An Investigation of Medicinal Plants Traditionally

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BIOLOGICAL ACTIVITIES OF MEDICINAL PLANTS TRADITIONALLY USED TO TREAT SEPTICAEMIA IN THE EASTERN CAPE, SOUTH AFRICA

ROBERT FRED CHINYAMA

Submitted in fulfilment of the requirements for the degree of

MAGISTER TECHNOLOGIAE BIOMEDICAL TECHNOLOGY

in the

Faculty of Health Science

at the

Nelson Mandela Metropolitan University

December 2009

SUPERVISOR: Dr N. SMITH

CO-SUPERVISOR: Mrs E. BAXTER

DECLARATION

I, the undersigned, hereby declare that the research work contained in this study is my own original work, and all the sources I have used or quoted have been indicated and acknowledged by means of complete references.

………………………………….

ABSTRACT

Over the past 25 years, there has been a resurgence of worldwide scientific research in the fields of ethnopharmacology. The Western world has acknowledged the continued use of traditional medicines by the majority of third world countries, and the need for novel drug development. Hence, much of the pharmaceutical research in recent years has focused on the ethnobotanical approach to drug discovery (Light et al., 2005).

In South Africa, as in most developing parts of the world, traditional herbal medicine still forms the backbone of rural healthcare. The government health services in South Africa provide only western medical care although the majority of the population consult traditional healers for some or all of their healthcare needs (McGaw et al., 2005).

Medicinal plants like Harpephyllum caffrum are used as blood purifiers or emetics (Watt and Breyer-Brandwijk, 1962), and also for treating acne and eczema. The antimicrobial activity of this plant can be used to treat septicaemia, which is ranked the sixth leading cause of death among neonates and the eighth leading cause of death for infants through the first year of life (Heron, 2007).

In this study, the plants investigated for antimicrobial activity were Harpephyllum caffrum, Hermannia cuneifolia, Chironia baccifera, Rhigozum obovatum, Felicia muricata and Pentzia incana. These plants were tested against ATTC (American Type Culture Collection) strains and microorganisms isolated from clinical isolates of patients suffering from septicaemia. The assay methods used included the agar diffusion method using the Mast multipoint inoculator, the microtitre dilution method were used to determine the minimum inhibitory concentration, thin layer chromatography fingerprints accompanied by bioautographic assay were used to detect the inhibition of bacterial growth by active compounds separated from plant extracts and the Ames test was required to assess the possibility of bacterial mutagenesis upon the exposure to plant extracts which can lead to carcinogenicity.

In agar diffusion method, extracts of Harpephyllum caffrum inhibited nine strains of Candida albicans, three species of Acinetobacter and four strains of E.faecalis. Extracts of Hermannia cuneifolia inhibited four strains of B.cereus and three strains of Staphylococcus aureus. Extracts of Chironia baccifera inhibited one strain of Acinetobacter and five strains of E.faecalis.

Extracts of plants Rhigozum obovatum, Felicia muricata, and Pentzia incana showed no antimicrobial activity.

In the microtitre dilution method used to determine the minimum inhibitory concentration (MIC), the results were different from the agar diffusion method. More activity was observed. Extracts of Harpephyllum caffrum inhibited three strains of E.coli, six strains of S.aureus, three species of Acinetobacter and one strain of Klebsiella pneumonia. Extracts of Hermannia cuneifolia inhibited four strains of B.cereus, three strains of S.aureus, two strains of K.oxytoca and one species of Acinetobacter. Extracts of Chironia baccifera inhibited three strains of S.aureus, one strain of MRSA, one species of Acinetobacter and one strain of S.haemolyticus. The MIC values ranged from 0.049 to 6.25mg/ml.

Using the thin layer chromatography fingerprints, bioautography showed the presence of various inhibitory chemical compounds. Methanol and acetone extracts of Harpephyllum caffrum, separated very well and showed various inhibition zones on exposure to Candida albicans, Enterococcus faecalis and Staphylococcus aureus. The different inhibition zones were recorded as Rf values ranging from 0.25 to 0.95. The zones indicate the different inhibiting chemical compounds present in the plant. Petroleum ether, ethyl acetate, chloroform and formic acid were the solvents used in the assay in the ratio 8:7:5:1, respectively.

In the Ames test (Maron and Ames, 1983) the methanol and acetone extracts of Harpephyllum caffrum and Hermannia cuneifolia were negative which means they were devoid of any mutagenic properties. Methanol extracts of Harpephyllum caffrum showed similar results in the Ames assay as reported by Verschaeve and Van Staden (2008).

