EVALUATION of Uvaria Chamae AQUEOUS and ETHANOLIC EXTRACTS FOR

EVALUATION OF Uvaria chamae AQUEOUS AND ETHANOLIC EXTRACTS FOR

ACTIVITY AGAINST MULTIDRUG RESISTANT BACTERIA

ONAKPA, IDACHABA E.

PG/M.Sc/07/42926

DEPARTMENT OF MICROBIOLOGY

UNIVERSITY OF NIGERIA, NSUKKA

AUGUST, 2010

TITLE PAGE

EVALUATION OF Uvaria chamae AQUEOUS AND ETHANOLIC EXTRACTS FOR

ACTIVITY AGAINST MULTIDRUG RESISTANT BACTERIA

ONAKPA, IDACHABA EMMANUEL

PG/M.Sc/07/42926

TO THE SCHOOL OF POST GRADUATE STUDIES,

UNIVERSITY OF NIGERIA, NSUKKA

IN PARTIAL FULFILMENT OF THE REQIREMENTS

FOR THE AWARD OF MASTER’S DEGREE

(M.Sc) IN MEDICAL MICROBIOLOGY

SUPERVISOR: DR. (MRS) I.M. EZEONU

AUGUST, 2010

CERTIFICATION

Mr. Onakpa, Idachaba Emmanuel, a postgraduate student in the Department of

Microbiology, majoring in Medical Microbiology, has satisfactorily completed the requirements for course work and research for the degree of Master in Science (M.Sc) in Microbiology. The work is embodied in his dissertation original and has not been submitted in part or full for either diploma or degree of this university or any other university.

Dr. (Mrs.) I. M EZEONU Dr. (Mrs.) I.M. EZEONU Head, Supervisor, Department of Microbiology, Department of Microbiology University of Nigeria, University of Nigeria, Nsukka. Nsukka.

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DEDICATION

This work is dedicated to my mother –Mrs. Habiba Onakpa –for being a constant source of quiet strength.

ACKNOWLEDGEMENT

My gratitude first goes to God Almighty for His gift of life and health. I wish also to acknowledge with humility the motherly support of my supervisor Dr. (Mrs.) I.M. Ezeonu without whom this work would have been impossible. Words fail me to express my gratitude for her patience, attention and motivation as she was always there to guide and direct me.

I wish to thank Mrs. Nwuike of Medical Diagnostic Laboratory of the Microbiology

Department, University of Nigeria, Nsukka and the staff of the Microbiology Section of the

Enugu State University Teaching Hospital, Parklane Enugu for providing me with the clinical specimens used for the work. My thanks also go to Dr. E.A Eze, Mr. Ngene and Mr. Uchenna

Nwodo for also furnishing me with the other strains of bacteria used.

I must also not fail to thank Prof. C.U. Iroegbu for his constant and often critical observations. He gave me reasons to believe in myself, my work and its implications for the ageless annals of scientific collections and hence, the need to work diligently. I wish to also thank Prof. J.C. Ogbonna, Prof. (Mrs.) J.I Okafor and Dr. J.I Ihedioha for their cooperation, understanding and assistance throughout the duration of this research.

Finally, I wish to thank my mother –Mrs. Habiba Onakpa, my father and siblings viz:

Onakpa Aruwa, Asibi, Okutepa, Tijani, Shaibu, Idoko, Opaluwa, Uchogwu and Adi (egg) for their financial and moral support.

ABSTRACT

The antimicrobial activities of ethanolic and aqueous extracts of Uvaria chamae root bark, stem bark and leaves were evaluated. The root bark extract of the plant is used in ethnomedicine as a prescription for piles, haematuria, treatment of fevers classed locally as „yellow- fever‟, and almost any indisposition accompanied by jaundice. Thirty eight bacterial strains resistant to a variety of routinely used antibiotics were used for the assays; four strains were from American

Type Culture Collection (ATCC), one strain from Scottish Salmonella Reference Laboratory

(SSRL), one strain from Northern Regional Research Laboratory (NRRL) and thirty two were clinical isolates. Agar well diffusion technique was used for the antibacterial assays. Generally, the extracts had antimicrobial activity against most of the test organisms. Inhibition was a direct function of concentration of extracts with 100% of organisms inhibited at 250mg/ml, albeit to different degrees. The alcoholic extracts were significantly (P<0.05) more active than the aqueous extracts, with the ethanolic stem bark extract having higher activity than the root bark and leaf extracts. The minimum inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC) varied with test isolates as well as with various extracts. For instance, the

MIC of the ethanolic extracts of the stem bark varied from 7.81 mg/ml for Proteus mirabilis to

100 mg/ml for Klebsiella with their respective MBC‟s varying from 37.03 mg/ml to 200 mg/ml.

The ethanolic extracts were successively partitioned using n-hexane(A), chloroform (B), ethyl acetate (C), acetone (D), and methanol (E). Five fractions were obtained for each of root bark, stem bark, and leaves and were designated Ar to Er, As to Es and AL to EL respectively. Fractions

Ds, Es, and Cr had activity against 100, 90 and 73% of test strains respectively. The least active fractions were El (27%), Al (36%), Dl (45%), Ar (45%) and As (45%). Acute toxicity studies using LD50 assays showed the alcoholic stem bark extract to be virtually non-toxic ( LD50

>5000mg/kg body weight). Phytochemical analyses of crude extracts and fractions indicated the presence of flavonoids, saponins, tannins, alkaloids amongst others in relative proportion.

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TABLE OF CONTENTS

Title Page… … … … … … … … … … i

Certification… … … … …. … … … … ii

Dedication… … … … … … … … … … iii

Acknowledgement… … … … … … … … … iv

Abstract.. … … … … … … … … … v

Table of Contents.. … … … … … … … … vii

List of Tables .. … … ...... … x

List of Figures … … .. … … …. … … … xi

CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW… 1

1.1 Specific aims and objectives … … … … … .. 3

1.2 Literature review … … … … … … .. 3

1.2.1 Historical development of ethno medicine … …. … … 3

1.2.2 Antibiotic resistance.. … …. … … … … … 8

1.2.3 Natural products in drug discovery … … …. … …. 9

1.2.4 Plant and antibacterial production … … …. …. …. 11

1.2.5 Safety issues in herbal medicine: Implications for the health professions 14

1.2.6 Taxonomy and systematic of Uvaria.. … … … .. 17

1.2.7 The Plant- Uvaria chamae … .. … … … … 20

1.2.8 Uvaria chamae in alternative medicine.. … … … … 20

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CHAPTER TWO: MATERIALS AND METHODS… … …. …. 24

2.1 Collection of plant materials. .. … … … … … 24

2.2 Extraction… … … … … … … .. … 26

2.3 Microorganisms… …. …. … .. … … … 26

2.4 Test for sensitivity or resistance to standard antibiotics… … … 27

2.5 Determination of antimicrobial activity of crude extracts.. … … 28

2.6 Determination of minimum inhibitory concentration… … … 29

2.7 Determination of minimum bactericidal concentration… …. … 29

2.8 Solvent-solvent fractionation… …. …. …. … … 29

2.9 Antibacterial assay of extract fractions… … … … … 32

2.10 Phytochemical evaluation of crude extracts and fractions.. … … 32

2.10.1 Test for saponins… … … … … … … … 32

2.10.2 Test for tannins… … … … … … …. … 33

2.10.3 Test for alkaloids.. .. … … … … … … 33

2.10.4 Test for flavonoids.. … … … … … .. … 34

2.10.5 Test for fats and oils… … … … … … … 34

2.10.6 Test for reducing sugar… … … … … … … 34

2.10.7 Test for glycosides … … … … … … … 35

2.11 Acute toxicity studies.. … … … … … .. 35

2.12 Statistical Analysis… … … … … … … … 36

CHAPTER THREE: RESULTS.. … … … … .. … 37

3.1 Antimicrobial susceptibility profile of test bacteria … .. … 37

3.2 Aqueous and ethanolic extractions.. … … .. … .. 40

3.3 Antimicrobial activities of crude extract… .. …. … … 42

3.4 Minimum inhibitory and minimum bactericidal concentrations of extracts 53

3.5 Yield from solvent-solvent fractionation… . … … … 56

3.6 Antimicrobial activities of extract fractions… … … … 58

3.7 Phytochemical composition of crude extract and fractions… …. 84

3.8 Acute toxicity profile.. … … … … … … 89

CHAPTER FOUR: DISCUSSION … … …. …. … .. 90

REFERENCES … … … … … ...... 97

APPENDICES … … …. …...... 102

LIST OF TABLES

Table 1: Plant derived widely employed in western medicine… … … … 7

Table 2: Resistance pattern of Gram negative organisms to conventional antibiotics.. 38

Table 3: Resistance pattern of Gram positive organisms to conventional antibiotics.. 39

Table 4: Minimum inhibitory comcentration… …. …. … … … 54

Table 5: Minimum bactericidal concentration… … …. … … … 55

Table 6: Preliminary phytochemical screening of crude extracts… … … … 85

Table 7: Phytochemical screening of root bark fractions … … … … … 86

Table 8: Phytochemical screening of stem bark fractions …. …. … … 87

Table 9: Phytochemical screening of leaf fractions … …. … … … 88

LIST OF FIGURES

Figure 1: Extraction of crude plant parts …. …. …. .. 41

Figure 2: Activity of crude aqueous root bark extract … … .. 43

Figure 3: Activity of crude alcoholic root bark extract … .. .. 45

Figure 4: Activity of crude aqueous stem bark extract … .. .. 47

Figure 5: Activity of crude alcoholic stem bark extract … … ... 48

Figure 6: Activity of crude aqueous leaf extract … … .. .. 50

Figure 7: Activity of crude alcoholic leaf extract … … .. .. 52

Figure 8: The percentage recovery of crude extract fractions using solvent – solvent

Fractionation … … .. … .. … … 57

Figure 9: Activity of root bark acetone fraction … … .. .. 59

Figure 10: Activity of stem bark acetone fraction … … .. .. 61

Figure 11: Activity of leaf acetone fraction … … .. .. 63

Figure 12: Activity of root bark ethyl acetate fraction … … .. .. 65

Figure 13: Activity of stem bark ethyl acetate fraction … … .. .. 67

Figure 14: Activity of leaf ethyl acetate fraction … … .. .. 69

Figure 15: Activity of root bark methanol fraction … … .. .. 71

Figure 16: Activity of stem bark methanol fraction … … .. .. 73

Figure 17: Activity of leaf methanol fraction … … .. .. 75

Figure 18: Activity of leaf chloroform fraction … … .. .. 77

Figure 19: Activity of root bark n – heaxane fraction … … .. .. 79

Figure 20: Activity of stem bark n – heaxane fraction … … .. .. 81

Figure 21: Activity of leaf n – heaxane fraction … … .. .. 83

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APPENDICES

Appendix 1: A. Analysis of variance for crude aqueous root bark extract …. … 102

B. differences in mean inhibition amongst bacterial isolates … … 102

Appendix 2: A. Analysis of variance for crude alcoholic root bark extract … 103

B. differences in mean inhibition amongst bacterial isolates .. … 103

Appendix 3: A. Analysis of variance for crude aqueous stem bark extract .. … 104

B. differences in mean inhibition amongst bacterial isolates …. … 104

Appendix 4: A. Analysis of variance for crude alcoholic stem bark extract .. 105

B. differences in mean inhibition amongst bacterial isolates .. .. 105

Appendix 5: A. Analysis of variance for crude aqueous leaf extract .. … 106

B. differences in mean inhibition amongst bacterial isolates . … 106

Appendix 6: A. Analysis of variance for crude alcoholic leaf extract … …. 107

B. differences in mean inhibition amongst bacterial isolates .. … 107

Appendix 7: A. Analysis of variance for root bark acetone fraction .. .. 108

Appendix 8: A. Analysis of variance for stem bark acetone fraction … …. 109

B. differences in mean inhibition amongst bacterial isolates … …. 109

Appendix 9: A. Analysis of variance for leaf acetone fraction … …. 110

Appendix 10: A. Analysis of variance for root bark ethyl acetate fraction … …. 110

Appendix 11: A. Analysis of variance for stem bark ethyl acetate fraction … …. 111

Appendix 12: A. Analysis of variance for leaf ethyl acetate fraction … …. 111

Appendix 13: A. Analysis of variance for root bark methanol fraction … …. 112

Appendix 14: A. Analysis of variance for stem bark methanol fraction … …. 113

Appendix 15: A. Analysis of variance for leaf methanol fraction … …. 113

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Appendix 16: A. Analysis of variance for root bark n – hexane fraction … …. 114

Appendix 17: A. Analysis of variance for stem bark n – hexane fraction … …. 115

Appendix 18: A. Analysis of variance for leaf n – hexane fraction … …. 115

Appendix 19: A. Analysis of variance for leaf chloroform fraction … …. 116

CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW

Multiple drug resistance is a condition enabling a disease-causing organism to grow in the presence of distinct drugs or chemicals of a wide variety of structure and function targeted at eradicating them. In spite of production of new antibiotics by pharmaceutical industries, resistance continues to be a public health problem worldwide.

In developing countries, infective diseases are thought to account for approximately one- half of all deaths and in the industrialized nations, incidence of epidemics due to drug resistant microorganisms and the emergence of hitherto unknown disease-causing microbes, pose enormous public health concerns (Iwu et al., 1999). Consequent upon the increasing problem of antibiotic resistance, ethnomedicine is gaining popularity as an alternative to orthodox medicine.

The tradition of collecting, processing and applying plant and plant-based medication has been handed down from generation to generation. In many African countries, traditional medicine, with medicinal plants as their most important components, are sold in market places or prescribed by traditional healers (without accurate dose values) in their homes (Herdberg and Staugard, 1989). Extracts from these plants are currently used for the treatment of various infectious diseases from minor skin infections to gastrointestinal disturbances and more serious infectious diseases such as typhoid fever and malaria. A vast knowledge of and experience with medicinal plants has accumulated over generations. This information has been annotated in herbals, herbarium notes, ethno botanical studies and pharmacopoeias and is still part of the oral tradition of

2 many ethnic groups. In the search for plant drugs for medicinal/pharmaceutical needs, it would be obviously unwise not to make use of this ethno-pharmacological experience.

Moreover, the World Health Organization (WHO), has observed that up to 80% of the rural populace in the developing countries depend on herbal or alternative medicine and requested member countries to explore safe indigenous medicine for their national health care (Sofowora, 1984). It is possible that antimicrobial compounds from plants may inhibit bacteria by a different mechanism than the presently used antibiotics and may have clinical values in the treatment of resistant bacterial strains.

The rate of increase in scientific enquiry into the use, effects and constituents of medicinal plants has risen steeply in Nigeria over the last decades; for instance, the inhibitory activities of Acalypha wilkesiana, Phyllanthus discoideus and Trema guineensis amongst others on different bacteria have been reported (Akinyemi et al.,

2006; Aladesanmi et al., 2007; Ogbulie et al., 2007; Ogbonnia et al., 2008). Other herbs abound in native medicine in the country and hence, more studies on their therapeutic properties and safety should be emphasized, especially those used in the control of antibiotic resistant microbes.

Uvaria chamae, a small tree of about 4.5 meters high is native to West and

Central Africa where it grows in wet and dry forests. The fruit carpels are in finger-like clusters and the sweet pulp of the yellow fruit is widely eaten. The properties are mainly astringent and it is used in native medicine for piles, amenorrhea and haematuria amongst others. This study was aimed at determining the antimicrobial activities and safety of extracts of U. chamae.

1.1 Specific Aims and Objectives

• To evaluate the activity of crude aqueous and ethanolic extracts from the root

bark, stem bark and leaves of Uvaria chamae on multiple drug resistant bacteria.

• To partition extracts into composite fractions and hence determine their relative

activities.

• To determine the phytochemical constituents of crude extracts and fractions.

• To evaluate the toxicity of extracts and hence their safety.

1.2.0 LITERATURE REVIEW

1.2.1 Historical development of ethnomedicine

It is sometimes suggested that the field of medicinal plants and/or crude drugs has already been exploited to the extent that further research would yield only “academic results” and no novel drugs. In contrast to this point of view, however, some believe that modern research has only scratched the surface of this field (Wijesekera, 1991). Carl Von linnẻ for instance believed that only about 10,000 species exist in the plant kingdom; modern estimates vary between 280,000 and 700,000 species.

In the written record, the study of herbs dates back over 5,000 years to the

Sumerians, who described well-established medicinal uses for such plants as laurel, caraway, and thyme. Ancient Egyptian medicine of 1000 B.C. are known to have used garlic, opium, castor oil, coriander, mint, indigo, and other herbs for medicine and the

Old Testament also mentions herb use and cultivation, including mandrake, vetch, caraway, wheat, barley, and rye. Indian Ayurveda medicine has used herbs such as turmeric possibly as early as 1900 B.C (Aggarwal et al., 2007). The first Chinese herbal

4 book, the Shennong Bencao Jing, compiled during the Han Dynasty but dating back to a much earlier date, possibly 2700 B.C., lists 365 medicinal plants and their uses - including ma-Huang, the shrub that introduced the drug ephedrine to modern medicine.

