 2.87 (d) 41.4 2.81 19 38.9 CH 1.32 (dd,14.3,6.7) 40.1 1.30 20 30.3 C 30.9 21 33.1 CH2 2.22 (d, 5.7) 33.8 2.18 22 32.1 CH2 1.79 (m) 32.4 1.79

23 29.4 CH3 0.89(s) 29.7 0.89 24 16.3 CH3 0.99 (s) 16.7 0.97 25 15.8 CH3 0.93(s) 15.5 0.98 26 16.5 CH3 0.97 (s) 17.2 0.91 27 25.6 CH3 1.15 (s) 25.9 1.18 28 180.1 COOH 178.6 29 33.9 CH3 0.79 (s) 33.1 0.79 30 23.5 CH3 0.90 (s) 23.6 1.00

4.4 Melting point determination for compound 1, compound 2 and compound 3

Table 4.7: Melting point for compound 1, compound 2 and compound 3 Melting temperature at Melting temperature at Average melting point which the solid started which the solid stopped in °C to melt in ° C to melt in° C Compound 1 316 319 317.5 Compound 2 304 306 305 Compound 3 334 336 335

The average melting point for compound 1 (Betulinic acid), compound 2 (oleanolic acid) and compound 3 (oleanolic acetate acid) was 317.5°C, 305°C and 335°C respectively. According to Sai Prakash et al., (2012), the melting point range for Betulinic acid was 316°C to 318°C, this confirmed that compound 1 is Betulinic acid and also indicated that compound 1 was fairly pure since a narrow melting point range of 3°C. The melting point for oleanolic acid range was 304°C to 306 °C (Joshi et al., 2013), this confirmed that compound 2 is oleanolic acid and also indicated compound 2 was fairly pure since it had a narrow melting point range of 2°C. The melting point for oleanolic acetate acid range was between 334 °C to 336 °C (Joshi et al., 2013) , this confirmed that compound 3 was oleanolic acetate acid and indicated that compound 3 was fairly pure since it had a narrow melting point range of 2°C.

4.5 Antibacterial activity of compound 1, 2 and 3

Antibacterial activity was done on compound 1 (betulinic acid), compound 2 (oleanolic acid) and compound 3 (oleanolic acetate acid) against two bacterial strains namely Staphylococcus aureus and Pseudomonas aeruginosa. Compound 2 exhibited the highest zone of inhibition of (14.2 ±0.07) mm against both bacterial strains. Compound 3 exhibited the lowest zone of inhibition of (6.5± 0.21) mm against S. aureus and (8.1±0.14) mm against P. aeruginosa (Table 4.8).

Table 4.8: Zone of inhibition of compound 1, 2 and 3 against Pseudomonas aeruginosa and Staphylococcus aureus

Zone of inhibition in mm Organism Compound Compound Compound Control 1 µg/ml) 2 ( 0.1 3 ( 0.1 Positive control Negative control µg/m) µg/ml) (CIPRO) (DMSO) S.aureus 12.3±0.14 14.2±0.07 6.5±0.21 41.67±0.24 0 P.aeruginosa 12.3±0.14 14.2±0.07 8.1±0.14 40.1±0.14 0 The diameters corresponding to zone of inhibition were measured for compound 1, 2 and 3 (in mm). The letters, Cipro = Ciproflaxin, DMSO = Dimethylsulfoxide

Compound 1 and 2 showed moderate antibacterial activity since they had moderate zone of inhibition of (12.1± 0.014) mm and (14.2 ±0.07) mm) respectively against both bacterial strains because they were not within the range for standard antibiotics such as ampicillin (inhibition diameter 16-22 mm), oxacillin (inhibition diameter 17-23 mm), doxycycline (inhibition diameter 18-24 mm), and tetracycline (inhibition diameter 18-25 mm), as reported by the Clinical and Laboratory Standards Institute – CLSI (2007).