Establishing the antimicrobial activity of these plants contribute to the systematic scientific investigation of indigenous South African medicinal plants.

iii

ACKNOWLEDGEMENTS

I, the author, would like to express my sincerest gratitude and appreciation to the following people for their contribution in one way or the other towards the completion of this study.

Dr N. Smith for her patient guidance, encouragement, support and assistance during the duration of this project.

Mrs E. Baxter for her loving guidance, encouragement, support and instructions throughout the course of this study. Furthermore, for sourcing medicinal plants every time they were needed.

Mrs L. Beyleveld and Mrs B. Jordan for the ordering of supplies and for their kindness and support during the study.

The National Research Foundation and Nelson Mandela Metropolitan University for the financial assistance.

Malawi Union of the Seventh-Day Adventist Church and Malamulo College of Health Sciences for their sponsorship and financial support during the period of study.

My wife Thandiwe and my children Josephine, Wilson and Yamiko for their unconditional love, perseverance and endurance during the time when I was away from them.

Finally, I thank the Almighty God, for the good health, wisdom to study and for enabling the above mentioned individuals to be so kind to me. God’s name be praised.

TABLE OF CONTENTS

DECLARATION ...... i ACKNOWLEDGEMENTS ...... iv TABLE OF CONTENTS ...... v LIST OF FIGURES ...... viii LIST OF TABLES ...... ix LIST OF ABBREVIATIONS ...... x

CHAPTER 1: INTRODUCTION ...... 1 1.1 Aim ...... 3 1.2 Objectives ...... 3

CHAPTER 2: LITERATURE REVIEW ...... 4 2.1 Introduction ...... 4 2.2 Septicaemia ...... 4 2.2.1 Bacteriology ...... 6 2.2.2 Size and shape of bacteria ...... 6 2.2.3 Mycology ...... 7 2.2.4 Prevention, diagnosis and treatment ...... 8 2.2.5 Antibiotic therapy ...... 9 2.2.6 Antibiotic resistance ...... 10 2.3 Bacteria and fungi selected for investigation ...... 11 2.4 Traditional Herbal Medicines ...... 11 2.5 Medicinal plants and their importance ...... 13 2.5.1 Economic importance of traditional medicine ...... 16 2.6 Medicinal plants under investigation ...... 19 2.6.1 Chironia baccifera ...... 20 2.6.1.1 Active ingredients ...... 21 2.6.1.2 Pharmacological effects ...... 21 2.6.2 Harpephyllum caffrum ...... 22 2.6.2.1 Active ingredients ...... 22 2.6.2.2 Pharmacological effects ...... 23

2.6.3 Hermannia cuneifolia ...... 23 2.6.3.1 Economic and cultural value ...... 24 2.6.4 Pentzia incana (Karoo bush) ...... 24 2.6.5 Rhigozum obovatum ...... 25 2.6.5.1 Description ...... 26 2.6.5.2 Distribution ...... 26 2.6.5.3 Derivation of name and historical aspects ...... 27 2.6.5.4 Uses and cultural aspects ...... 27 2.6.6 Felicia muricata ...... 27 2.7 Traditional healers’ beliefs and practices ...... 27 2.8 Poisonous plants...... 29 2.9 Mutagenic effect ...... 31 2.10 The concept of Western medicine versus Traditional medicine ...... 33 2.11 Methods of diagnosis and treatment of disease ...... 34 2.12 Methods of preparation and administration of traditional remedies ...... 35 2.13 The study area ...... 36

CHAPTER 3: RESEARCH METHODOLOGY ...... 37 3.1 Introduction ...... 37 3.2 Sampling technique ...... 37 3.3 Plant preparation, extraction and methods...... 38 3.3.1 Preparation ...... 38 3.3.2 Plant Extraction ...... 39 3.3.3 Assays ...... 41 3.3.3.1 Agar diffusion method ...... 43 3.3.3.2 Determination of minimum inhibitory concentration ...... 44 3.3.3.3 Thin Layer Chromatography (TLC) fingerprints ...... 45 3.3.3.3.1 Materials and Techniques ...... 45 3.3.3.4 The Ames test ...... 46

CHAPTER 4: RESULTS ...... 48 4.1 Introduction ...... 48

4.2 Agar diffusion method ...... 48 4.3 Minimum inhibitory concentration (MIC) ...... 67 4.4 Thin Layer Chromatography...... 72 4.4 The Ames test ...... 76