Medical schools known as Bimaristan began to appear from the 9th century in the medieval Islamic world, which was generally more advanced than medieval Europe at the time. The Arabs venerated Greco-Roman culture and learning, and translated tens of thousands of texts into Arabic for further study (Castleman, 2001). As a trading culture, the Arab travellers had access to plant material from distant places such as China and

India. Herbals, medical texts and translations of the classics of antiquity filtered in from east and west. Muslim botanists and Muslim physicians significantly expanded on the earlier knowledge of materia medica. For example, al-Dinawari described more than 637 plant drugs in the 9th century (Fahd, 1996), and Ibn al-Baitar described more than 1,400 different plants, foods and drugs, over 300 of which were his own original discoveries, in the 13th century (Boulanger, 2002). The experimental scientific method was introduced into the field of materia medica in the 13th century and this allowed the study of materia medica to evolve into the science of pharmacology (Huff, 2003).

Many of the pharmaceuticals currently available to physicians have a long history of use as herbal remedies, including opium, aspirin, digitalis, and quinine. The World

Health Organization (WHO) estimates that 80 percent of the world's population presently uses herbal medicine for some aspect of primary health care (WHO, 2010). Herbal medicine is a major component in all traditional medicine systems, and a common element in Siddha, Ayurvedic, homeopathic, naturopathic, traditional Chinese medicine, and Native American medicine.

The use of, and search for, drugs and dietary supplements derived from plants have accelerated in recent years. Pharmacologists, microbiologists, botanists, and natural- products chemists are combing the Earth for phytochemicals and leads that could be developed for treatment of various diseases. In fact, according to the World Health

Organization, approximately 25% of modern drugs used in the United States have been derived from plants (WHO, 2010).

Plants in popular medicine

Plants have served as a source of new pharmaceutical products and inexpensive starting materials for the synthesis of many known drugs. Such plants as chives, ginger, garlic, African bread fruit, yarrow, lady‟s mantle, angelica, thyme, germander, garden heliotrope, dandelion, evening primrose, German chamomile, hyssop and curry have dominated interests for centuries because of their superior medicinal qualities. Although the first chemical substance to be isolated from plants was benzoic in 1560, the search for useful drugs of known structure did not begin until 1804 when morphine was separated from Papaver somniferum L (Pium) (Angeh, 2006). Since then many drugs from higher plants have been discovered, but less than 100 with defined structures are in common use.

Less than half of these (Table 1) are accepted as useful drugs in industrialized countries

(Farnsworth, 1984). Considering the great number of chemicals that have been derived from plants as medicine, scientific evaluation of plants used traditionally for the treatment of bacterial infections seems to be a logical step of exploiting the antimicrobial compounds that may be present in plants. Plant-based antimicrobials represent a vast untapped source of medicines with enormous therapeutic potential (Cowan, 1999). They

6 are supposedly effective in the treatment of infectious diseases while simultaneously mitigating many of the side effects that are often associated with synthetic antimicrobials

(Iwu et al., 1999).

Table 1: Plant-derived drugs widely employed in Western medicine (Adapted from

Farnsworth, 1984)

Acetyldigoxin Ephidrine Pseudoephedrine Xanthotoxin Aescin Hyoscyamine Quinidine Ajmalicine Khellin Quinine Allantoin Lanatoside Reserpine Atropine Leurocristine Rescinnamine Bromelain Lobeline Scillarens A & B Caffeine Morphine Scopolamine Codeine Narcotine Sennosides A & B Colchicines Ouabain Sparteine Danthron Papain Strychnine Deserpidine Papaverine Tetrahydrocannabinol Digitoxin Physostigmine Threobromine Digoxin Picritoxin Theophylline L-Dopa Pilocarpine Tubocurarine Emetine Protoveratrines A & B Vincaleukoblastine

1.2.2 Antibiotic resistance

Almost since the beginning of the antibiotic era, bacterial resistance has been seen as one of the major obstacles to successful treatment (Iwu et al., 1999). Microbial resistance to antibiotics in the clinic emerged soon after their first use in the treatment of infectious disease, and continues to pose a significant challenge for the health sector.

Resistance has now firmly emerged as a problem in the wider community. At the end of the 1960s the Surgeon General of the United States stated that: “we could close the book on infectious diseases”. At the time he uttered these words the emergence of resistance did not seem to affect therapeutic options. Although S. aureus had become resistant to benzylpenicillin and was showing resistance to thethincillin, it remained sensitive to gentamycin and infections could therefore still be treated. At the start of the next century, things looked very different. Already at least three bacterial species, capable of causing life-threatening illness (Enterococcus faecalis, Mycobacterium tuberculosis and

Pseudomonas aeruginosa), had become resistant to every one of the 100 antibiotics, available except for vancomycin (Iwu et al., 1999). Vancomycin was the antibiotic of last resort for treatment of resistant infections for many years, but in recent years scientists have found strains of Streptococcus pneumoniae and S. aureus to be resistant to this antibiotic. This is attested by spread, with associated deaths, of infection by methicillin- resistant Staphylococcus aureus and the increased prevalence of drug-resistant S. pneumoniae in patients suffering from pneumonia. Antimicrobial resistance is driven by inescapable evolutionary pressures and is therefore predictable and inevitable. The emergence in recent years of vancomycin-resistant S. aureus, punctuates this assertion.

Hardly any group of antibiotics has been introduced to which some bacterium has not developed resistance (Iwu et al., 1999).

Recent reports have shown a marked increase in antibiotic resistance of food- poisoning bacteria due to non-rational and excessive use of antibiotics as therapeutic agents or as growth promoters in livestock. Another factor of resistance potentially lies in the use of antibiotic resistant genes as selection markers in genetically modified organisms (GMOs) (Angeh, 2006). The main safety issue of concern is the release of these resistant genes to sensitive organisms when these GMOs are introduced into the environment.

Due to emergence of drug resistant bacteria, the search for new antimicrobial compounds with improved activity is necessary (Harold and Heath, 1992). Many indigenous plants are used in treating bacterial related diseases. Only a small fraction of these indigenous plants has been investigated (Carr and Rogers, 1987).

1.2.3 Natural products in drug discovery

Medicinal plants use is widespread. Life saving and essential drugs from medicinal plants such as morphine, digoxin, aspirin, and emetine were introduced into modern therapeutics several centuries ago. However, plants have been used as drugs for over millennia by human beings. Plants historically have served as models in drug development for some major reasons: the first being that plants are unique chemical factories capable of synthesizing large numbers of highly complex and unusual chemical substances. It has also been estimated by the World Health Organization (WHO) that about 80% of the population of the developing countries rely exclusively on plants to

10 meet their health care needs (Farnsworth et al., 1985). The second reason is that biologically active substances derived from plants have served as templates for synthesis of pharmaceuticals; while the third reason concerns the fact that highly active secondary plant constituents have been instrumental as pharmacological tools to evaluate physiological processes (Farnsworth, 1984). There are numerous illustrations of plant- derived drugs.

Despite the expense involved in the development of a drug today, at least US$230 million and a time span between 10 – 20 years (Farnsworth, 1984), nature remains the most reliable and most important source of novel drug molecules. Nature provides 80% of all pharmacological and therapeutic lead compounds and the National Cancer Institute

(NCI) estimates that over 60% of the compounds currently in pre-clinical and clinical development in its laboratories are of natural origin. Thus, higher plants remain an important and reliable source of potentially useful chemical compounds not only for direct use as drugs, but also as unique prototypes for synthetic analogues and as tools that can be used for a better understanding of biological processes (Farnsworth, 1984).

Literally thousands of phytochemicals with inhibiting effects on microorganisms have shown in vitro activity. One may argue that these compounds have not been tested in vivo and therefore activity cannot be claimed, but one must take into consideration that many, if not all, of these plants have been used for centuries by various cultures in the treatment of diseases. Another argument could possibly be that at very high concentrations, any compound is likely to inhibit the growth of microorganisms. Firstly, if this is the case, the high concentrations required would no doubt have serious side effects on the patient unfortunate enough to contract an illness. Secondly, these

11 compounds are compared with those of standard antibiotics already available in the market. This means that the concentrations used must compare favourably to those that have already passed the test (Cowan, 1999; Angeh, 2006).

Asiaticode, an antimicrobial compound isolated from Centella asiatica (used traditionally in skin diseases and leprosy), has been studied in normal as well as delayed- type wound healing. The results indicated significant wound healing in both models.

Another compound, cryptolepine, isolated from Crytolepis sanguinolenta and active against Campylobacter species, has been used traditionally in Guinea Bissau in the treatment of hepatitis and in Ghana for the treatment of urinary and upper respiratory tract infections and malaria (Angeh, 2006).

1.2.4 Plants and antibacterial production

An antibiotic has been defined as a chemical compound derived from or produced by living organisms, which is capable, in small concentrations of inhibiting the growth of microorganisms (Evans, 1989). This definition limited antibiotics to substances produced by microorganism but the definition could now be extended to include similar substances present in higher plants. Plants have many ways of generating antibacterial compounds to protect them against pathogens. External plant surfaces are often protected by biopolymers e.g. waxes, and fatty acid esters such as cutin and suberin. In addition, external tissues can be rich in phenolic compounds, alkaloids, diterpenoids, steroids glycoalkaloids and other compounds, which inhibit the development of fungi and bacteria

(Kuc, 1985). Cell walls of at least some monocotyledons also contain antimicrobial proteins, referred to as thionins.

Plant cells containing sequestered glycosides release them when ruptured by injury or infection. These glycosides may have antimicrobial activity against the invading pathogens or may be hydrolysed by glycosidases to yield more active aglycones. In the case of phenolic compounds, these may be oxidized to highly reactive, antimicrobial quinones and free radicals. Thus, damage to a few cells may rapidly create an extremely hostile environment for a developing pathogen. This rapid, but restricted disruption of a few cells after infection can also result in the biosynthesis and accumulation of phytoalexins, which are low molecular weight antimicrobial compounds, which accumulate at sites of infection (Kuc, 1985). Some phytoalexins are synthesized by the malonate pathway others by the mevalonate, or shikimate pathways, whereas still others require participation of two or all three of the pathways. Phytoalexins are degraded by some pathogens and by the plant; thus they are transient constituents and their accumulation is a reflection of both synthesis and degradation rates.

Biopolymers are also often associated with the phytoalexin accumulation at the site of injury or infection. These biopolymers include: lignin, a polymer of oxidized phenolic compounds; callose, a polymer of β-1,3-linked glucopyranose; hydroxyproline- rich glycoproteins, and suberin. They provide both mechanical and chemical restriction of development of pathogens (Kuc, 1985). The macromolecule produced after infection or certain forms of physiological stress includes enzymes, which can hydrolyse the walls of some pathogens including chitinases, β-1,3-glucanases and proteases. Unlike the phytoalexins and structural biopolymers, the amounts of these enzymes increase systematically in infected plants even in response to localized infection. These enzymes are part of a group of stress or infection-related proteins commonly referred to as

13 pathogenesis-related (PR) proteins. The function of many of these proteins is unknown.

Some may be defense compounds while others may regulate the response to infection

(Rao and Kuc, 1990).

Another group of systematically produced biopolymer defense compounds comprises the peroxidases and phenoxidases (Rao and Kuc, 1990). Both can oxidize phenols to generate protective barriers to infection, including lignin. Phenolic oxidation products can also cross-link to carbohydrates and proteins in the cell walls of plants and fungi to restrict further microbial development. Peroxidases also generate hydrogen peroxidase, which is strongly antimicrobial. Associated with peroxidative reactions after infection is the transient localized accumulation of hydroxyl radicals and super oxide anion, both of which are highly reactive and toxic to cells (Angeh, 2006).

Plants therefore have several mechanisms to counter antimicrobial attack. Some of the antimicrobial compounds in plants may be exploited for use against bacterial diseases in man. Plants have developed an arsenal of weapons to survive attack by microbial invasions. These include both physical barriers as well as chemical ones, i.e. the presence or accumulation of antimicrobial metabolites. These are either produced in the plant (prohibitins) or induced after infection, the so-called phytoalexins. Since phytoalexins can also be induced by abiotic factors such as UV irradiation, they have been defined as antibiotics formed in plants via a metabolic sequence induced either biotically or in response to chemical or environmental factors (Angeh, 2006).

1.2.5 Safety issues in herbal medicine: implications for the health professions

Adverse effects of herbal medications may be intrinsic or extrinsic. The patient's age, genetic constitution, nutritional state, concomitant diseases and concurrent medication may affect the risk and severity of adverse events, as can consumption of large amounts or a wide variety of herbal preparations, or long-term use (De Smet, 1995).

Intrinsic effects are those of the herb itself and are characterised, as for pharmaceuticals, as type A (predictable, dose-dependent) and type B (unpredictable, idiosyncratic) reactions. Yohimbine, an alkaloid found in Pausinystalia yohimbe bark that has 2-adrenoceptor antagonist activity, is taken for male impotence, and can cause hypertension and anxiety in a predictable, dose-related manner (type A reaction); it has also been associated with the serious idiosyncratic reactions of bronchospasm and increased mucus production when taken in normal doses by a patient with severe allergic dermatitis (type B) (De Smet and Smeets, 1994).Type A reactions with herbal preparations also include effects with deliberate overdose or accidental poisoning and interactions with pharmaceuticals.

Extrinsic effects are not related to the herb itself, but to a problem in commercial manufacture or extemporaneous compounding. Potential failures to adhere to a code of

Good Manufacturing Practice, while not specific to herbal medicine, can occur, particularly in developing countries where such a code is not in place. This makes it more difficult for medical practitioners and other health professionals to assess the adverse effects of herbal preparations compared with pharmaceuticals.

Misidentification: It is difficult to track and identify adverse effects of herbal ingredients, as the plants can be named in four different ways -- the common English

15 name, the transliterated name, the Latinized pharmaceutical name, and the scientific name. It is essential that plants are referred to by their binomial Latin names for genus and species; misidentification can occur when other names are used. For example, the scientific name of the Chinese herb that is variously transliterated as "dong quai", "dong guai", "danggui" and "tang kuei" is Angelica polymorpha (formerly sinensis). The common English name "angelica" and the Latinized name "Radix Angelica" could refer either to this species, which is used in Australia, or to the European species Angelica archangelica, depending on the country of origin (Drew and Myers, 1997).

Misidentification can result in erroneous associations being made, with potential clinical implications. Plant material can be misidentified at the time of the manufacturer's bulk purchase or when wild plants are picked.

Lack of standardisation: The therapeutic/toxic components of plants vary depending on the part of the plant used, stage of ripeness, geographic area where the plant is grown, and storage conditions. Therefore, batch-to-batch reproducibility of plant material should be assessed in the production of marketed products, but, in practice, product variation in herbal medicines can be significant. The content of ginsenoside, the glycosylated steroid to which most of the biological activity of ginseng (Panax ginseng) has been ascribed, was examined in 50 commercial brands of ginseng sold in 11 countries. In 44 of these products, the concentration of ginsenoside ranged from 1.9% to

9% w/w; six products contained no ginsenoside, and one of these six contained large amounts of ephedrine (for which a Swedish athlete was accused of doping).

Contamination: During growth and storage, crude plant material can become contaminated by pesticide residues, microorganisms, aflatoxins, radioactive substances

16 and heavy metals; lead, cadmium, mercury, arsenic and thallium have been reported as contaminants of some overseas herbal preparations. In a case series of five patients in the

United Kingdom with lead poisoning from Asian traditional remedies, the preparations implicated contained 6%-60% w/w lead by weight (Bayly et al., 1995).The Australian

Code of Good Manufacturing Practice specifies detection of microorganisms and leaves estimation of other contaminants (not specified in internationally recognized pharmacopoeial standards) to the discretion of manufacturers.

Substitution: A report of nine cases of rapidly progressive interstitial nephritis in young women taking a Belgian slimming treatment led to the discovery that Aristolochia fangchi, containing the nephrotoxic component aristolochic acid, had been introduced in place of Stephania tetrandra. Eighty cases have now been identified and more than half of these patients developed terminal renal failure (Drew and Myers, 1997)

Adulteration: The intentional use of pharmaceutical adulterants has been reported. Cases of acute interstitial nephritis, reversible renal failure, loss of blood pressure control and peptic ulceration have been reported with a product called "Tung

Shueh" pills, taken for arthritic complaints. The product contained mefenamic acid and diazepam, neither of which was included on the label. Adulterants can also be added by unethical herbalists compounding preparations for individual patients. In a recent court case, a Chinese herbalist was prosecuted for adding a steroid cream to a herbal preparation, which produced severe facial erythema in a patient.

Incorrect preparation/dosage: The processing of crude plant material carried out by a manufacturer, CAM practitioner or the patient is a major determinant of the pharmacological activity of the finished product. A Western Australian patient had a

17 heart attack when he failed to follow a herbalist's instructions to boil aconite (a restricted plant in Australia) in three pints of water for one hour and take the decanted liquid; the patient increased the dose and shortened the boiling time. Boiling changes the alkaloid composition, rapidly reducing the plant's toxicity, and can substantially reduce microorganism contamination.

Another point to consider is that the activity of crude plant material may differ from that of the purified constituents, as some constituents may modify the toxicity of others.

1.2.6 Taxonomy and Systematics of Uvaria

The plant Uvaria belongs to the family Annonaceae. Annonaceae, also called the custard apple family, is a family of flowering plants consisting of trees, shrubs or rarely lianas. With about 2300 to 2500 species and more than 130 genera, it is the largest family in Magnoliales. Only four genera, Annona, Rollinia, Uvaria and Asimina produce edible fruits. The family is concentrated in the tropics, with few species found in temperate regions. About 900 species are Neotropical, 450 are Afrotropical, and the other species

Indomalayan (Wikipedia, 2010)

Monophyly and inter-familial systematic have been well supported for

Annonaceae by a combination of morphological and molecular evidence (Doyle et al.,

2004). The APG II system places Annonaceae as most closely related to the small

Magnoliid family Eupomatiaceae.