Since compound 1, 2 and 3 showed antibacterial activity, they were considered for minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) as seen in table 4.9. Table 4.9 : Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the compound 1, compound 2 and compound 3

Microorganisms Compound 1 Compound 2 Compound 3 MIC in µg/ml MBC in MIC in MBC in MIC in MBC in µg/ml µg/ml µg/ml µg/ml µg/ml S. aureus 0.00625 0.0125 0.003125 0.003125 0.05 0.0125 P.aeruginosa 0.0125 0.0125 0.0125 0.0125 0.0125 0.0125

The Minimum Inhibitory Concentration (MIC) for compounds 1, 2 and 3 ranged from 0.003125µg/ml to 0.05 µg/ml. Compound 1, 2 and 3 had the same MIC of 0.0125 against P.aeruginosa. Compound 2 had the lowest MIC of 0.003125µg/ml against S.aureus. This means compound 2 is more effective against S.aureus than compound 1 and 3. The minimum bactericidal concentration (MBC) ranged from 0.003125 µg/ml to 0.0125 µg/ml. Compound 1, 2

63 and 3 had the same MBC of 0.0125 µg/ml against P. aeruginosa. Compound 2 had the lowest MBC of 0.003125 µg/ml against S .aureus meaning it requires a less dosage for effective treatment.

Compound 1 (betulinic acid) exhibited moderate zone of inhibition of (14.2 ±0.07) mm against both bacterial strains, MIC of 0.0003125 µg/ml against S. aureus and MBC of 0.0125 µg/ml against S . aureus which was in line with the studies done by Sallau et al., 2016. This concluded that compound 1 had a moderate antibacterial activity. Also compound 2 (oleanolic acid) showed moderate zone of inhibition of (12.1± 0.014) mm against both bacterial strains, MIC of 0.00625 µg/ml against S. aureus and MBC of 0.0125 µg/ml against both bacterial strains which was similar to the studies done by Kuete et al., 2007. This conclude that compound 2 had a moderate antibacterial activity. Compound 3 (oleanolic acetate) exhibited the lowest zone of inhibition because of its structure where by the acetate group prevents the blinding of the protein molecule in the parasite.

CHAPTER FIVE: CONCLUSION AND RECOMMENDATION

5.1 CONCLUSION In this study, three compounds have been isolated and characterized from the stem bark of Psorospermum febrifugum as: Betulinic acid (1), oleanolic acid (2) and oleanolic acetate acid (3). This is the first time these compounds are reported in this plant. Compound 1(Betulinic acid) and 2 (oleanolic acid) exhibited a moderate range of antibacterial activities against the two tested bacterial stains while compound 3 (oleanolic acetate acid) exhibited the lowest antibacterial activity against the two tested bacterial strains. This coupled with the findings from the bioassay studies on the crude extract, justify the use of Psorospermum febrifugum in traditional medicine. Furthermore, standardization of herbal formulations from Psorospermum febrifugum bark can be possible by using the characterized compounds as markers in the improved traditional medicine.

5.2. RECOMMENDATION

This study encompasses only the stem bark of Psorospermum febrifugum since one of the aims is to validate the use of the plant for the treatment of bacterial infections. The following are therefore recommended:

i. Other twenty one fractions of the ethyl acetate extract from the stem bark of Psorospermum febrifugum could be further isolated and elucidated to get to get more antibacterial compounds.

ii. Toxicity and cytotoxicity should be carried on ethyl acetate extract of stem bark of Psorosperumum febrifugum to determine if the extract is harmful or not and to what extend does it cause the harm. iii. Other pharmacological studies could be conducted on the crude extracts to explore the efficacy of the plant as medicinal plant.

CHAPTER SIX: APPENDICES

Figure 6.1: TLC plates showing the different forms of the solvent system for the ethyl acetate extract

Figure 6.2: Packing the column using silica gel as the stationary phase

Figure 6.3: TLC plate showing a pure sample for compound 1 and compound 2 respectively

Figure 6.4: TLC plate showing a pure compound for compound 3

Figure 6.5: Plates showing the zone of inhibition for compound 1, compound 2 and compound 3 against S aureus

Figure 6.6: Plates showing zone of inhibition for compound 1, compound 2 and compound 3 against P.aeruginosa

Figure 6.7: Plates showing the MIC for compound 1 against S aureus and P.aeruginosa

Figure 6.8: Plates showing the MIC for compound 2 against S. aureus and P.aeruginosa

Figure 6.9: Plates showing the MIC for compound 3 against S. aureus and P.aeruginosa

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