CHAPTER 5: DISCUSSION AND CONCLUSION ...... 78

REFERENCES ...... 86

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LIST OF FIGURES

Figure 2.1: Chironia baccifera ...... 21 Figure 2.2: Harpephyllum caffrum ...... 22 Figure 2.3: Hermannia cuneifolia ...... 23 Figure 2.4: Hermannia cuneifolia leaves and seeds ...... 24 Figure 2.5: Pentzia incana ...... 25 Figure 2.6: Rhigozum obovatum ...... 26 Figure 2.7: Felicia muricata ...... 27 Figure 3.1: Powdered leaves of Rhigozum obovatum ...... 39 Figure 3.2: Centrifuged extracts in the respective solvents ...... 40 Figure 3.3: Centrifuged extracts in evaporating beakers ...... 40 Figure 3.4: Methods used for antimicrobial investigation versus No. of Publications ...... 42 Figure 3.5: Mast Multipoint Inoculator ...... 43 Figure 3.6: Salmonella typhimurium reaction on API Kit ...... 47 Figure 4.1: Agar plate containing extracts of Pentzia incana showing slight inhibition of bacteria ...... 48 Figure 4.2: Agar plates containing extracts of P.incana, F.muricata & H.cuneifolia ...... 49 Figure 4.3: Agar plate containing methanol extracts of H.cuneifolia compared with the control plate ...... 50 Figure 4.4: Agar plate containing acetone extracts of H.cuneifolia compared with the control plate ...... 50 Figure 4.5: Agar plate containing aqueous extracts of H.cuneifolia compared with the control plate ...... 51 Figure 4.6: Microtitre plate showing colour intensity ...... 67 Figure 4.7: Microtitre plate showing MIC values ...... 67 Figure 4.8: Plant extracts spotted on TLC silica plates ...... 73 Figure 4.9: TLC silica plates spotted with extracts developing in air tight glass jar ...... 73 Figure 4.10: Developed TLC silica plates after evaporating and drying ...... 74 Figure 4.11: F1 to F6 are TLC silica plates after the bioautographic assay viewed under UV light ...... 75

viii

LIST OF TABLES

Table 2.1: Comparison of Gram-positive and Gram-negative cell walls ...... 7 Table 2.2: Western versus Traditional Diagnosis and Treatment ...... 34 Table 3.1: Microtitre plate layout with plant extracts ...... 44 Table 4.2: Summary of microorganisms inhibited by extracts of Hermannia cuneifolia ...... 54 Table 4.3: Antimicrobial results of Pentzia incana extracts ...... 55 Table 4.4: Antimicrobial results of Rhigozum obovatum extracts ...... 57 Table 4.5: Antimicrobial results of Felicia muricata extracts ...... 60 Table 4.6: Antimicrobial results of Harpephyllum caffrum extracts ...... 63 Table 4.7: Antimicrobial results of Chironia baccifera extracts ...... 65 Table 4.8: Mean MIC results of 39 selected microorganisms tested with extracts of H.caffrum . 68 Table 4.9: Mean MIC results of 39 selected microorganisms tested with extracts of C.baccifera ...... 69 Table 4.10: Mean MIC results of 39 selected microorganisms tested with extracts of H.cuneifolia ...... 71

Table 4.11: Rf values of major zones present in chromatograms of methanol and acetone Harpephyllum caffrum extracts...... 76 Table 4.12: Number of histidine+ revertants in Salmonella typhimurium strain TA100 ...... 76

LIST OF ABBREVIATIONS

A A - Acetone A.baumanii – Acinetobacter baumanii A.haemolyticus – Acinetobacter haemolyticus AIDS - Acquired Immunodeficiency Syndrome A.lwoffi – Acinetobacter lwoffii APC – Activated Protein C (drotrecogin alfa activated) API – Analytical Profile Index AST – Antimicrobial Susceptibility Test ATCC – American Type Culture Collections

B B.cereus - Bacillus cereus

C C.albicans – Candida albicans C.lusitaniae – Candida lusitaniae C.krusei – Candida krusei CNS – Coagulase Negative Staphylococcus CSF – Cerebral Spinal Fluid C.baccifera - Chironia baccifera CPFA – Coupled Plasma Filtration Adsorption CRRT – Continous Renal Replacement Therapy

D DMSO – Dimethyl Sulphoxide

E Ed. - Editor ed. - Edition E.agglomerans – Enterococcus agglomerans

E.cloacae – Enterococcus cloacae E.faecalis - Enterococcus faecalis E.coli – Escherichia coli