Canellales

Piperales

Magnoliales Myristicaceae

Magnoliaceae

Degeneriaceae

Himantandraceae

Eupomatiaceae

Annonaceae

Laurales

The bark, leaves and roots of some species are used in folk medicine.

Pharmaceutical research has found antifungal, bacteriostatic, antimalarial, and especially cytostatic capability of some chemical constituents of the leaves and bark. A large number of chemical compounds, including flavonoids, alkaloids and acetogenins have been extracted from the seeds and many other parts of these plants. Flavonoids and alkaloids have shown antibacterial properties, and have been used for treatment of medical conditions, such as skin disease, intestinal worms and inflammation of the eye.

Pharmaceutical products are currently in animal and human cell-line trials (Suedee, 2007;

Cuendet, 2008). Acetogenins are thought to have anti-HIV and anti-cancer effects. A wide variety of products have been developed and are available for cancer treatment

(Wikipedia, 2010).

Plant root extracts from the family Annonaceae have been reported to have potent cytotoxic and antimicrobial activity and members of the genus Uvaria, have been found to contain compounds such as acetogenins useful as pesticides, and recently as

19 anticarcinogenic agent. Two acetogenins were reported to be chemo-therapeutically active. These acetogenin compounds were isolated in substantially pure forms from a plant in the Annonaceae family, Annona bulata Rich. The two compounds, bullantacin and bullantacinone, were found to exhibit anti tumor and pesticidal activity and were characterized as being new members of an unusual class of compounds. The compounds are composed of tetrahydrofuran rings having two adjacent hydroxyl groups (Bashengezi and Constantin, 1997). These authors in the same report disclosed an annonaceous acetogenin -asimicin extracted from the root bark of the plant Asimina triloba. Asimicin was used in the control of pests. However, the report noted that the most potent form of the Asimina triloba extract is taken from the bark of the plant, not the root. The extract appears to be a tetrahydrofuranoid fatty acid lactone having variable side chain; however the disclosure is directed to the substantially pure compound, asimicin. Reported literature has shown that the compound, Uvaritin, from Uvaria acuminata, another annonaceous plant, demonstrated anti-tumor activity (Cole et al., 1976). Also, the extract

Uvaricin, taken from the purified root extracts of Uvaria acuminata, was shown to have an in vitro activity against lymphocytic leukemia in mice (Jolad et al., 1982).

In fact, other papers contain numerous reports that plant extracts from the

Annonaceae family have been investigated for their medicinal and toxicological effects.

In one pharmacological screening, substantial antibacterial, antifungal and antihelminthic activities were observed using extracts of the root barks of Uvaria narum wall, and

Uvaria hookeri king (Bashengazi and Constantin, 1997).

In a study, species from Uvaria originating from Tanzanian plants were tested for their in vitro activity against the multi-drug resistant K strain of Plasmodium falciparum,

20 the causative agent for the disease malaria. In that study, extracts from the stem and root barks of Uvaria lucida and Uvaria sp (Pande), were reported as having anti-malarial activity. Among the compounds isolated in the study were Uvaretin, Diuvaretin, and

8‟9‟– dihydroxy-3- farnesy-lindole as the most active compounds. The particular Uvaria species consisted of nine varieties from Tanzania and include: U. dependens, U. faulknerae, U. kirkii, U. leptocaldon, U. lucida, Uvaria sp (Pande), U. scheffieri, and U. tanzaniae (Nkunya et al., 1991).

1.2.7 The plant -Uvaria chamae

This is a climbing large shrub or small tree of about 4.5 meters high, native to tropical West and Central Africa where it grows in wet and dry forests and coastal scrublands. It is called “Mmimi ohia”, “Kas kaifi”, and “Akisan” amongst the Ibo, Hausa and Yoruba of Nigeria respectively. The fruit carpels are in finger-like clusters, the shape giving rise to many vernacular names translated as „bush banana‟ or the like implying wildness. The Sierra Leone Krio names „finger‟ and „finger-root‟ for the roots are also from the fruit shape. The fruits are yellow when ripe and have a sweet pulp which is widely eaten. All parts of the plants are fragrant.

1.2.8.1 Uvaria chamae in alternative medicine

The various vernacular names mostly cover more than this one species. The root and root-bark have a widespread reputation. The latter yields an oleo-resin and is taken internally for catarrhal inflammation of mucous membranes, bronchitis and gonorrhoea in

Nigeria, while at one time a fluid extract entered into the composition of a stock hospital prescription in Ghana for dysentery (Burkill, 1985). The properties are mainly astringent

21 and styptic, and it is used in native medicine for the treatment of piles, useful also for menorrhagia (for which it is taken mixed with Guinea grains and added to food), epistaxis, haematuria, haematemesis and haemoptysis. In Sierra Leone and Lagos the root is credited with having purgative and febrifugal properties. In Sierra Leone the root or the root-bark is boiled with spices and the decoction drunk for fevers classed locally as

„yellow-fever‟, including almost any indisposition accompanied by jaundice, and in the

Ivory Coast it enters into a treatment for a form of jaundice.

In the Casamance of Senegal, leaves and roots are macerated for internal use as a cough mixture, and mixed with those of Annona senegalensis, dried and pulverized are considered strong medicine for renal and costal pain (Burkill, 1985). The roots are used in the Casamance for healing sores, and a concoction called n‟taba in the Bayot dialect is reputed to cure infantile rickets. In Nigeria a root-decoction is also held to be stomachic and vermifugal, and is used as a lotion; sap from the root and stem is applied to wounds; and the root is made into a drink and a body-wash for oedematous conditions. Amongst the Fula peoples of Senegal, the root has a reputation as the „Medicine of Riches‟, and is taken for conditions of lassitude and senescence. It is also considered to be a „woman‟s medicine‟ used for amenorrhoea and to prevent miscarriage, and in Togo a root-decoction is given for the pains of childbirth.

In Ghana severe abdominal pain is treated by a root-infusion with native pepper in gin, and the root with Guinea grains (probably Piper guinense Schum. & Thonn.

(Piperaceae)) is used in application to the fontanelle for cerebral diseases. The sap of leaves, roots and stems is widely used on wounds and sores (Burkill, 1985) and is said to promote rapid healing. A leaf-infusion is used as an eyewash and a leaf-decoction as a

22 febrifuge. The crushed seeds with those of Piper guinense are rubbed on the body. The crushed root, along with Capsicum or other rubefacient substances, is rubbed on as a local counter-irritant.

Antihepatotoxic and trypanocidal activities of a root bark extract derived from

Uvaria chamae have been tested in vivo and in vitro (Madubunyi et al., 1996).

Intraperitoneal injection of the ethanol extract into mice showed no significant effect on pentobarbitone-induced hypnosis. Pento-barbitone-induced sleep in tetrachloromethane

(CCL4)-poisoned rats was significantly reduced by oral administration of the extract (60 mg/kg). The elevation of serum GOT, GPT, alkaline phosphatase and urea induced by

CCL4 intoxication in rats was also significantly reduced by the ethanol extract. In the same study, U. chamae ethanol extract showed a significant trypanocidal effect which was comparable to that of diminazine aceturate. Reduction of existing parasitaemia in mice infected with Trypanosoma brucei brucei was found to be dose-dependent.

Volatile constituents of the leaves and root barks of Uvaria chamae were investigated in detail. Sesquiterpenes and aromatic compounds were the predominant constituents of the leaf and root bark oils, respectively. The major constituent of the leaf oil was found to be O-cadinene while thymoquinoldimethyl ether and benzyl benzoate were the major components of the root oil (Oguntimein et al 1989). The antimicrobial activities of a number of cytotoxic C-benzylated flavonoids from Uvaria chamae have been determined. The minimum inhibitory concentration values of these flavonoids and their derivatives against Staphylococcus aureus, Bacillus subtilis, and Mycobacterium smegmatis compare favorably with those of streptomycin sulfate, an inhibitor of protein synthesis (Hufford and Lasswel, 1978).

Although the exact mechanism of action of root bark compounds taken from the

Annonaceae family is not known, some insight has been provided (Bashengezi and

Constantin, 1997). Annonaceous acetogenins, natural products from these plants, were shown to be very potent inhibitors of the NADH-ubiquinone reductase (Complex 1) activity of mammalian mitochondria, and exhibit a high potential for interfering with the production of energy within mammalian cells. The acetogenins, Rolliniastatin-1 and

Rolliniastatin-2, were compared against classical inhibitors of Complex 1 and were shown to be more powerful in terms of both their inhibitory constant and the protein dependence of their titer in bovine submitochondrial particles. Although the above results only apply to Rolliniastatin-1 and Rolliniastatin-2, squamocin and otivarin also showed an inhibitory constant lower than that of the classical inhibitor. Another potential mechanism of action may be similar to that of annonaceous alkaloids. These compounds show a selective toxicity against DNA repair and recombination-deficient mutants of the yeast–Saccharomyces cerevisiae.

Studies on the efficacy of medicinal plants on resistant bacteria have been published (Nascimento et al., 2000; Salman et al., 2005; Abu-Shanab et al., 2006; Betoni et al., 2006; Pereira et al., 2006). However, there is no reference in literature reporting that the use of plant root, leaf and stem extracts or compounds generated from the plant

Uvaria chamae were tried for efficacy against multi-drug resistant bacteria.

CHAPTER TWO

MATERIALS AND METHODS

2.1 Collection of plant materials

Fresh leaves, stem bark and root bark of Uvaria chamae were collected in the forests of Odolu in Igalamela/Odolu LGA, Kogi State, Nigeria where they were found growing. The plant known locally as “Aloko” was identified by traditional users and authenticated by Mr. Ugwuozo, curator of the herbarium of University of Nigeria,

Nsukka. Voucher specimen was then deposited at the herbarium for future reference.

The plant materials were air-dried at room temperature until well dried and hence breaks easily. Leaves were examined and old, insect damaged, fungus-infested leaves and twigs were removed.

Plate 1: photograph of Uvaria chamae plant

2.2 Extraction

The extraction method used was a modification of Akinyemi et al (2005). In line with traditional methods of preparation, shredded plant materials were put in sterile bottles containing either distilled water or 80% ethanol.

Aqueous extract: Three hundred grams of ground plant materials were soaked in sterile amber coloured bottles containing 3 liters of distilled water and left to stand for 24 hours with intermittent stirring after every 12 hours. The suspension was then filtered first through muslin cloth to remove coarse particles and then through Whatman (No 1) filter paper. The plant residue was re-extracted with the same solvent for a further 24 hours and then filtered. The resulting filtrate was evaporated to dryness before a steady stream of cool air.

Ethanol extract: Three hundred grams of each of the powdered plant materials

was put in amber coloured bottles containing 3 liters of 80% ethanol. Further processes

were as carried out in the aqueous extraction above. The resulting filtrate was

evaporated to dryness to remove excess alcohol.

2.3 Microorganisms

The clinical specimens used were collected from the microbiology section of

Enugu State University Teaching Hospital, Parklane Enugu, and Medical Diagnostic

Laboratory of Microbiology Department University of Nigeria, Nsukka. The microorganisms include: six(6) isolates of Staphylococcus aureus including ATCC

12600, three(3) isolates of Bacillus subtilis including ATCC 6051, three(3) of Bacillus cereus including NRRL 14724, one(1) of Enterococcus sp, four(4) of Pseudomonas

27 aeruginosa including ATCC 10145, three(3) of Klebsiella, six(6) of Escherichia coli including ATCC 11775, two(2) of Proteus mirabilis, two(2) of Enterobacter, six(6) of

Salmonella including Salmonella kintambo SSRL113 and two(2) of Shigella dysenteriae .

These organisms were obtained and identified by standard microbiological methods. The organisms were maintained on agar slants at 4oC and sub-cultured for 24 hours before use.

2.4 Test for sensitivity or resistance to standard antibiotics

The bacteria were tested for their sensitivity or resistance to a number of antibiotics. The concentrations of antimicrobial sensitivity testing discs used and interpretation of sizes of zones of inhibition (determination of resistance) was in accordance with performance standards for antimicrobial disc susceptibility tests (CLSI,

2006). The antibiotics tested and their concentrations were ampiclox (10 g/disc), amoxicillin (20 g/disc), cephalexin (30 g/disc), chloramphemicol (30 g/disc), ciprofloxacin (5 g/disc), erythromycin (15 g/disc), gentamicin (10 g/disc), nalidixic acid (30 g/disc), tarivid (5 g/disc), sparfloxacin (5 g/disc), tetracycline (30 g/disc), zinnacef (20 μg/disc), streptomycin (30 µg/disc), septrin (30 μg/disc), augmentin (30

μg/disc), pefloxacin (30 μg/disc), clindamycin (10 μg/disc), floxapen (30 μg/disc), nitrofurantoin (100 μg/disc) and rocephin (25 μg/disc).

Multidrug resistance was taken as resistance to at least 4 first line antibiotics

(Salman et al., 2005). These resistant organisms were stocked and used for the rest of the research.

2.5 Determination of antimicrobial activity of crude extracts

The agar well diffusion method was used (Ogbonnia et al., 2008). The dried extracts were reconstituted with sterile distilled water to obtain a stock solution of 250 mg/ml from which various concentrations of 250, 125, 100, 62.5, 31.25, 15.625 mg/ml were prepared.

Overnight broth culture of the respective bacteria strains having turbidity adjusted to 0.5 Macfarland standards were freshly prepared and 0.2 ml of each was aseptically transferred onto the surface of a sterile Muellar Hinton agar plate and distributed evenly with the aid of a bent sterile glass rod. Six wells (6 mm in diameter) were made equidistance in each of the plates using a sterile cork borer. A small aliquot (0.1 ml) of each concentration of the extracts was respectively introduced into a well using sterile automatic pipettes, with the stock solution in the centre well. The plates were allowed to stand for one hour for prediffusion of the extracts to occur (Esimone et al., 1998). The plates were incubated at 37oC for 24 hours. The solvent control (distilled water and 20%

DMSO for the aqueous and ethanolic extracts respectively) and the control antibiotic discs ciprofloxacin (5 μg/ml) for both gram-positive and gram-negative bacteria were used.

Following incubation, diameters of the zones of inhibition were measured. The antibacterial activity was expressed as the mean zone of inhibition diameters (mm) produced by the plant extracts. All experiments were repeated in triplicates. Overall, cultured bacteria with halos equal to or greater than 7 mm were considered susceptible to tested extract (Nascimento et al., 2000).

2.6 Determination of minimum inhibitory concentration (MIC)

The MIC of the extracts was determined by diluting the various concentrations

(7.81, 15.625, 31.25, 37.03, 40, 58.8, 62.5, 83.33, 100, 111.11, 125, 166.67, 200, and 250 mg/ml) of root bark, stem bark, and leaves respectively. Equal volume of the extracts and nutrient broth were mixed in the test tube. Specifically 0.1 ml of standardized inoculums of 1-2 X 107cfu/ml were added to each tube. The tubes were incubated aerobically at

37oC for 18-24 hours. Two control tubes were maintained for each test batch as follows: tubes containing extracts and the growth medium without inoculums (antibiotic control) and the tubes containing the growth medium, physiological saline and the inoculums

(organism control). MIC was determined as the lowest concentration of the extracts permitting no visible growth (no turbidity) when compared with the control tubes.

2.7 Determination of minimum bactericidal concentration (MBC)

The MBC was determined by sub-culturing the test dilution on fresh solid medium and further incubating at 37oC for 18-24 hours. The lowest concentration of MIC tubes with no visible bacterial growth on solid medium was regarded as MBC.

2.8 Solvent-solvent fractionation

The high activity antibacterial extract (alcoholic extracts) were partitioned successively with n-hexane, chloroform, ethyl acetate, acetone and methanol to obtain their corresponding fractions: n-hexane soluble fraction (A), chloroform soluble fraction

(B), ethyl acetate soluble fraction (C), acetone soluble fraction (D), and methanol soluble fraction (E). Forty grams of the dried crude alcoholic extract was initially extracted with

100 ml of n-hexane. The solvent was allowed to extract for 1 hour with shaking before being decanted. The same quantity of solvent was added to the marc and shaken for an hour again. The process repeated three times. The marc was allowed to dry and the process of extraction was repeated with chloroform, ethyl acetate, acetone and methanol.

The extracts were filtered through Whatman (No. 1) filter paper and solvents removed by air-drying before a cool stream of air. For quantitative determination, extractants were placed in pre-weighed flasks and masses recovered with each solvent determined.

Separation and isolation of the bioactive compounds was performed as shown.

80% alcoholic extracts

Marc partitioned with n-hexane

Marc partitioned with chloroform N-hexane soluble fraction (A)

Marc partitioned with ethyl acetate Chloroform soluble fraction (B)

Marc partitioned with acetone Ethyl acetate soluble fraction (C)

Marc partitioned with methanol Acetone soluble fraction (D)

Methanol soluble fraction (D)

Diagrammatic summary of fractionation routes

2.9 Antibacterial assay of extract fractions

The different fractions recovered were re-dissolved at different concentrations

(0.625, 1.25, 2.50, 5.0, and 10 mg/ml) in 20% DMSO for determination of their antimicrobial profile. The n-hexane, chloroform and ethyl acetate fractions were insoluble in 20% DMSO and hence, 60% DMSO was used. The assay was conducted as previously described for the crude extracts.

Plates containing 20 and 60% DMSO were prepared to serve as negative control depending on the fraction. The plates were incubated at 37oC for 18 hours and diameters of zones of inhibition measured. Cultured bacteria with halos equal to or greater than 7 mm were considered susceptible to tested fraction. Where bacteria were seen to have been inhibited by DMSO, zone of inhibition was total inhibition diameter minus diameter due to negative control.

2.10 Phytochemical evaluation of the crude extracts and fractions

Phytochemical screening was carried out based on procedures outlined by

Harbone (1984) and Evans (1989).

2.10.1 Test for Saponins

Twenty milliliters of distilled water was added to 0.25 g of extract and boiled in a hot water bath for 2 minutes. The mixture was filtered while hot and allowed to cool and the filtrate used for the following tests.

(a) Frothing test: - 5 ml of the filtrate was diluted with 15 ml of distilled water and

shaken vigorously. A stable froth (foam) upon standing indicated the presence of

saponins.

(b) Emulsion test: - To the frothing solution was added 2 drops of olive oil and the

contents shaken vigorously. The formation of emulsion indicated the presence of

saponins.

parts of A and B) and the contents were heated in a water bath. A reddish

precipitate which turned brick red on further heating with sulphuric acid indicated

the presence of saponins.

2.10.2 Test for Tannins

A quantity (1 g) of the extracts were boiled with 20 ml of water, filtered and used for following tests.

(a) Ferric chloride Test: - To 3 ml of the filtrate, few drops of ferric chloride were added.

A transient greenish to black precipitate indicated the presence of tannins.

(b) Lead acetate Test: - To 1 ml of the filtrate was added 3 drops of lead acetate solution.

The presence of tannins was indicated by reddish colour.

2.10.3 Test for alkaloids

A quantity (0.2 g) of extract was boiled with 5 ml of 2% hydrochloric acid in a water bath for 3 minutes. The mixture was filtered and 0.1 ml of each filtrate treated with

2 drops 1% picric acid. A yellow precipitate indicated the presence of alkaloids.

2.10.4 Test for Flavonoids

Ten milliliters of ethyl acetate was added to 0.2 g of the extract and heated in a water bath for 3 minutes. The mixture was cooled, filtered and the filtrate was used for the following tests.

(a) Ammonium Test: - 4 ml of filtrate was shaken with 1 ml of dilute ammonia

solution. The layers were allowed to separate. A yellow colour in the ammoniacal

layer was indicative of the presence of flavonoids.

(b) 1% Aluminium chloride solution test: - Another 4 ml portion of the filtrate was

shaken with 1 ml of 1% aluminium chloride solution. The layers were allowed to

separate. A yellow colour in the aluminium chloride layer indicated the presence

of flavonoids.

2.10.5 Test for fats and oils

A small amount (0.1 g) of extract was pressed between filter paper and the paper observed. A control was also prepared by placing 2 drops of olive oil filter paper.

Translucency of the filter paper indicated the presence of fats and oils.

2.10.6 Test for reducing sugar

Five milliliters of a mixture of equal parts of Fehling‟s solution I and II were added to 5 ml of extracts and heated in a water bath for 5 minutes. A brick red precipitate indicated the presence of reducing sugar.

2.10.7 Tests for glycosides

Five milliliters of dilute sulphuric acid was added to 0.1 g of the extract in a test tube and boiled for 15 minutes in a water bath, then cooled and neutralized with 20% potassium hydroxide solution. 10 ml of a mixture of equal parts of Fehling‟s solution I and II was added and boiled for 5 minutes. A more dense brick red precipitate indicated the presence of glycosides.

2.11 Acute toxicity studies (LD50)

Young adult albino rats of both sexes weighing 180 – 220 g were used. They were kept in clean cages and maintained under laboratory conditions of temperature, humidity and light and were allowed free access to food (standard laboratory diet) and water. All animals were kept for about a week before the commencement of experiment for proper acclimatization.

LD50 determination was conducted using modified method of Lorke (1983) and

Salawu et al., (2008). The evaluation was done in two phases. In the first phase, three groups, of three rats each were treated with 10, 100, and 1000 mg extract/kg body weight orally. The rats were observed for clinical signs and symptoms of toxicity within 24 hours and death within 72 hours. Another two groups of three rats were administered with normal saline and 20% DMSO to serve as control and observed also for clinical signs.

Based on the results of phase one, (no sign of toxicity), another set of three fresh rats were each treated with 1600, 2900 and 5000 mg extract/kg body weight orally in the

36 second phase. Clinical signs and symptoms of toxic effects and mortality were then observed for seven days. The weights were also recorded.

2.12 Statistical analysis

Results were expressed as the mean ± standard error of mean (S.E.M). Statistical analysis of data was carried out using two-way analysis of variance (ANOVA).

Differences in means were considered to be significant when P<0.05 using Fisher‟s Least

Significance Difference (F-LSD).

CHAPTER THREE

RESULTS

3.1 Antimicrobial susceptibility profile of test bacteria

Of the 38 organisms (25 Gram negatives and 13 Gram positives) used in the study, 8 (21.05%) were resistant to 4 to 6 antibiotics, 17 (44.74%) were resistant to 7 to 9 antibiotics, 7 (18.42%) resisted 10 to 12 antibiotics while 6 (15.79%) resisted all 13 antibiotics (Tables 2 and 3) and among the Gram negatives, the highest degree of resistance was observed among Klebsiella (82%), Pseudomonas spp (81%), E. coli

(74.83%), before Enterobacter (61.5%), Salmonella (53.83%), Proteus mirabilis

(46.5%) and Shigella dysenteriae (42.5%) (Table 2). Among the Gram positives on the other hand, resistance was in the order Bacillus spp > S. aureus > Enterococcus sp (Table

3).

In the Gram negative bacteria, the antibiotics to which there was most resistance were augmentin and septrin (all were resistant) followed by nalidixic acid (24 isolates resistant), amoxicillin and nitrofurantoin (21 isolates resistant). The least resisted antibiotics were ciprofloxacin (5 isolates resistant), pefloxacin and gentamycin (7 isolates resistant to each). For the Gram positives, all the isolates were resistant to ampiclox, erythromycin, zinnacef, septrin and amoxicillin while only 4 isolates (30.77%) resisted ciprofloxacin and 5 (38.46%) resisted gentamycin (Table 3).

TABLE 2: RESISTANCE PATTERN OF GRAM NEGATIVE ORGANISMS TO

CONVENTIONAL ANTIBIOTICS

A N T I B I O T I C S TOTAL S/N ORGANISMS AU CN PEF OFX S SXT TE CH SP NA CPX AM N Resistanc e (%) 1 Salmonella 1 + - + + - + - + + + - + + 69.23 2 Salmonella 2 + - - + - + - + +/- + +/- + + 69.23 3 Salmonella 3 + - - - - + - + - +/- - + + 46.15 4 Salmonella 4 + - - + - + + + - +/- - + + 61.54 5 Salmonella 5 + - - - - + + + - + - - + 46.15 6 Salmonella 6 + - - - - + - - - + - + - 30.76 7 P. aeruginosa1 + + + + + + + + + + + + + 100 8 P. aeruginosa 2 + + + + + + + + + + + + + 100 9 P. aeruginosa 3 + - - - + + + + + + - - + 61.54 10 P. aeruginosa 4 + - - +/- + + + + - - - + + 61.54 11 E. coli 1 + + + + - + +/- + + + - - + 76.92 12 E. coli 2 + - - +/- + + - - - +/- - + + 53.85 13 E. coli 3 + + + + + + + + + + + + + 100 14 E. coli 4 + - - - + + + - + + - + + 61.54 15 E. coli 5 + - - +/- + + + +/- + + - + +/- 76.92 16 E. coli 6 + + - - + + + + + + - + + 76.92 17 Enterobacter 1 + - - + - + +/- +/- +/- +/- - + + 69.23 18 Enterobacter 2 + - - - - + +/- - + + - + + 53.85 19 Klebsiella 1 + + + + + + + + + + + + + 100 20 Klebsiella 2 + + + - + + + +/- - + - + +/- 76.92 21 Klebsiella 3 + - + - +/- + + + +/- +/- - + - 69.23 22 P. mirabilis 1 + - - - - + + + - + - - - 38.46 23 P. mirabilis 2 + - - - - + + + +/- +/- - + - 53.85 24 S. dysenteriae1 + - - - - + - - - + - + + 38.46 25 S. dysenteriae2 + - - - - + + - - + - + + 46.15 Total Resistance (%) 100 28 32 48 48 100 76 76 60 96 20 84 84

Key: +: resistant; -; susceptible; +/-: intermediate

AU: augmentin; CN: gentamycin; PEF: pefloxacin; OFX: ofloxacin; S: streptomycin; SXT: septrin; TE: tetracycline; CH: chloramphenicol; SP: sparfloxacin; NA: nalidixic acid; CPX: ciprofloxacin; AM: amoxicillin; N: nitrofurantoin.

TABLE 3.2: RESISTANCE PATTERN OF GRAM POSITIVE ORGANISM

C O N V E N T I O N A L D R U G S Total resistan S/ ORGANISM APX E FX Z CX R CN PEF S SXT CPX AM CD ce (%) N S 1 S. aureus 1 + + - + - - + - - + - + + 54.85 2 S. aureus 2 + + + + + + + + + + + + + 100 3 S. aureus 3 + + + + - - - - - + +/- + + 61.54 4 S. aureus 4 + + - + - - - - - + - + + 46.15 5 S. aureus 5 + + + + + +/- - - - + +/- + - 69.23 6 S. aureus 6 + +/- + + - - - - - + - + + 54.85 7 B. subtilis 1 + + + + + + + + +/- + +/- + + 100 8 B. subtilis 2 + + - + - + + - +/- + - + - 61.54 9 B. subtilis 3 + + + + +/- - - + - + - + - 61.54 10 B cereus 1 + + + + + - - - - + - + + 61.54 11 B. cereus 2 + + - + + + - + + + - + + 76.92 12 B. cereus 3 +/- + - + + + + + + + - + + 84.62 13 Enterococcu + + + + + + - + + + - + + 84.62 s sp Total 100 100 61.54 100 61.5 53.9 38.46 46.2 46.15 100 30.8 100 77

resistance (%)

Key: +: Resistant; -: susceptible; +/-: intermediate

APX: ampiclox; E: erythromycin; FX: floxapen; Z: zinnacef; CX: cephalexin; R: rocephin; CN: gentamycin; PEF: pefloxacin; S: streptomycin; SXT: septrin; CPX: ciprofloxacin; AM: amoxicillin; CD: clindamycin.

3.2 AQUEOUS AND ETHANOLIC EXTRACTIONS

From 300 g of dried plant parts extracted, 25.98 g (8.66%), 21.39 g (7.31%) and 29.52 g

(9.84%) for the root bark, stem bark and leaves, respectively, were recovered following aqueous extraction. The yields for ethanolic extraction were 34.05 g (11.35%), 29.01 g

(9.67%) and 39.33 g (13.11%) for the root bark, stem bark and leaves respectively (fig. 1)

Root bak Stem bark Leaves

6 %RECOVERY 4

0 Ethanolic extracts Aqueous extracts

S O L V E N T S

FIG. 1 YIELDS OF ETHANOLIC AND AQUEOUS EXTRACTION OF PLANT PARTS

3.3 ANTIMICROBIAL ACTIVITIES OF CRUDE EXTRACTS

Figure 2 shows the results of the antimicrobial screening of different concentrations of extracts. Generally, average zone of inhibition varied directly with increase in extract concentration. All, tested strains of S. aureus, B. cereus, and

Enterococcus sp were inhibited from 62.5 mg/ml concentration, with a mean inhibition zone diameter (IZD) of 13.4, 14.5, 15 mm, respectively. Pseudomonas aeruginosa, S. aureus, B. cereus and B. subtilis were inhibited at concentrations as low as 31.25 mg/ml.

The most resistant organism was Klebsiella where complete inhibition only occurred at

125 mg/ml concentration. Differences in inhibition were significant at concentrations

62.5 mg/ml and below, especially against S. aureus, Salmonella, Proteus, P. aeruginosa,

Enterococcus sp and B. cereus. Only against P. aeruginosa and Enterococcus sp was there a significant (P<0.05) inhibitory difference at concentrations 125 and 250 mg/ml.

The inhibition of Enterococcus sp, P. aeruginosa and S. aureus at the 250 mg/ml concentration were significantly (P<0.05) higher than the antibiotic (ciprofloxacin) control.

The organisms – S. aureus, P. aeruginosa, E. coli, Klebsiella, and Enterococcus sp apparently inhibited by the antibiotic (ciprofloxacin) control looking at the inhibitionzone diameters (Figs. 2 – 7) are actually resistant according to the performance standards to the antimicrobial disk susceptibility tests (CLSI, 2006).

43 30 25 20 15 10 5

Averagezone (mm) inhibition of 0

E. coli Proteus Shigella Klebsiella S. aureus B. cereus B. subtilis Salmonella P. aeruginosa Enterobacter Organisms Enterococcus sp

15.625 mg/ml 31.25 mg/ml 62.5 mg/ml 100 mg/ml 125 mg/ml 250 mg/ml cpx control

FIG 2 ACTIVITY OF CRUDE AQUEOUS ROOT BARK EXTRACT ON TEST BACTERIA

Figure 3 shows the result of the activity of crude alcoholic root bark extract. All tested strains S. aureus, B. cereus, E. coli, P. aeruginosa, Proteus and Shigella were inhibited at 62.5 mg/ml while all strains of Salmonella, B. subtilis, Klebsiella,

Enterobacter and Enterococcus sp were inhibited at 100 mg/ml and above. One strain of

S. aureus; Salmonella and 66.7% of P. aeruginosa were inhibited at 31.25 mg/ml. High activities (inhibition diameter ≥20 mm) were recorded on E. coli, Proteus, P. aeruginosa,

Shigella, B. cereus, S. aureus and Enterococcus sp at concentration 250 mg/ml. The lowest activities were against Klebsiella and B. subtilis.

At 250 mg/ml concentration, there was no significant (P>0.05) difference in the inhibition of B. cereus, Enterococcus sp, S. aureus and P. mirabilis. No significant differences were recorded between inhibition at concentrations 125 and 250mg/ml for B. cereus, Enterococcus sp, Proteus mirabilis, S. aureus and Shigella. Only against

Enterobacter, P. mirabilis and Shigella were there significant differences (P<0.05) at 100 and 125 mg/ml concentrations. The inhibition of Enterococcus sp, P. aeruginosa and S. aureus at the 250 mg/ml concentration were also significantly (P<0.05) higher than the antibiotic (ciprofloxacin) control.

45 30

5 AVERAGE ZONE OF INHIBITION (MM)AVERAGE 0

E. coli -5 Proteus Shigella Klebsiella S. aureus B. cereus Salmonella B. subtilis P. aeruginosa Enterobacter Enterococcus sp ORGANISMS

15.625 mg/ml 31.25 mg/ml 62.5 mg/ml 100 mg/ml 125 mg/ml 250 mg/ml cpx control

FIG 3 ACTIVITY OF CRUDE ALCOHOLIC ROOT BARK EXTRACT ON TEST BACTERIA

For aqueous stem bark extract, Proteus and 40% of tested strains of Salmonella were inhibited at 31.25 mg/ml concentration. There was 100% inhibition of S. aureus, P. aeruginosa, Klebsiella, Enterobacter, and B. subtilis at 62.5 mg/ml while E. coli,

Shigella, Enterococcus sp, B. cereus were inhibited only at 100 mg/ml concentration. The inhibition of P. aeruginosa, Klebsiella, Enterococcus sp and S. aureus at the 250 mg/ml concentration were significantly (P<0.05) higher than the antibiotic (ciprofloxacin) control (Fig. 4).

For the alcoholic stem bark extract, the highest activity was against Proteus with inhibition significantly (P<0.05) higher than other test organisms. All Proteus isolates and 60% of S. aureus strains were inhibited at 15.625 mg/ml while B. subtilis was inhibited at 31.25 mg/ml (Fig. 5).

Generally, no significant (P>0.05) differences were found between the inhibition of P. aeruginosa, B. cereus, Enterococcus sp, E. coli and Salmonella. At lower concentrations (15.625, 31.25, 62.5 mg/mg) where inhibition occurred, significant

(P<0.05) concentration effects were recorded except against P. mirabilis, B. cereus and

E. coli.

47 30

5 AVERAGE ZONE OF INHIBITION (MM) AVERAGEINHIBITION OF ZONE 0

E. coli Proteus Shigella Klebsiella S. aureus B. cereus B. subtilis Salmonella P. aeruginosa Enterobacter Enterococcus sp ORGANISMS

15.625 mg/ml 31.25 mg/ml 62.5 mg/ml 100 mg/ml 125 mg/ml 250 mg/ml cpx control FIG. 4 ACTIVITY OF CRUDE AQUEOUS STEM BARK EXTRACT ON TEST BACTERIA

48 30

AVERAGE ZONE AVERAGE OF INHIBITION (MM) 5

E. coli Proteus Shigella Klebsiella S. aureus B. cereus B. subtilis Salmonella P. aeruginosa Enterobacter Enterococcus sp ORGANISMS

15.625 mg/ml 31.25 mg/ml 62.5 mg/ml 100 mg/ml 125 mg/ml 250 mg/ml cpx control

FIG. 5 ACTIVITY OF CRUDE ALCOHOLIC STEM BARK EXTRACT ON TEST BACTERIA 49

Figure 6 shows the results obtained for the aqueous leaf extract. All tested strains of S. aureus, E. coli, Salmonella, and Enterobacter were inhibited at concentration of 100 mg/ml and above. P. aeruginosa, and Proteus were inhibited from 62.5 mg/ml, B. cereus and B. subtilis from 31.25 mg/ml while Klebsiella and Shigella were only inhibited at 250 mg/ml. Zone of inhibition against P. aeruginosa at 100 mg/ml was significantly (P<0.05) higher than recorded zones for other organisms at equivalent and higher concentrations.