F F.muricata – Felicia muricata FTIR - Fourier transform-infrared

G g - Grams

H H.caffrum – Harpephyllum caffrum H.cuneifolia – Hermannia cuneifolia HIV – Human Immunodeficiency Virus HPF – High Power Field HPLC - High Performance Liquid Chromatograph

I INT – р-iodonitrotetrazolium violet

K K.oxytoca – Klebsiella oxytoca K.pneumoniae – Klebsiella pneumoniae

L L.pseudomesenteroides – Leuconostoc pseudomesenteroides

M M – Methanol MDR – Multi-drug Resistance MH – Mueller-Hinton MIC – Minimum Inhibitory Concentration MOF – Multiple Organ Failure MRSA – Methicillin Resistant Staphylococcus aureus

MRSE – Methicillin Resistant Staphylococcus epidermidis

N nm - Nanometer NMMU - Nelson Mandela Metropolitan University

P PAS – Physiologically Active Substances PBS - Phosphate buffered saline P.incana – Pentzia incana P.stuartii – Providentia stuartii P.orizihabitans – Pseudomonas orizihabitans P.stutzeri – Pseudomonas stutzeri P.aeruginosa – Pseudomonas aeruginosa

R Rƒ - Distance of travel of the zone divided by the distance of the mobile phase front. rpm - Revolution per minute R.obovatum – Rhigozum obovatum

S Salm – Salmonella SATMERG – South African Traditional Medicines Research Group SIRS – Systemic Inflammatory Response Syndrome S.aureus – Staphylococcus aureus S.haemolyticus – Staphylococcus haemolyticus S.hominis - Staphylococcus hominis S.group b – Beta-hemolytic Streptococcus group b S.bovis – Streptococcus bovis

T TLC – Thin Layer Chromatography TCM - Traditional Chinese Medicine xii

TM – Traditional Medicine

U UA – Urine analysis UV – Ultra-violet light

W W – Water WBC – White Blood Cells WHO – World Health Organization

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CHAPTER 1: INTRODUCTION

The amount of medicinal plants and herbal medicinal products used worldwide has risen dramatically in the last decades (Reich and Schibli, 2006). Southern Africa has over 30,000 species of higher plants, mostly endemic, of which about 3,000 are used in traditional medicine (Van Wyk and Gericke, 2000). The use of plants in herbal medicine is an age old practice and is still prevalent all over the world, while the dependence on plants as the source of medicine is still very common in developing countries, where traditional medicine plays a major role in health care delivery (Adhikarla, 1984; Farnsworth, 1984a).

In most developing countries, where coverage by health services is limited, it is to the traditional practitioner or to folk medicine that the majority of the population turns when sick. The treatment they receive is largely based on the use of medicinal plants. Early in this century, the greater part of medical therapy in the industrialized countries was dependent on medicinal plants but, with the growth of the pharmaceutical industry, their use fell out of favour. Even so, 25% of all prescriptions dispensed between 1959 and 1980 from community pharmacies in the United States contained plant extracts or active principles prepared from higher plants (Farnsworth, 1984b). Now the pendulum is swinging back and the value of medicinal plants in treatment is receiving increasing attention worldwide.

According to data from the World Health Organization [WHO] (2006), plants are sources of biologically active compounds, used by about 80% of the world population, both in natural form as teas, and as manufactured drugs. In wealthy countries, growing numbers of patients rely on alternative medicine for preventive or palliative care. In France, 75% of the population has used complementary medicine at least once; in Germany, 77% of pain clinics provide acupuncture; and in the United Kingdom, expenditure on complementary or alternative medicine stands at US$ 2,300 million per year (WHO, 2009). Traditional medicine is an integral part of the South African cultural life, a position that is unlikely to change to any significant degree in the coming years. For instance, it is estimated that between 12 and 15 million South Africans still depend on traditional herbal medicines from as many as 700 indigenous plant species (Brandt et al., 1995; Meyer et al., 1996).

There has been an increasing incidence of microbial infections in recent years, largely due to the increase in AIDS-related opportunistic fungal pathogens and the emergence of resistance 1 microbial species (Silva et al., 2001; Afolayan et al., 2002). Bacterial infections are prevalent in developing countries due to factors such as inadequate sanitation, poor hygiene and overcrowded living conditions (Rasoanaivo and Ratsimamanga-Urveg, 1993).