Differences between P. mirabilis and S. aureus, Enterobacter and E. coli were significant

(P<0.05) at 125 mg/ml concentration. The inhibition of S. aureus and E. coli at the 250 mg/ml concentration were equivalent to that of the antibiotic (ciprofloxacin) control while the inhibition of P. aeruginosa was significantly (P<0.05) higher.

5 AVERAGE ZONE AVERAGE OF INHIBITION (MM)

E coli Proteus Shigella Klebsiella S aureus B cereus B subtilis Salmonella P aeruginosa Enterobacter Enterococcus sp ORGANISMS

15.625 mg/ml 31.25 mg/ml 62.5 mg/ml 100 mg/ml 125 mg/ml 250 mg/ml cpx control

FIG 6 ACTIVITY OF CRUDE AQUEOUS LEAF EXTRACT ON TEST BACTERIA 51

For the alcoholic leaf extract, generally, the highest activity was at 250 mg/ml concentration, but there were no significant (P>0.05) differences at this concentration between Enterococcus sp, P. aeruginosa, Proteus mirabilis, Enterobacter, E. coli and

Shigella (Fig. 7). At lower concentrations, significant (P<0.05) differences were recorded between B. subtilis and B. cereus, Shigella and Salmonella and between P. mirabilis and

P. aeruginosa. Significant concentration effects were recorded especially at concentrations 31.25 and 62.5 mg/ml against B. cereus, E. coli, Enterobacter, P. aeruginosa, Salmonella and Shigella.

There was 100% inhibition of S. aureus, E. coli, Salmonella, P. aeruginosa, B. cereus, Enterobacter, and Enterococcus sp at 100 mg/ml. Shigella and Proteus were inhibited at 62.5 mg/ml, Klebsiella at 125 mg/ml and B. subtilis at 31.25 mg/ml. Two strains of Salmonella and a strain of B. cereus were inhibited at 31.25 mg/ml while 80%

S. aureus and E. coli, 67% of P. aeruginosa and a strain of Enterobacter were inhibited at

62.5 mg/ml concentration.

5 AVERAGE(MM) INHIBITION ZONEOF 0

E. coli Proteus Shigella Klebsiella S. aureus B. cereus B. subtilis Salmonella P. aeruginosa Enterobacter ORGANISMS Enterococcus sp 15.625 mg/ml 31.25 mg/ml 62.5 mg/ml 100 mg/ml 125 mg/ml 250 mg/ml cpx control FIG. 7 ACTIVITY OF CRUDE ALCOHOLIC LEAF EXTRACT ON TEST BACTERIA 53

3.4 MINIMUM INHIBITORY AND MINIMUM BACTERICIDAL

CONCENTRATIONS OF EXTRACTS

The minimum inhibitory concentrations (MIC) of the extracts ranged from 7.81 mg/ml to

200 mg/ml. Generally, the alcoholic extracts had lower MIC than the aqueous extracts

(Table 4). The alcoholic stem bark extract had the best activity (lowest MIC values) with

MIC ranging from 7.81 mg/ml (for Proteus) to 100 mg/ml (for Klebsiella). The least activity (highest MIC) was recorded for the aqueous leaf extract with MIC ranging from

40 mg/ml to 200 mg/ml.

From the results Proteus mirabilis was the most sensitive organism to the extracts

(MIC 7.81 mg/ml to 62.5 mg/ml) while Klebsiella was the least sensitive (MIC 58.8 to

200 mg/ml)

MBC values ranged from 37.03 to 250 mg/ml with the lowest and highest values for Proteus and Klebsiella, respectively (Table 5).

TABLE 4: MINIMUM INHIBITORY CONCENTRATION (mg/ml) OF PLANT

EXTRACTS

M I C V A L U E S

ROOT BARK STEM BARK LEAF

Organisms Aqueous Alcoholic Aqueous Alcoholic Aqueous Alcoholic S. aureus 83.33 40 83.33 58.8 100 62.5 E. coli 83.33 62.5 83.33 83.33 100 100 P. aeruginosa 62.5 40 83.33 83.33 100 100 B. cereus 15.625 40 83.33 40 100 83.33 Salmonella 62.5 100 62.5 58.8 83.33 58.8 Klebsiella 100 100 58.8 100 200 166.67 Enterobacter 83.33 83.33 62.5 58.8 83.33 62.5

Proteus mirabilis 40 40 31.23 7.81 62.5 58.8

Shigella dysenteriae 100 58.8 83.33 58.8 200 62.5 Bacillus subtilis 62.5 83.33 58.8 31.25 40 31.25

Enterococcus sp 62.5 83.33 83.33 40 100 83.33

TABLE 5: MINIMUM BACTERICIDAL CONCENTRATION (mg/ml) OF

PLANT EXTRACTS

M B C V A L U E S

ROOT BARK STEM BARK LEAF

Organisms Aqueous Alcoholic Aqueous Alcoholic Aqueous Alcoholic S. aureus 100 62.5 100 62.5 125 83.33 E. coli 111.11 100 100 100 125 111.11

P. aeruginosa 100 62.5 111.11 100 125 125 B. cereus 58.8 62.5 111.11 62.5 125 100 Salmonella 100 125 100 100 111.11 111.11 Klebsiella 200 125 125 200 250 200 Enterobacter 111.11 111.11 100 100 125 100

Proteus mirabilis 62.5 62.5 58.8 37.03 100 100

Shigella dysenteriae 125 100 111.11 83.33 250 100 Bacillus subtilis 100 100 83.33 58.8 62.5 58.8

Enterococcus sp 100 100 111.11 62.5 125 100

3.5 YIELD FROM SOLVENT – SOLVENT FRACTIONATION

Among the solvents, n –hexane had the least yield -3.025% (1.21 g), 2.3% (0.92 g), and 1.65% (0.66 g) –fractions for the root bark, stem bark and leaf extracts respectively. Acetone was the best extractor; recovering 60.7% (24.28 g), 76.225%

(30.49 g), and 66.775% (26.71 g) of crude extracts of root bark, stem bark and leaves respectively. Other solvents –chloroform, ethyl acetate and methanol –were used and components of various masses recovered as shown (Fig. 8). Certain components were insoluble in any of the five solvents used and hence were classified as “mass lost”. These include 7.15%, 4.15% and 7.85% of the crude extracts of root bark, stem bark and leaves respectively.

57 57

90 Root bark Stem bark Leaves

80 70

60 50

40 30

Fraction recovery (%) recovery Fraction 20 10

n-hexane acetone methanol chloroform mass lost ethyl acetate Different Solvent Fractions

Fig. 8 Percentage yield of Crude Extract Fractions using Solvent - Solvent Fractionation

58 58

3.6 ANTIMICROBIAL ACTIVITY OF EXTRACT FRACTIONS

Figure 9 shows the results obtained for the root bark acetone fraction. S. aureus,

P. aeruginosa, Klebsiella, Proteus mirabilis were all inhibited by fraction at the lowest concentration level of 0.625 mg/ml. The highest activity was against S. aureus and

Klebsiella and significant (P<0.05) difference was recorded between the inhibition of these organisms and the others. Also well inhibited were P. aeruginosa and Proteus mirabilis whose overall inhibition were significantly (P<0.05) higher than the other test strains except S. aureus and Klebsiella. Against Klebsiella, the difference in inhibition between the lowest (0.625 mg/ml) and the highest (10 mg/ml) concentration was not significant indicating a high average activity. No activity was recorded against

Salmonella, Enterobacter, Shigella dysenteriae, and B. subtilis.

The organisms – S. aureus, P. aeruginosa, E. coli, Klebsiella, and Enterococcus sp apparently inhibited by the antibiotic (ciprofloxacin) control looking at the inhibition zone diameters (Figs. 9 – 21) are actually resistant according to the performance standards for the antimicrobial disk susceptibility tests (CLSI, 2006).

59 30

AVERAGE ZONE OF INHIBITION (MM) AVERAGEINHIBITION OF ZONE 0

E. coli S. aureus B. cereus Klebsiella Salmonella P. aeruginosa Enterobacter Bacillus subtilis Proteus mirabilis Enterococcus sp TEST ORGANISMS Shigella dysenteriae

0.625 mg/ml 1.25 mg/ml 2.5 mg/ml 5 mg/ml 10 mg/ml cpx control FIG. 9 ACTIVITY OF ROOT BARK ACETONE FRACTION ON TEST BACTERIA 60

Figure 10 shows the results of the antibacterial screening of different concentrations of the stem bark acetone fraction. This fraction had the widest spectrum of activity inhibiting all test strains. P. aeruginosa, Klebsiella, and Proteus mirabilis were inhibited at the lowest concentration (0.625 mg/ml). Overall, highest activity was against

P. aeruginosa and Proteus mirabilis but significant (P<0.05) difference was recorded between these organisms and other test strains. There was no significant (P<0.05) difference between the inhibition of Klebsiella and S. aureus. B. cereus, Enterobacter and

S. dysenteriae were only inhibited at 10 mg/ml concentration while Salmonella, B. subtilis and Enterococcus sp were inhibited at 5 mg/ml.

61 30 25 20 15 10 5

0 AVERAGE ZONE OF INHIBITION (mm) AVERAGEINHIBITION OF ZONE

E. coli S. aureus B. cereus Klebsiella Salmonella P. aeruginosa Enterobacter Bacillus subtilis Proteus mirabilis Enterococcus sp Shigella dysenteriae TEST ORGANISMS

0.625 mg/ml 1.25 mg/ml 2.5 mg/ml 5 mg/ml 10 mg/ml cpx control FIG. 10 ACTIVITY OF STEM BARK ACETONE FRACTION ON TEST BACTERIA 62

For the leaf acetone fraction, highest activity was against Enterobacter which overall, was significantly (P<0.05) higher than other test strains. There was no inhibition of E. coli, B. cereus, Salmonella, Shigella dysenteriae, B. subtilis and Enterococcus sp and fraction also had low activity against P. aeruginosa, Klebsiella and Proteus mirabilis

(Fig. 12). Leaf acetone fraction had overall significantly (P>0.05) lower activity against most test strains in comparison with the acetone soluble fractions of the root bark and stem bark extracts.

30 25 20 15 10 5

0 AVERAGE ZONE OF INHIBITION (mm) AVERAGEINHIBITION OF ZONE E. coli S. aureus B cereus Klebsiella Salmonella P. aeruginosa Enterobacter Bacillus subtilis Proteus mirabilis Enterococcus sp Shigella dysenteriae

TEST ORGANISMS 0.625 mg/ml 1.25 mg/ml 2.5 mg/ml 5 mg/ml 10 mg/ml cpx control

FIG. 11 ACTIVITY OF LEAF ACETONE FRACTION ON TEST BACTERIA 64

Figure 12 shows the activity of root bark ethyl acetate fraction. Fraction profoundly inhibited S. aureus, P. aeruginosa and Proteus mirabilis and this overall, was significantly (P<0.05) higher than the average inhibition of other organisms. Inhibited to a lesser extent were E. coli, Salmonella, B. subtilis and Enterococcus sp. B. cereus,

Enterobacter, and S. dysenteriae however were not inhibited. At concentrations 5 and 10 mg/ml, differences in inhibition of Klebsiella, Proteus, Enterococcus sp, E. coli, P. aeruginosa, S. aureus and Salmonella were not significant (P>0.05). However, there was significant (P<0.05) concentration effects overall.

65 35 30 25 20 15 10

AVERAGE ZONE OF INHIBITION (MM)AVERAGE 5 0

E. coli S. aureus B. cereus Klebsiella Salmonella P. aeruginosa Enterobacter Bacillus subtilis Proteus mirabilis Enterococcus sp TEST ORGANISMS Shigella dysenteriae 0.625 mg/ml 1.25 mg/ml 2.5 mg/ml 5 mg/ml 10 mg/ml cpx control FIG. 12 ACTIVITY OF ROOT BARK ETHYL ACETATE FRACTION ON TEST BACTERIA 66

For the stem bark ethyl acetate fraction, highest activity was against Klebsiella where inhibition at 2.5 mg/ml was significantly (P<0.05) higher than for other organisms at equivalent and higher concentrations. Fraction also had a high activity against P. aeruginosa, Proteus mirabilis and S. aureus. No inhibition however, was recorded against B. cereus, Salmonella, Enterobacter, Shigella dysenteriae, B. subtilis and

Enterococcus sp (Fig. 13).

67 30

AVERAGE ZONE OF INHIBITION (MM) AVERAGEINHIBITION OF ZONE 5

E. coli S. aureus B. cereus Klebsiella Salmonella P. aeruginosa Enterobacter Proteus mirabilis Bacillus subtilisEnterococcus sp Shigella dysenteriae TEST ORGANISMS 0.625 mg/ml 1.25 mg/ml 2.5 mg/ml 5 mg/ml 10 mg/ml cpx control FIG. 13 ACTIVITY OF STEM BARK ETHYL ACETATE FRACTION ON TEST BACTERIA 68

Figure 14 shows the activity of leaf ethyl acetate fraction. Fraction was active albeit moderately, against P. aeruginosa, Klebsiella and E. coli. Overall, there was no significant (P>0.05) difference in the inhibition of Klebsiella and P. aeruginosa. Where activity was recorded there was generally significant (P<0.05) concentration effects.

Enterococcus sp, B. subtilis, B. cereus, Shigella and Salmonella were however not inhibited.

69 30 25 20 15 10 5

AVERAGE ZONE OF INHIBITION (MM) AVERAGEINHIBITION OF ZONE 0

E. coli S. aureus B. cereus Klebsiella Salmonella P. aeruginosa Enterobacter Bacillus subtilis Proteus mirabilis Enterococcus sp TEST ORGANISMS Shigella dysenteriae 0.625 mg/ml 1.25 mg/ml 2.5 mg/ml 5 mg/ml 10 mg/ml cpx control FIG 14 ACTIVITY OF LEAF ETHYL ACETATE FRACTION ON TEST BACTERIA 70

Root bark methanol fraction was most active against B. subtilis where inhibition was significantly (P<0.05) higher than against other test strains and no other organism except Proteus mirabilis was inhibited at any concentration below 5 mg/ml. Also well inhibited were S. aureus, P. aeruginosa and Klebsiella (Fig. 15).

30 25 20 15 10 5

0 AVERAGE(MM) INHIBITION ZONEOF

E. coli S. aureus B. cereus Klebsiella B. subtilis Salmonella Enterobacter P. aeruginosa Proteus mirabilis Enterococcus sp Shigella dysenteriae TEST ORGANISMS 0.625 mg/ml 1.25 mg/ml 2.5 mg/ml 5 mg/ml 10 mg/ml cpx control

FIG. 15 ACTIVITY OF ROOT BARK METHANOL FRACTION ON TEST BACTERIA 72

For stem bark methanol fraction, highest activity was against S. aureus, P. aeruginosa and Klebsiella and inhibition of these organisms was significantly (P<0.05) higher than inhibition of other test strains. This fraction had a broad spectrum of activity being active against most test organisms except S. dysenteriae (Fig. 16).

30 25 20 15 10 5

AVERAGE ZONE OF INHIBITION (MM) AVERAGEINHIBITION OF ZONE 0

E. coli S. aureus B. cereus Klebsiella B. subtilis Salmonella P. aeruginosa Enterobacter Proteus mirabilis Enterococcus sp Shigella dysenteriae

TEST ORGANISMS 0.625 mg/ml 1.25 mg/ml 2.5 mg/ml 5 mg/ml 10 mg/ml cpx control FIG. 16 ACTIVITY OF STEM BARK METHANOL FRACTION ON TEST BACTERIA 74

Figure 17 shows the activity of Leaf methanol fraction. Highest activity was against Proteus mirabilis where activity was recorded even at the lowest concentration

(0.625 mg/ml). Overall, differences in inhibition between Proteus and Pseudomonas and between Pseudomonas and Klebsiella were significant (P<0.05)`. Fraction was not active against S. aureus, E. coli, B. cereus, Salmonella, Enterobacter, Shigella, Bacillus subtilis and Enterococcus sp.

75 30

AVERAGE ZONES OF INHIBITION (MM) AVERAGE INHIBITION ZONESOF 0

E. coli S. aureus B. cereus Klebsiella B. subtilis Salmonella P. aeruginosa Enterobacter Proteus mirabilis Enterococcus sp Shigella dysenteriae TEST ORGANISMS

0.625 mg/ml 1.25 mg/ml 2.5 mg/ml 5 mg/ml 10 mg/ml cpx control

FIG. 17 ACTIVITY OF LEAF METHANOL FRACTION ON TEST BACTERIA 76

Figure 18 shows the activity of leaf chloroform fraction at different concentrations. The organisms most inhibited were E. coli and P. aeruginosa and it was only at 10 mg/ml concentration were there a significant (P<0.05) difference between the inhibition of these organism. S. aureus and Klebsiella were inhibited at 2.5 mg/ml while

Proteus mirabilis and B. subtilis were inhibited at 5 mg/ml. However, B. cereus,

Salmonella, Enterobacter, Shigella dysenteriae and Enterococcus sp were not inhibited.

AVERAGE ZONE OF INHIBITION (MM) INHIBITION OF ZONE AVERAGE 5

E. coli S. aureus B. cereus Klebsiella Salmonella P. aeruginosa Enterobacter Bacillus subtilis Proteus mirabilis Enterococcus sp Shigella dysenteriae

TEST ORGANISMS 0.625 mg/ml 1.25 mg/ml 2.5 mg/ml 5 mg/ml 10 mg/ml cpx control FIG. 18 ACTIVITY OF LEAF CHLOROFORM FRACTION ON TEST BACTERIA

Figure 19 shows the activity of root back n-hexane fraction. S. aureus and

Klebsiella were profoundly inhibited with the inhibition zone diameter for the least concentration (0.625 mg/ml) greater than 15 mm. Proteus mirabilis was also well inhibited but at lower concentrations of 0.625 to 2.5 mg/ml, inhibition was significantly

(P>0.05) lower than the inhibition of S. aureus and Klebsiella. There was no inhibition of

Enterococcus sp, Bacillus subtilis, Shigella dysenteriae, Enterobacter, Salmonella and B. cereus.

30 25 20 15 10 5

0 AVERAGE ZONE OF INHIBITION (MM) AVERAGEINHIBITION OF ZONE E. coli S. aureus B. cereus Klebsiella Salmonella P. aeruginosa Enterobacter Bacillus subtilis Proteus mirabilis Enterococcus sp Shigella dysenteriae TEST ORGANISMS

0.625 mg/ml 1.25 mg/ml 2.5 mg/ml 5 mg/ml 10 mg/ml cpx control FIG. 19 ACTIVITY OF ROOT BARK N-HEXANE FRACTION ON TEST BACTERIA 80

For stem bark n-hexane fraction, highest activities were recorded against S. aureus, Klebsiella and Proteus. There was also a low to moderate activity against E. coli and P. aeruginosa. However, other test organisms –B. cereus, Salmonella, B subtilis,

Enterobacter, Shigella and Enterococcus sp –were not inhibited (Fig. 20).

81 30

AVERAGE(MM) INHIBITION ZONEOF 0

E. coli S. aureus B. cereus Klebsiella Salmonella P. aeruginosa Enterobacter Bacillus subtilis Proteus mirabilis Enterococcus sp Shigella dysenteriae TEST ORGANISMS

0.625 mg/ml 1.25 mg/ml 2.5 mg/ml 5 mg/ml 10 mg/ml cpx control FIG. 20 ACTIVITY OF STEM BARK N-HEXANE FRACTION ON TEST BACTERIA

Figure 21 shows the activity of the leaf n-hexane fraction. A relatively high activity was recorded against Proteus mirabilis, and low to moderate activities against S. aureus, P. aeruginosa and Klebsiella. This fraction was inactive against E. coli, B. cereus, Salmonella, Enterobacter, Shigella dysenteriae, B. subtilis and Enterococcus sp.

83 30 25 20 15 10 5

0 AVERAGE ZONE AVERAGE OF INHIBITION (MM)

E. coli S. aureus B. cereus Klebsiella Salmonella P. aeruginosa Enterobacter Bacillus subtilis Proteus mirabilis Enterococcus sp Shigella dysenteriae TEST ORGANISMS 0.625 mg/ml 1.25 mg/ml 2.5 mg/ml 5 mg/ml 10 mg/ml cpx control FIG. 21 ACTIVITY OF LEAF N-HEXANE FRACTION ON TEST BACTERIA 84

3.7 PHYTOCHEMICAL COMPOSITION OF CRUDE EXTRACTS AND

FRACTIONS

The phytochemical analyses of the crude extracts and fractions revealed the presence of saponins, tannins, flavonoids, alkaloids, glycosides, reducing sugars and fats and oil in varying proportions as shown in Tables 6 to 9.

Table 6 Preliminary phytochemical screening of crude extracts

Phytochemicals Root bark Stem bark Leaves

1 Saponins + + +

2 Tannins + + +

3 Flavonoids +++ +++ +

4 Alkaloids ++ ++ +

5 Glycosides + + +

6 Reducing sugar + + +

7 Fats and oils + + +

KEY:

- = Absent, + = slightly present, ++ = fairly present, +++ = abundantly present,

Table 7 Phytochemical screening of root bark fractions

Phytochemicals n -hexane Chloroform Ethyl Acetone Methanol

acetate

1 Saponins N + +++ +++ +++

2 Tannins N - - + ++

3 Flavonoids N + +++ - -

4 Alkaloids N - - + +

5 Glycosides N + +++ + +

6 Fats and oils + - - - -

KEY:

- = Absent, + = slightly present, ++ = fairly present, +++ = abundantly present,

N = not determined

Table 8 Phytochemical screening of stem bark fractions

Phytochemicals n-hexane Chloroform Ethyl Acetone Methanol

acetate

1 Saponins N + ++ +++ +++

2 Tannins N - - + ++

3 Flavonoids N - +++ - -

4 Alkaloids N - - + +

5 Glycosides N + +++ + +

6 Fats and oils + - - - -

KEY:

- = Absent, + = slightly present, ++ = fairly present, +++ = abundantly present,

N = not determined

Table 9 Phytochemical screening of leaf fractions

Phytochemicals n-hexane Chloroform Ethyl Acetone Methanol

acetate

1 Saponins N ++ + + +

2 Tannins N - - + ++

3 Flavonoids N - ++ - -

4 Alkaloids N + + +++ +++

5 Glycosides N + +++ + +

6 Fats and oils + + - - -

KEY:

- = Absent, + = slightly present, ++ = fairly present, +++ = abundantly present,

N = not determined

3.8 ACUTE TOXICITY PROFILE

In the first phase of experimentation, the rat treated with 1000 mg/kg body weight of extract showed reduced locomotor activity after 4 hours of extract administration but became active again after 20 hours. The rats at the other dose levels (10 and 100 mg/kg body weight) were not likewise affected. No death occurred.

In the second phase, the three rats also showed reduced locomotor activity, were irritable and tended to huddle together between the 4th to the 20th hours of dose administration. These symptoms disappeared after 24 hours. No other signs of toxicity were recorded. There was no mortality at the highest dose level of 5000 mg/kg body weight. Therefore, the oral LD50 is greater than 5000 mg/kg.

CHAPTER FOUR

4.0 DISCUSSION

Widespread antibiotic usage exerts a selective pressure that acts as a driving force in the development of antibiotic resistance. The association between increased rates of antimicrobial use and resistance has been documented for nosocomial infections as well as for resistant community acquired infections. As resistance develops to "first-line" antibiotics, therapy with new, broader spectrum, more expensive antibiotics increases, but is followed by development of resistance to the new class of drugs. The results of in vitro antibiotic susceptibility testing, guide clinicians in the appropriate selection of initial empiric regimens and, drugs used for individual patients in specific situations. The selection of an antibiotic panel for susceptibility testing is based on the commonly observed susceptibility patterns, and is revised periodically.

In this study, the test bacterial strains showed differences in their resistance pattern to different class of antibiotics. Twenty antibiotics were used for screening. Of the

38 organisms used in the study 23.68% were resistant to ciprofloxacin; 31.57% to gentamycin, 34.21% to pefloxacin and 47.36% to streptomycin. Ciprofloxacin was hence chosen as the control antibiotic in the antibacterial assay for the crude extracts and fractions.

All 38 organisms were resistant to septrin. The 25 gram negatives showed 100% resistance to augmentin, 84% resistance to amoxicillin and 76% resistance to

91 nitrofurantoin. High resistance was also observed to nalidixic acid and chloramphenicol.

All the 13 strains of gram-positive bacteria used were resistant to zinnacef, amoxicillin, erythromycin and ampiclox. Equally high was the 85% resistance of gram positive organisms to clindamycin. The bacteria in were not only resistant to the routinely used antibiotics in Nigeria such as ampiclox, erythromycin, septrin, amoxicillin etc, but also showed resistance to the newer generation antibiotics like the fluoroquinolones e.g. ciprofloxacin (23.68%) and ofloxacin (48% amongst gram negatives). The resistance properties of these isolates showed the worsening situation of antibiotics resistance in the

Nigerian environment and lend credence to the search for substances that could be added to or replace the antibiotics in current clinical use, which are becoming less useful with every passing hour as previously suggested by other investigators (Aladesanmi et al.,

2007). The better activities observed for the extracts of fractions of U. chamae may provide an answer to this phenomenon.

The results obtained in this study indicated that the crude ethanolic and aqueous extracts of U. chamae inhibited the growth of a majority of the test isolates. This is an indication that the extracts posses substances that can inhibit the growth of some microorganisms. However, the observed inhibitory effects were more with the ethanolic extracts of the plant. Thus, ethanol is a better extraction solvent than water for the extraction of plant‟s active principles. This is in agreement with the findings of Obi and

Onuoha (2000) who stated that ethanol is a better extraction solvent than water. The traditional medicine practitioners may have observed this in the past that they almost always recommend the use of ethanol (local gin) for the maceration and extraction of plants for native medicine. The results have indicated that in general the ethanolic stem

92 bark and root bark extracts had greater inhibitory effects on the isolates than the leaf extracts. This was evident on all test organisms except B. subtilis. This is of importance to the traditional medical practitioners. Thus, they could use more of the root bark and stem bark extracts for treatment of infections.

Comparisons of the overall activities of the crude alcoholic extracts confirmed the higher activity of stem bark extracts against 72.7% of test strains with significantly

(P<0.05) higher inhibitions against S. aureus, B. subtilis, Klebsiella, Enterobacter,

Enterococcus sp and Proteus mirabilis. Against E. coli, P aeruginosa and B. cereus the difference in the inhibition of the alcoholic root bark and stem bark extracts were not significant (P>0.05). The aqueous root bark extracts had significantly (P<0.05) higher activity against E. coli, Enterococcus sp and S. dysenteriae while the aqueous stem bark extract had significantly (P<0.05) higher activity against Salmonella, Klebsiella and

Proteus mirabilis.

The low minimum inhibitory concentration (MIC) exhibited by the root bark and stem bark extracts against S. aureus, P. aeruginosa, B. cereus and E. coli is of great significance as these organisms show high resistance to conventional antibiotics

(Singleton, 1999). In a region where the cost of Medicare is so high, they can be used as alternatives to orthodox antibiotics as they are much cheaper.

The observed antibacterial effects on the isolates corroborate their use in traditional medicine. Ethanolic and water preparations of the root bark, stem bark and leaves of the plant are used by traditional medical practitioners in the treatment of cough, stomach upsets such as diarrhea and urinary tract infections. Preparations from the roots,

93 stem and leaves are also applied to wounds and sores to promote rapid healing (Igoli et al., 2005).

Plant materials were fractionated with n-hexane, ethyl acetate, chloroform, acetone and methanol. Hexane as the starting solvent was aimed at defating the crude extract by extracting non-polar compound, thus paving the way for subsequent solvents to extract intermediate polarity antibacterial compound (Angeh, 2006). The quantity of materials extracted from each plant part varied with different solvents used. In this study, acetone extracted the highest quantity of extract from the root bark, stem bark and leaves of U. chamae. Other solvents like hexane and methanol might extract a wide range of compounds, but acetone will always extract compounds with a wider range of polarities

(Angeh, 2006).

Fractions showed varying inhibitory activities against different microorganisms.

Inhibition was not affected by Gram status and hence, different strains of both Gram positive and Gram negative organisms were inhibited with effectiveness dependent on fraction and organisms. Methanol and acetone fractions were soluble in 20% DMSO; other fractions were only dissolved at higher concentration of DMSO (60%). Stem bark acetone fraction was active against the widest range of organisms showing low to high activity against all the test strains (Fig. 10). Stem bark methanol fraction was also inhibitory to a wide range of organisms except Shigella dysenteriae (fig. 16). Activity increased with increasing concentration and most organisms were inhibited to varying extents. Ethyl acetate root and stem bark fractions though ineffective against B. cereus,

Enterobacter and Enterococcus sp showed profound activities against S. aureus, P. aeruginosa, Proteus mirabilis and Klebsiella. Average activities against some of these

94 organisms were significantly (P<0.05) higher than all others (Figures 12 and 13). N- hexane root and stem bark fractions were also profoundly inhibitory to S. aureus, proteus and Klebsiella with an inhibition diameter of 17 mm at 0.625 mg/ml against S. aureus

(Fig. 19).

Chloroform fraction was the least active of all the fractions. The stem and root bark chloroform fractions were profoundly active against S. aureus with an inhibition zone of 33 mm at 2.5 mg/ml for the stem bark and 18-30 mm at 0.625 – 10 mg/ml concentrations. No other organism was inhibited by any of these fractions. The leaf chloroform fraction showed low to moderate activity against most organisms except

Enterococcus sp, Shigella, Salmonella, Enterobacter and B cereus (Fig. 18).

As shown in figures 2 to 7, crude extracts had comparatively low antimicrobial activities against Klebsiella but upon fractionation, most fractions were profoundly inhibitory against the organisms, with highest activities by ethyl acetate stem bark and root bark n-hexane fractions. This phenomenon may be due to antagonism between components of the crude extracts.

The observed activity may be due to the presence of potent phytoconstituents in the extracts. The differences in activity among fractions may also be due to differences in concentration and distribution of phytochemicals derived from their relative solubilities.

Present investigation into the phytochemical profile of U. chamae crude extracts and fractions confirmed the presence of alkaloids, tannins, saponins, flavonoids, glycosides and fats and oils which might be responsible for their relative antimicrobial properties

(Tables 5 – 8). This is consistent with other reports which have shown these phytochemicals to possess antibacterial properties (Cowan, 1999; Draughon, 2004).

Acetone and methanol fractions being very polar were able to extract many compounds

(Angeh, 2006) and this could be responsible for the wide spectrum of activity against different strains of microorganisms. Only flavonoids and fats and oils were not detectable. The high activity ethyl acetate fractions had a disproportionately high concentration of saponins, flavonoids and glycosides in comparison with the other fractions and may account for its profound activity on S. aureus. The n-hexane fractions have the least number of detectable antibacterial compounds with most major phytochemicals absent except fats and oils this may account for n-hexane fractions being active on only 4 of the 11 tested strains.

The chloroform extracts were the least active and the most difficult to dissolve.

Only low proportion of saponins and glycosides were detected. In fact, the leaf chloroform fractions showed low to moderate activity against some of the tested strains but the root and stem bark fractions showed activity only on S. aureus. In the distribution of phytochemicals, the leaves have higher proportion of saponins and also contain alkaloids; this may account for its higher activity (table 9).

The root and stem bark fractions are of comparable activity almost showing equivalent efficacy against most test organisms. The stem bark acetone and methanol fractions were slightly more active than the root bark fractions. The root bark ethyl acetate fraction had greater activity against S. aureus and Pseudomonas aeruginosa but the stem bark was more active against Klebsiella and Proteus mirabilis. The leaf fractions were generally less efficacious when compared to either of the root or stem bark fractions except the leaf chloroform fraction that was relatively more active (Figure 18). Leaf

96 methanol fraction was the least active of all the leaf extracts being only slightly active against Klebsiella, P. aeruginosa and Proteus mirabilis.

The toxicity of the U. chamae extracts was evaluated. A scale proposed by Lorke

(1983) roughly classifies substances according to their LD50 as follows: very toxic

(LD50<1 mg/kg), toxic (LD50 up to 10 mg/kg), less toxic (LD50 up to 100 mg/kg) and only slightly toxic (up to 1000 mg/kg). Substances with LD50 values greater than 5000 mg/kg are practically non-toxic. Based on this proposal, the high oral LD50 (>5000 mg/kg body weight) obtained suggests that the U. chamae stem bark extract is practically non- toxic through this route and is therefore safe in the rats and in its traditional use orally for treatment of the diseases it is indicated for.

The results from this study show that extracts of U. chamae, particularly the stem bark extracts have high and broad spectrum antimicrobial activity against multiple antibiotic resistant bacterial strains. They therefore represent an alternative remedy for infections due to these resistant strains. The plant should also be investigated further for development of new antimicrobials.

REFERENCES

Abu-Shanab, B., Adwan, G., Jarrar, N., Abu-Hijleh, A. and Adwan, K. (2006). Antibacterial activity of four plant extracts used in Palestine in folkloric medicine against methicillin-resistant Staphylococus aureus. Turk, J. Biol 30: 195-198.

Aggarwal, B.B., Sundaram, C., Malani, N. and Ichikawa H. (2007). "Curcumin: the Indian solid gold". Adv. Exp. Med. Biol. 595: 1–75.

Akinyemi, K.O., Oladapo, O., Okwara, C.E., Ibe, C.C. and Fasure, K.A. (2005). Screening of Crude extracts of six medicinal plants used in South-West Nigerian unorthodox medicine for anti-meticillin resistant Staphylococcus aureus activity. BMC Complement Altern Med. 5: 6-13.

Akinyemi, K.O., Oluwa, O.K. and Omonigbehin, E.O. (2006). Antimicrobial activity of crude extracts of three medicinal plants used in South-West Nigerian folk medicine on some food born bacterial pathogens. Afr. J. Trad., Complement Alternative med. 3(4): 13-22.

Aladesanmi, A.J., Iwalewa, E.O., Adebajo, A.C., Akinkunmi, E.O., Taiwo, B.J., Olorunmola, F.O. and Lamikanra, A. (2007). Antimicrobial and antioxidant activities of some Nigerian medicinal plants. Afr. J. Trad. Complement Alternative med. 4(2): 173-184.

Angeh, J.E. (2006). Isolation and characterization of antibacterial compounds present in Members of Combretum section, Hypocrateropsis (PhD theses). University of Pretoria etd.