Bloodstream infections cause substantial morbidity and mortality. Increasing rates of antimicrobial resistance, changing patterns of antimicrobial usage and the wide application of new medical technologies may change the epidemiology and outcome of bloodstream infections. According to the Journal of Clinical Microbiology, 44% of the total number of deaths as a percentage of septic episodes due to infection is caused by Candida species, 41% by anaerobes, 36% by Pseudomonas aeruginosa, 29% by Enterococci, 28% by Coagulase Negative Staphylococci, 23% by Staphylococcus aureus, 20% by Escherichia coli and Streptococcus viridans, 16% by Klebsiella species, 14% by Enterobacter species and 7% by Streptococcus pneumoniae (Diekema et al., 2003).

Sepsis remains the major cause of mortality worldwide, claiming millions of lives each year. The past decade has seen major advances in the understanding of the biological mechanisms involved in this complex process. Unfortunately, no definitive therapy yet exists that can successfully treat sepsis and its complications (Tetta et al., 2003b). Despite vast resources spent on sepsis research, the condition remains complex and poorly understood, and to this day treatment consists essentially of critical organ support. Multiple therapies have failed in the past (Tetta et al., 2003a).

It is believed that most of the plants used by Africans in the Eastern Cape in the preparation of herbal remedies possess pharmacologically active compounds (Coetzee, 2000). For example, in a study by Fourie et al., (1992) at least 31% of 300 plants screened showed marked activity, 21% were considered inactive, and 48% moderately active. On the other hand, Brown (1969) was of the opinion that plant remedies should be regarded with scepticism because the efficacy of many had not been proven.

Despite the availability of different approaches for the discovery of therapeutic agents, natural products still remain as one of the best reservoirs of new structural types (Hostettmann, 1999). To this effect, in the constant effort to improve the efficacy and ethics of modern medicinal practice, researchers are increasingly turning their attention to folk medicine as a source of new drugs (Wayne, 1998; Hoareau and Dasilva, 1999).

1.1 Aim

The purpose of this study is to investigate the biological activities of medicinal plants. Challenges in biological screening remain a key focus in drug discovery from medicinal plants (Balunas and Kinghorn, 2005).

1.2 Objectives

• To determine the antimicrobial activity of the plant extracts. • To determine the minimum inhibitory concentration (MIC) of the plant extracts. • To analyze the thin layer chromatography fingerprints found in the selected medicinal plants. • To evaluate the mutagenicity of the plant extracts.

CHAPTER 2: LITERATURE REVIEW

2.1 Introduction

The purpose of this chapter is to discuss: a) Septicaemia with regard to its definition, causes, severity and risk factors, classification, prevention, diagnosis and treatment. b) Traditional herbal medicine, its importance, its toxicity and mutagenic effect, how it is prepared and administered, as well as its comparison to western medicine. c) Medicinal plants under investigation, including botanical classifications and vernacular names, macroscopical morphology and geographical distributions. d) Traditional healers’ methods of diagnosis, beliefs and practices. e) The study area. Furthermore, a brief summary of the known major phytochemical constituents of each of the plant species under investigation is also given, as well as the ethnopharmacological application of these plants by various cultures, and previous scientific research conducted on the efficacy of some plants in treating the conditions for which they are used.

2.2 Septicaemia

Septicaemia is defined as a very serious condition with severe toxaemia and shock (Reid and Robert, 2005). It is also known as sepsis or blood poisoning. Sepsis is a systemic immune response that leads to multiple organ failure (MOF) (Wenzel, 2002). Severe sepsis and septic shock are the most common causes of MOF (Beal and Cerra, 1994). MOF remains the most frequent cause of death in patients admitted to the intensive care unit, with a mortality rate exceeding 50% (Baue et al., 1998; Wenzel, 2002). Sepsis occurs when the bloodstream becomes infected with bacteria. If the bacteria continue to multiply, the condition progresses to septic shock, blood pressure plummets and organ systems begin to shut down. Over 600,000 cases of septicaemia occur in the United States each year, and approximately two-thirds of these cases are diagnosed in hospitalized patients (Longe and Frey, 2006).

Sepsis is ranked as the sixth leading cause of death among neonates and the eighth leading cause of death for infants through the first year of life (Heron, 2007). The incidence of neonatal sepsis is 1 to 5 per 1,000 live births (Fanaroff and Martin, 2006). Although no significant sex difference

4 has been reported, it was noted as early as the 1960s that male infants had a higher incidence of neonatal sepsis than females, which may be related to X-linked immunoregulatory genes (Fanaroff and Martin, 2006). Other risk factors for neonatal sepsis include a history of immune deficiency disorders such as severe combined immunodeficiency syndrome and some inborn errors of metabolism such as galactosemia, which may present in the first week of life with Escherichia coli sepsis or urosepsis (Robinson et al., 2008).