Bashengezi and Constantin M. (1997). Purified extract of Uvaria brevistipitata and a process for obtaining the purified extract therefor. United States Patent 5, 607, 673. Assignee: C.S.S.A.H.A., Inc. (Chicago, IL).

Bayly, G.R., Braithwaite, R.A. and Sheehan, T.M.T. (1995). Lead poisoning from Asian traditional remedies in the West Midlands -- report of a series of five cases. Hum Exp Toxicol. 14: 24-28.

Betoni, J.E.C., Mantovani, R.P., Barbosa, L.N., Di Stasi, L.C. and Fernandes Jr. A. (2006). Synergism between plant extract and antimicrobial drugs used on Staphylococcus aureus diseases. Memorias do Instituto Oswaldo Cruz. 101(4).

Boulanger, D. (2002). "The Islamic Contribution to Science, Mathematics and Technology", OISE Papers, in STSE Education. 3.

Burkill, H.M .(1985). Entry for Uvaria chamae P. Beauv. (Family Annonaceae). The useful plants of West Africa. 1.

Carr, J.D. and Rogers, C.B. (1987). Chemosystematic studies of the genus Combretum (Combretaceae). 1. A convenient method of identifying species of this genus by a comparison of the polar constituents extracted from leaf material. South African Journal of Botany. 53: 173-176.

Castleman, M. (2001). The New Healing Herbs: The Classic Guide to Nature's Best Medicines Featuring the Top 100 Time-Tested Herbs. Rodale. p. 15. Clinical and Laboratory Standards institute (CLSI). (2006). 26(1).

Cole J.R., Torrance, S.J., Weidhoff, R.M., Arora S.K., Bates R.B. (1976). J. Org. Chem. 41(10): 1852-1855.

Cowan, M.M. (1999). Plant Products as Antimicrobial Agents. Clinical Microbiology Reviews 12: 564-582.

Cuendet, M. (2008). "Dietary administration of Asimina triloba extract increases tumor latency in N-methyl-N-nitrosourea-treated rats". Pharmaceutical Biology. 46:1- 2.

De Smet, P.A.G.M. (1995). Health risks of herbal remedies. Drug Saf. 13: 81-93.

De Smet, P.A.G.M. and Smeets O.S.N.M. (1994). Potential risk of health food products containing yohimbe extracts. BMJ. 309: 958.

Doyle, J.A., Sauquet, H. and Scharaschkin, T. (2004). "Phylogeny, molecular and fossil dating, and biogeographic history of Annonaceae and Myristicaceae (Magnoliales)". International Journal of Plant Sciences. 165(4): 55-67.

Draughon, F.A. (2004). Use of botanicals as biopreservatives in foods. Food Technology. 58(2): 20 – 28.

Drew, A.K. and Myers S.P. (1997). Safety issues in herbal medicine: implications for the health professions. The Medical Journal of Australia. 166: 538-541.

Esimone, C.O., Adiukwu, M.U. and Okonta, J.M. (1998). Preliminary antimicrobial screening of the ethanolic extract from the lichen Usnea subfloridans. (L). Journal of Pharmaceutical Research and Development. 3(2): 99 – 102.

Evans, W.C. (1989). Trease and Evans‟ Textbook of Pharmacognosy. 13th Edition. Bailliere, Tindall, London, 26-35.

Fahd, T. (1996). "Botany and agriculture" in Morelon and Rashed. pp. 813-52.

Farnsworth, N.R. (1984). The role of medicinal plants in drug development. In: Natural products and Drugs Development, eds. P. Krogsgaard-Larsen, S.B. Christensen, H. Kofod, 8-98. Balliere, Tindall and Cox, London.

Farnsworth, N.R., Akerele., O. and Bingel, A.S. (1985). Medicinal plants in therapy. Bull. World Health Organization. 63: 965-981.

Harbone, J.B.C. (1984). Phytochemical Methods. Chapman and Hall, London. p. 279.

Harold, R.L. and Heath, I.B. (1992). Journal of Cell Sciences. 102(3): 611-627.

Herdberg, M. and Staugard, P. (1989). Traditional medicine in Botswana. Traditional Medicinal Plants. Gaborone Ipeleggeng.

Huff, T. (2003). The Rise of Early Modern Science: Islam, China, and the West. Cambridge University Press. P. 218. ISBN 0521529948.

Hufford, C.D. and Lasswell, W.L. Jr. (1978). Antimicrobial activities of constituents of Uvaria chamae. J. Org. Chem. 41(7): 1297-1298.

Igoli, J.O., Ogaji, T.A., Tor-Anyiin and Igoli, N.P. (2005). Traditional medicine practice amongst the Igede people of Nigeria. Part 2. African J. Traditional Complementary and Alternative Medicine. 2(2): 134 – 152.

Iwu, M.W., Duncan, A.R. and Okunji, C.O. (1999). New antimicrobials of plant origin. In: Perspective on New crops and new uses. ASHS Press, Alexandria, VA. 457- 462.

Jolad, S.D., Hoffmann, J.J., Schram, K.H. and Cole, J.R. (1982). Uvaricin, a new antitumor agent from Uvaria acuminata (Annonaceae). J. Org. Chem. 47: 3151-3153.

Kuc, J. (1985). Increasing crop productivity and value by increasing disease resistance through non-genetic techniques. In: Forest potentials: productivity and value; ed. R. Ballard. Weyerhaeuser Company Press, Centralia. P.147-190.

Lorke, D. (1983). A new approach to practical acute toxicity testing. Archives of Toxicology. 54: 275 – 287.

Madubunyi, I.I., Njoku, C.J., Ibeh, E.O. and Chime A.B. (1996). Antihepatototic and trypanocidal effects of the root bark extract of Uvaria chamae. Int. J. Pharmacog. 34(1): 34-40.

100

Nascimento G.G.F., Locatelli, J., Freitas, P.C. and Silva, G.L. (2000). Antibacterial activity of plant extracts and phytochemicals on antibiotic resistant bacteria. Braz. J. Microbiol. 31(4). Nkunya, M., Moshi, M., Joseph, C. and Innocent, E. (1991). In vitro antibacterial activities of extracts and compounds from Uvaria scheffleri. Pharmaceutical Biology, 1744-5116. 42(4): 267-273.

Obi, V.I. and Onuoha, C. (2000). Extraction and characterization methods of plants and plant products. In: Biological and Agricultural Techniques. Ogbulie, J.N. and Ojiako, O.J. (eds). Websmedia Publications, Owerri. P. 271-286.

Ogbonnia, S.O., Enwuru, N.V., Onyemenem, E.U., Oyedele, G.A. and Enwuru, C.A. (2008). Phytochemical evaluation and antibacterial profile of Treculia Africana Decne bark extract on gastro-intestinal bacterial pathogens. Afr. J. Biotech. 7(10): 1385-1389.

Ogbulie, J.N., Ogueke, C.C. and Nwanebu, F.C. (2007). Antibalterial properties of Uvaria chamae, Congronema latifolium, Garcinia kola, Vernonia Anymgdalina and Aframomium melegueta. Afr. J. Biotech. 6(13): 1549-1553.

Oguntimein, B., Ekundayo, O., Laako, I. and Hiltunen R. (1989). Volatile constituents of Uvaria chamae leaves and root bark. Planta medica. 55(3): 312-313(14).

Pereira, E.M., Machado T.B., Leal, I.C.R., Jesus, D.M, Damaso, C.R.A., Pinto A.V., Giambiagi-demarval M., Kuster, R.M. and Dos Santos, K.R.N. (2006). Tabebuia avellanedae naphthoquinones: activity against methicillin-resistant staphylococcal strains, cytotoxic activity and in vitro dermal irritability analysis. Annals of clinical microbiology and Antimicrobials. 5:5 – 10.

Rao, N. and Kuc, J. (1990). Induced systemic resistance in plants. In: the fungal spore and disease initiation in plants and animals, G.T. Cole, H.C. Hoch. Plenum Press. Newyork.

Salawu, O.A., Aliyu, M. and Tijani, A.Y. (2008). Haematological studies on the ethanolic stem bark extract of Pterocarpus erinaceus poir (fabaceae). Afr. J. Biotechnology. 7(9): 1212 – 1215.

Salman, M.T., Khan, R.A., Shukla, I. (2005). In vitro antimicrobial activity of Nigella sativa oil against multi-drug resistant bacteria. Dept. of Pharmacology, Jawaharlal Nehru medical college, Aligarh Muslim University. India.

Singleton, P. (1999). Bacteria in Biology, Biotechnology and Medicine. 4th edn. John Wiley and Sons Ltd, New York.

Sofowora, A. (1984). Medicinal plants and tradition in Africa medicine. Part 2.

101

Suedee, A. (2007). "Constituents of Polyalthia jucunda and their cytotoxic effect on human cancer cell lines." Pharmaceutical Biology. 45(7):575-579.

World Health Organization (WHO) .(2010). Traditional medicine. http://www.who.int/mediacentre/factsheets/fs134/en/.

Wikipedia, the free encyclopedia. (2010). Annonaceae. Wikipedia foundation, Inc.

Wijesekera, R.O.B. (1991). The prospect of screening. Medicinal plant industry. CRC press, pp 224 (1st ed.)

102

APPENDIX I

A: ANALYSIS OF VARIANCE FOR CRUDE AQUEOUS ROOT BARK EXTRACT

Variate: Zone_of_inhibition_mm

Source of variation d.f. s.s. m.s. v.r. F pr. Concentration 5 13829.437 2765.887 728.52 <.001 Test_organisms 10 1576.240 157.624 41.52 <.001 CAqRBE.Test_organisms 50 1931.204 38.624 10.17 <.001 Residual 198 751.723 3.797 Total 263 18088.603

Grand mean 10.43

Table of Means for Test_organisms B. cereus B. subtilis E. coli 12.71 12.94 9.58 Enterobacter Enterococcus sp Klebsiella 7.67 12.00 4.94 P. aeruginosa Proteus mirabilis S. aureus 13.08 11.29 11.96 Salmonella Shigella dysenteriae 9.85 8.75

Least significant differences of means for overall inhibition of test organisms (5% level) l.s.d. 1.109

B: DIFFERENCES IN MEAN INHIBITION AMONGST BACTERIAL ISOLATES

B. cereus B. sub E. coli Entero Entero Kleb P. aeru Pro S. aureus Salmo Shigella tilis bacter coccus siella ginosa teus nella

B. cereus -

B. subtilis 0.23* -

E. coli -3.13 -3.36 -

Enterobacter -5.04 -5.27 -1.91 -

Enterococcus -0.71* -0.94* 2.42 4.33 -

Klebsiella -7.77 -8.00 -4.64 -2.73 -7.06 -

P. aeruginosa 0.37* 0.14* 3.50 5.41 1.08* 8.14 -

Proteus -1.42 -1.65 1.71 3.62 -0.71* 6.35 -1.79 -

S. aureus -0.75* -0.98* 2.38 4.29 -0.04* 7.02 -1.12* 0.67* -

Salmonella -2.86 -3.09 0.27* 2.18 -2.15 4.91 -3.08 -1.44 -2.11 -

Shigella -3.96 -4.19 -0.83* 1.08* -3.25 3.81 -4.33 -2.54 -3.21 -1.10* -

Not significant: *

103

APPENDIX 2

A. ANALYSIS OF VARIANCE FOR CRUDE ALCOHOLIC ROOT BARK EXTRACT

Variate: Zone_of_inhibition_mm

Source of variation d.f. s.s. m.s. v.r. F pr. CARBE 5 16025.057 3205.011 970.65 <.001 Test_organisms 10 1097.984 109.798 33.25 <.001 CARBE.Test_organisms 50 1696.855 33.937 10.28 <.001 Residual 198 653.778 3.302 Total 263 19473.673

CARBE: Extract concentration Grand mean 10.42

Table of Means for Test_organisms

B. cereus B. subtilis E. coli 12.33 8.14 11.62 Enterobacter Enterococcus sp Klebsiella 8.17 10.17 6.56 P. aeruginosa Proteus mirabilis S. aureus 12.38 10.92 12.42 Salmonella Shigella dysenteriae 9.10 12.79

Least significant differences for overall inhibition of test organisms (5% level)

l.s.d. 1.034

B: DIFFERENCES IN MEAN INHIBITION AMONGST BACTERIAL ISOLATES

B. cereus B. sub E. coli Entero Entero Kleb P. aeru Pro S. aureus Salmo Shigella tilis bacter coccus siella ginosa teus nella

B. cereus -

B. subtilis -4.19 -

E. coli -0.71* 3.48 -

Enterobacter -4.16 0.03* -3.45 -

Enterococcus -2.16 2.03 -1.45 2.0 -

Klebsiella -5.77 -1.58 -5.06 -1.61 -3.61 -

P. aeruginosa 0.05* 4.24 0.76* 4.21 2.21 5.82 -

Proteus -1.41 2.78 -0.70* 2.75 0.75* 4.36 -1.46 -

S. aureus 0.09* 4.28 0.80* 4.25 2.25 5.86 0.04* 1.50 -

Slmonella -3.23 0.96* -2.52 0.93* -1.07 2.54 -3.28 -1.82 -3.32 -

Shigella 0.46* 4.65 1.17 4.62 2.62 6.23 0.41* 1.87 0.37* 3.69 -

*: Not Significant

104

APPENDIX 3

A. ANALYSIS OF VARIANCE FOR CRUDE AQUEOUS STEM BARK EXTRACT

Variate: Zone_of_inhibition_mm

Source of variation d.f. s.s. m.s. v.r. F pr. CAqSBE 5 14228.564 2845.713 1058.95 <.001 Test_organisms 10 1161.488 116.149 43.22 <.001 CAqSBE.Test_organisms 50 1858.384 37.168 13.83 <.001 Residual 198 532.082 2.687 Total 263 17780.518

CAqSBE: Extract concentration

Grand mean 10.25

Table of Means for Test_organisms B. cereu B. subtilis E. coli 7.33 10.83 7.46 Enterobacter Enterococcus sp Klebsiella 11.42 9.46 10.55 P. aeruginosa Proteus mirabilis S. aureus 11.28 14.58 11.31 Salmonella Shigella dysenteriae 11.08 7.42

Least significant differences for overall inhibition of test organisms (5% level)

l.s.d. 0.933

B: DIFFERENCES IN MEAN INHIBITION AMONGST BACTERIAL ISOLATES

B. cereus B. sub E. coli Entero Entero Kleb P. aeru Pro S. aureus Salmo Shigella tilis bacter coccus siella ginosa teus nella

B. cereus -

B. subtilis 3.50 -

E. coli 0.13* -3.37 -

Enterobacter 4.09 0.59* 3.96 -

Enterococcus 2.13 -1.37 2.00 -1.96 -

Klebsiella 3.22 -0.28* 3.09 -0.87* 1.09 -

P. aeruginosa 3.95 0.45* 3.82 -0.14* 1.82 0.73* -

Proteus 7.25 3.75 7.12 3.16 5.12 4.03 3.3 -

S. aureus 3.98 0.48* 3.85 -0.11* 1.85 0.76* 0.03* -3.27 -

Salmonella 3.75 0.25* 3.62 -0.34* 1.62 0.53* -0.2* -3.5 -0.23* -

Shigella 0.09* -3.41 -0.04 -4.0 -2.04 -3.13 -3.86 -7.6 -3.89 -3.66 -

*: Not significant

105

APPENDIX 4

A. ANALYSIS OF VARIANCE FOR CRUDE ALCOHOLIC STEM BARK EXTRACT

Variate: Zone_of_inhibition_mm

Source of variation d.f. s.s. m.s. v.r. F pr. CASBE 5 12540.264 2508.053 422.72 <.001 Test_organisms 10 2477.037 247.704 41.75 <.001 CASBE.Test_organisms 50 1292.953 25.859 4.36 <.001 Residual 198 1174.770 5.933 Total 263 17485.025

CASBE: Extract concentration

Grand mean 12.38

Table of Means for Test_organisms B. cereus B. subtilis E. coli Enterobacter 11.56 14.01 11.67 10.12 Enterococcus sp Klebsiella P. aeruginosa Proteus mirabilis 11.17 7.39 12.29 19.38 S. aureus Salmonella Shigella dysenteriae 16.33 11.33 10.88

Least significant differences for overall inhibition of test organisms (5% level)

l.s.d. 1.387

B: DIFFERENCES IN MEAN INHIBITION AMONGST BACTERIAL ISOLATES

B. cereus B. sub E. coli Entero Entero Kleb P. aeru Pro S. aureus Salmo Shigella tilis bacter coccus siella ginosa teus nella

B. cereus -

B. subtilis 2.45 -

E. coli 0.11* -2.34 -

Enterobacter -1.44 -3.83 -1.55 -

Enterococcus -0.39* -2.84 -0.5* 1.05* -

Klebsiella -4.17 -6.62 -4.28 -2.73 -3.78 -

P. aeruginosa 0.73* -1.72 0.62* 2.17 1.12* 4.9 -

Proteus 7.82 5.37 7.71 9.26 8.21 11.99 7.09 -

S. aureus 4.77 2.32 4.66 6.21 5.16 8.94 4.04 -3.05 -

Slmonella -0.23* -2.68 -0.34* 1.21* 0.16* 3.94 -0.94 -8.05 -5.0 -

Shigella -0.68* -3.13 -0.79* 0.76* -0.29* 3.49 -1.41 -8.5 -5.45 -0.45* -

*: Not significant

106

APPENDIX 5

A. ANALYSIS OF VARIANCE FOR CRUDE AQUEOUS LEAF EXTRACT

Variate: Zone_of_inhibition_mm

Source of variation d.f. s.s. m.s. v.r. F pr. CAqLE 5 10455.669 2091.134 903.03 <.001 Test_organisms 10 3304.327 330.433 142.69 <.001 CAqLE.Test_organisms 50 2696.616 53.932 23.29 <.001 Residual 198 458.507 2.316 Total 263 16915.120 CAqLE: Extract concentration

Grand mean 7.949

Table of Means for Test_organisms B. cereus B. subtilis E. coli 7.000 12.471 11.983 Enterobacter Enterococcus sp Klebsiella 6.250 7.333 2.275 P. aeruginosa Proteus mirabilis S. aureus 13.625 9.500 7.000 Salmonella Shigella dysenteriae 7.417 2.583

Least significant differences for overall inhibition of test organisms (5% level)

l.s.d. 0.8663

B: DIFFERENCES IN MEAN INHIBITION AMONGST BACTERIAL ISOLATES

B. cereus B. sub E. coli Entero Entero Kleb P. aeru Pro S. aureus Salmo Shigella tilis bacter coccus siella ginosa teus nella

B. cereus -

B. subtilis -0.5* -

E. coli -5.47 -4.98 -

Enterobacter -6.22 -5.73 -0.75* -

Enterococcus -5.14 -4.65 0.33* 1.08 -

Klebsiella -10.2 -9.7 -4.72 -3.97 -5.05 -

P. aeruginosa 1.16 1.65 6.63 7.38 6.3 11.4 -

Proteus -2.97 -2.48 2.5 3.25 2.17 7.22 -4.15 -

S. aureus -5.47 -4.98 0.00* 0.75* -0.33* 4.72 -6.63 -2.5 -

Slmonella -5.05 -4.56 0.42* 1.17 0.09* 5.15 -6.21 -2.08 0.42* -

Shigella -9.89 -9.4 -4.42 -3.67 -4.75 0.31* -11.0 -6.92 -4.42 -4.84 -

*: Not significant

107

APPENDIX 6

A. ANALYSIS OF VARIANCE FOR CRUDE ALCOHOLIC LEAF EXTRACT

Variate: Zone_of_inhibition_mm

Source of variation d.f. s.s. m.s. v.r. F pr. CALE 5 11675.108 2335.022 372.89 <.001 Test_organisms 10 1016.667 101.667 16.24 <.001 CALE.Test_organisms 50 1212.367 24.247 3.87 <.001 Residual 198 1239.857 6.262 Total 263 15144.000

CALE: Extract concentration

Grand mean 9.22

Table of Means for Test_organisms B. cereus B. subtilis E. coli 10.33 12.16 9.90 Enterobacter Enterococcus sp Klebsiella 8.00 7.67 4.14 P. aeruginosa Proteus mirabilis S. aureus 9.48 10.08 9.69 Salmonella Shigella dysenteriae 10.10 9.92

Least significant differences for overall inhibition of test organisms (5% level)

l.s.d. 1.425

B: DIFFERENCES IN MEAN INHIBITION AMONGST BACTERIAL ISOLATES

B. cereus B. sub E. coli Entero Entero Kleb P. aeru Pro S. aureus Salmo Shigella tilis bacter coccus siella ginosa teus nella

B. cereus -

B. subtilis 1.83 -

E. coli -0.43* -2.26 -

Enterobacter -2.33 -4.16 -1.9 -

Enterococcus -2.66 -4.49 -2.23 -0.33* -

Klebsiella -6.19 -8.02 -5.76 -3.86 -3.53 -

P. aeruginosa 0.85* -2.68 -0.42* 1.48 1.81 5.34 -

Proteus -0.25* -2.08 0.18* 2.08 2.41 5.94 0.6* -

S. aureus -0.64* -2.47 -0.21* 1.69 2.02 5.55 0.21* -0.39* -

Slmonella -0.23* -2.06 0.20* 2.1 2.43 5.96 0.68* 0.02* 0.41* -

Shigella -0.41* -2.24 0.02* 1.92 2.25 5.78 0.44* -0.16* 0.23* -0.18 -

*: Not significant

108

APPENDIX 7

A. ANALYSIS OF VARIANCE FOR ROOT BARK ACETONE FRACTION

Variate: Zone_of_inhibition_mm

Source of variation d.f. s.s. m.s. v.r. F pr. RBAF 4 418.818 104.705 81.11 <.001 Test_organisms 10 3828.873 382.887 296.60 <.001 RBAF.Test_organisms 40 540.582 13.515 10.47 <.001 Residual 55 71.000 1.291 Total 109 4859.273

RBAF: Extract concentration

Grand mean 5.55

Table of Means for Test_organisms B. cereus Bacillus subtilis E. coli 2.20 0.00 1.80 Enterobacter Enterococcus sp Klebsiella 0.00 5.00 14.80 P. aeruginosa Proteus mirabilis S. aureus 11.80 10.80 14.60 Salmonella Shigella dysenteriae 0.0 0.00

Least significant differences for overall inhibition of test organisms (5% level) l.s.d. 1.018

109

APPENDIX 8

A. ANALYSIS OF VARIANCE FOR STEM BARK ACETONE FRACTION

Source of variation d.f. s.s. m.s. v.r. F pr. SBAF 4 1237.691 309.423 261.82 <.001 Test_organisms 10 2678.473 267.847 226.64 <.001 SBAF.Test_organisms 40 477.709 11.943 10.11 <.001 Residual 55 65.000 1.182 Total 109 4458.873

SBAF :Extract concentration Grand mean 6.65

Table of Means for Test_organisms B. cereus Bacillus subtilis E. coli 1.60 4.60 5.40 Enterobacter Enterococcus sp Klebsiella 1.60 3.60 11.00

P. aeruginosa Proteus mirabilis S. aureus 15.20 14.40 10.60

Salmonella Shigella dysenteriae 3.40 1.80

Least significant differences for overall inhibition of test organisms (5% level)

l.s.d. 0.974 B: DIFFERENCES IN MEAN INHIBITION AMONGST BACTERIAL ISOLATES

B. cereus B. sub E. coli Entero Entero Kleb P. aeru Pro S. aureus Salmo Shigella tilis bacter coccus siella ginosa teus nella

B. cereus -

B. subtilis 3.0 -

E. coli 3.8 0.8* -

Enterobacter 0.0* -3.0 -3.8 -

Enterococcus 2.0 -1.0 -1.8 2.0 -

Klebsiella 9.4 6.4 5.6 9.4 7.4 -

P. aeruginosa 13.6 10.6 9.8 13.6 11.6 4.2 -

Proteus 12.8 9.8 9.0 12.8 10.8 3.4 -0.8* -

S. aureus 9.0 6.0 5.2 9.0 7.0 -0.4* -4.6 -3.8 -

Salmonella 1.8 -1.2 -2.0 1.8 -0.2* -7.6 -11.8 -11.0 -7.2 -

Shigella 0.2* -2.8 -3.6 0.2* -1.8 -9.2 -13.4 -12.6 -8.8 -1.6 -

*: Not significant

110

APPENDIX 9

A. ANALYSIS OF VARIANCE FOR LEAF ACETONE FRACTION

Variate: Zone_of_inhibition_mm

Source of variation d.f. s.s. m.s. v.r. F pr. LAF 4 426.3091 106.5773 229.87 <.001 Test_organisms 10 2239.2909 223.9291 482.98 <.001 LAF.Test_organisms 40 599.8909 14.9973 32.35 <.001 Residual 55 25.5000 0.4636 Total 109 3290.9909

LAF: Extract concentration Grand mean 3.809 Table of Means for Test_organisms B. cereus Bacillus subtilis E. coli 0.000 0.000 0.000 Enterobacter Enterococcus sp Klebsiella 12.800 0.000 5.100 P. aeruginosa Proteus mirabilis S. aureus 9.000 7.800 7.200 Salmonella Shigella dysenteriae 0.0 0.000

Least significant differences for overall inhibition of test organisms (5% level) l.s.d. 0.6103

APPENDIX 10

ANALYSIS OF VARIANCE FOR ROOT BARK ETHYL ACETATE FRACTION

Variate: Zone_of_inhibition_mm

Source of variation d.f. s.s. m.s. v.r. F pr. RBEAF 4 1446.945 361.736 61.50 <.001 Test_organisms 10 4289.655 428.965 72.93 <.001 RBEAF.Test_organisms 40 1185.255 29.631 5.04 <.001 Residual 55 323.500 5.882 Total 109 7245.355

RBEAF: Extract concentration

Grand mean 7.46

Table of Means for Test_organisms

B. cereus Bacillus subtilis E. coli 0.00 2.00 9.00 Enterobacter Enterococcus sp Klebsiella 0.00 5.40 11.20

111

P. aeruginosa Proteus mirabilis S. aureus 16.20 15.00 16.30 Salmonella Shigella dysenteriae 7.0 0.00

Least significant differences for overall inhibition of test organisms (5% level) l.s.d. 2.174

APPENDIX 11

ANALYSIS OF VARIANCE FOR STEM BARK ETHYL ACETATE FRACTION

Source of variation d.f. s.s. m.s. v.r. F pr. SBEAF 4 360.273 90.068 49.54 <.001 Test_organisms 10 6705.255 670.525 368.79 <.001 SBEAF.Test_organisms 40 559.927 13.998 7.70 <.001 Residual 55 100.000 1.818 Total 109 7725.455

SBEAF: Extract concentration

Grand mean 6.64

Table of Means for Test_organisms

B. cereus Bacillus subtilis E. coli 0.00 0.00 7.30 Enterobacter Enterococcus sp Klebsiella 0.00 0.00 20.40 P. aeruginosa Proteus mirabilis S. aureus 14.10 15.60 15.60 Salmonella Shigella dysenteriae 0.00 0.00

Least significant differences for overall inhibition of test organisms (5% level) l.s.d. 1.208

APPENDIX 12

ANALYSIS OF VARIANCE FOR LEAF ETHYL ACETATE FRACTION

Source of variation d.f. s.s. m.s. v.r. F pr. LeEAF 4 231.5091 57.8773 71.53 <.001 Test_organisms 10 2424.7636 242.4764 299.69 <.001 LeEAF.Test_organisms 40 364.6909 9.1173 11.27 <.001 Residual 55 44.5000 0.8091 Total 109 3065.4636

LeEAF: Extract concentration

112

Grand mean 4.482

Table of Means for Test_organisms

B. cereus Bacillus subtilis E. coli 0.000 0.000 8.300 Enterobacter Enterococcus sp Klebsiella 2.000 0.000 10.800 P. aeruginosa Proteus mirabilis S. aureus 11.500 9.400 7.300 Salmonella Shigella dysenteriae 0.000 0.000

Least significant differences for overall inhibition of test organisms (5% level) l.s.d. 0.8062

APPENDIX 13

ANALYSIS OF VARIANCE FOR ROOT BARK METHANOL FRACTION

Variate: Zone_of_inhibition_mm

Source of variation d.f. s.s. m.s. v.r. F pr. RBMF 4 967.4182 241.8545 491.21 <.001 Test_organisms 10 1056.6727 105.6673 214.61 <.001 RBMF.Test_organisms 40 945.7818 23.6445 48.02 <.001 Residual 55 27.0800 0.4924 Total 109 2996.9527

RBMF: Extract concentration

Grand mean 3.245

Table of Means for Test_organisms

B. cereus Bacillus subtilis E. coli 0.000 9.600 3.400 Enterobacter Enterococcus sp Klebsiella 0.000 0.000 4.600 P. aeruginosa Proteus mirabilis S. aureus 4.500 7.400 4.200 Salmonella Shigella dysenteriae 2.000 0.000

Least significant differences for overall inhibition of test organisms (5% level) l.s.d. 0.6289

113

APPENDIX 14

ANALYSIS OF VARIANCE FOR STEM BARK METHANOL FRACTION

Variate: Zone_of_inhibition_mm

Source of variation d.f. s.s. m.s. v.r. F pr. SBMF 4 1394.0364 348.5091 532.44 <.001 Test_organisms 10 2029.9636 202.9964 310.13 <.001 SBMF.Test_organisms 40 674.7636 16.8691 25.77 <.001 Residual 55 36.0000 0.6545 Total 109 4134.7636

SBMF: Extract concentration

Grand mean 5.782

Table of Means for Test_organisms

B. cereus Bacillus subtilis E. coli 2.000 8.400 4.000 Enterobacter Enterococcus sp Klebsiella 1.400 3.800 7.000 P. aeruginosa Proteus mirabilis S. aureus 12.800 10.600 11.600 Salmonella Shigella dysenteriae 2.000 0.000

Least significant differences for overall inhibition of test organisms (5% level) l.s.d. 0.7251

APPENDIX 15

ANALYSIS OF VARIANCE FOR LEAF METHANOL FRACTION Variate: Zone_of_inhibition_mm

Source of variation d.f. s.s. m.s. v.r. F pr. LMF 4 98.7273 24.6818 70.63 <.001 Test_organisms 10 1191.3636 119.1364 340.92 <.001 LMF.Test_organisms 40 375.2727 9.3818 26.85 <.001 Residual 55 19.2200 0.3495 Total 109 1684.5836

LMF: Extract concentration

Grand mean 1.682

Table of Means for Test_organisms

B. cereus Bacillus subtilis E. coli 0.000 0.000 0.000 Enterobacter Enterococcus sp Klebsiella

114

0.000 0.000 2.000 P. aeruginosa Proteus mirabilis S. aureus 6.000 10.500 0.000 Salmonella Shigella dysenteriae 0.000 0.000

Least significant differences of means (5% level) l.s.d. 0.5298

APPENDIX 16

ANALYSIS OF VARIANCE FOR ROOT BARK N-HEXANE FRACTION

Variate: Zone_of_inhibition_mm

Source of variation d.f. s.s. m.s. v.r. F pr. RBNHF 4 211.636 52.909 29.69 <.001 Test_organisms 10 6712.000 671.200 376.69 <.001 RBNHF.Test_organisms 40 404.364 10.109 5.67 <.001 Residual 55 98.000 1.782 Total 109 7426.000

RBNHF: Extract concentration

Grand mean 6.00

Table of Means for Test_organisms

B. cereus Bacillus subtilis E. coli 0.00 0.00 5.20 Enterobacter Enterococcus sp Klebsiella 0.00 0.00 18.20 P. aeruginosa Proteus mirabilis S. aureus 6.20 16.20 20.20 Salmonella Shigella dysenteriae 0.00 0.00

Least significant differences for overall inhibition of test organisms (5% level) l.s.d. 1.196

115

APPENDIX 17

ANALYSIS OF VARIANCE FOR STEM BARK N-HEXANE FRACTION

Variate: Zone_of_inhibition_mm

Source of variation d.f. s.s. m.s. v.r. F pr. SBNHF 4 424.9455 106.2364 127.02 <.001 Test_organisms 10 5575.5636 557.5564 666.64 <.001 SBNHF.Test_organisms 40 618.2545 15.4564 18.48 <.001 Residual 55 46.0000 0.8364 Total 109 6664.7636 SBNHF: Extract concentration Grand mean 5.418

Table of Means for Test_organisms

B. cereus Bacillus subtilis E. coli 0.000 0.000 3.600 Enterobacter Enterococcus sp Klebsiella 0.000 0.000 14.800 P. aeruginosa Proteus mirabilis S. aureus 6.400 16.400 18.400 Salmonella Shigella dysenteriae 0.0 0.000

Least significant differences for overall inhibition of test organisms (5% level) l.s.d. 0.8196

APPENDIX 18

ANALYSIS OF VARIANCE FOR LEAF N-HEXANE FRACTION

Variate: Zone_of_inhibition_mm

Source of variation d.f. s.s. m.s. v.r. F pr. LNHF 4 385.3091 96.3273 253.49 <.001 Test_organisms 10 1829.0182 182.9018 481.32 <.001 LNHF.Test_organisms 40 905.8909 22.6473 59.60 <.001 Residual 55 20.9000 0.3800 Total 109 3141.1182

LNHF: Extract concentration Grand mean 2.673

Table of Means for Test_organisms

B. cereus Bacillus subtilis E. coli 0.000 0.000 0.000 Enterobacter Enterococcus sp Klebsiella

116

0.000 0.000 6.800 P. aeruginosa Proteus mirabilis S. aureus 3.200 12.600 6.800 Salmonella Shigella dysenteriae 0.0 0.000

Least significant differences for overall inhibition of test organisms (5% level) l.s.d. 0.5525

APPENDIX 19

ANALYSIS OF VARIANCE FOR LEAF CHLOROFORM FRACTION

Variate: Zone_of_inhibition_mm

Source of variation d.f. s.s. m.s. v.r. F pr. LChE 4 424.1455 106.0364 191.21 <.001 Test_organisms 10 1428.4182 142.8418 257.58 <.001 LChE.Test_organisms 40 587.8545 14.6964 26.50 <.001 Residual 55 30.5000 0.5545 Total 109 2470.9182

LCHE: Extract concentration Grand mean 3.427

Table of Means for Test_organisms

B. cereus Bacillus subtilis E. coli 0.000 4.000 9.400 Enterobacter Enterococcus sp Klebsiella 0.000 0.000 5.400 P. aeruginosa Proteus mirabilis S. aureus 9.900 4.400 4.600 Salmonella Shigella dysenteriae 0.000 0.000

Least significant differences for overall inhibition of test organisms (5% level) l.s.d. 0.6674