Sepsis encompasses a complex mosaic of interconnected events that generates systemic inflammatory response syndrome (SIRS), sepsis syndrome, and septic shock (Tetta et al., 2003a). According to an observational study conducted by Dombrovskiy et al. (2006) entitled as “to determine the frequency of severe sepsis and drotrecogin alfa use in hospitalized patients in New Jersey”, patients with sepsis were identified using the following International Classification of Diseases, 9th Edition, Clinical Modification. The classifications were septicaemia, salmonella septicaemia, septicemic plague, anthrax septicaemia, meningococcal septicaemia, waterhouse- Friderichsen syndrome, herpetic septicaemia, gonococcal septicaemia, systemic candidiasis, and systemic inflammatory response syndrome due to infectious process without organ dysfunction. They defined severe sepsis as sepsis associated with organ dysfunction, as established by the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference (American College of Chest Physicians, 1992).

Septicaemia is common in Africa because of cultural practices like circumcisions. About 25% of the total world male population is circumcised and circumcision remains one of the oldest and commonest operations performed all over the world (Ben Chaim et al., 2005; Puig Sola et al., 2003; Wilkinson, 1997). The complications rates of the procedure range between 0.19% and 3.1% (Ben Chaim et al., 2005; Wiswell and Geschke, 1989; Manji, 2000; O’Brien et al., 1995). Neonatal circumcision as practiced is quite common worldwide. The people of pacific origin prefer their children to be circumcised between the ages of 6 and 10 years (Afsari et al., 2002).

Circumcisions undertaken in non-clinical settings can have significant risks of serious adverse events, including death. Among 50 patients admitted to hospital with post-circumcision complications in Nigeria and Kenya between 1981 and 1998, 80% had been circumcised by medically untrained traditional surgeons. One of these patients died from septicaemia (Magoha, 1999).

Among the Xhosa tribe in the Eastern Cape, South Africa, it is done in adolescence as an initiation rite to manhood (Mogotlane et al., 2004). Mayatula and Mavundla, (1997) found that an unsterilized unwashed blade was being used on a dozen or more initiates in a single session (Naude, 2002). Initiates were also significantly dehydrated during their 2 week period of seclusion in the belief that this reduces weeping of the wound, and after-care was in the hands of a traditional attendant with no basic medical training (Mayatula and Mavundla, 1997). The combination of dehydration and septicaemia could result in acute renal failure, gangrene, tetanus or even death (Mayatula and Mavundla, 1997; Naude, 2002). The Eastern Cape Provincial Department of Health recorded 2,262 hospital admissions, 115 deaths and 208 genital amputations involving circumcisions between 2001 and 2006 (Meissner and Buso, 2007). In a number of cases Helichrysum species were used to treat circumcision wounds. An investigation on Helichrysum aureonitens for antimicrobial activity and antimicrobial compound isolation yielded 3,5,7-trihydroxyflavone (Meyer and Afolayan, 1995; Meyer and Dilika, 1996; Afolayan and Meyer, 1997; Dilika et al., 1997), a compound with antimicrobial activity mainly against Gram-positive bacteria.

2.2.1 Bacteriology As a number of indications for the bacterial species relate to infection and infectious conditions, it is pertinent to include a brief review of bacteriology and mycology in an attempt to understand the antimicrobial actions of the plant extracts.

2.2.2 Size and shape of bacteria The majority of bacteria range from 0.20 to 2.0 micrometres (µm) in diameter and from 2 to 8µm in length. They are unicellular structures which may occur as cylindrical (rod shaped), spherical (coccoid) or spiral forms (Talaro and Talaro, 1996; Tortora et al., 1994; Hugo, 1992). Coccoid cells have a tendency to grow in aggregates. Other bacteria exist in assemblies of pairs (diplococci), groups of four (sarcinae), unorganized arrays like a bunch of grapes (staphylococci) and in chains like string of beads (streptococci). Rod shaped bacteria occasionally occurs in chains while spiral shaped bacteria normally occur singly (Talaro and Talaro, 1996; Tortora et al., 1994).

Bacteria can be divided into two main groups according to the characteristics of their cell wall. Table 2.1 below shows the comparison of Gram-positive and Gram-negative cell walls. 6

Table 2.1: Comparison of Gram-positive and Gram-negative cell walls

Characteristic Gram-positive Gram-negative Number of major layers 1 2 Chemical make-up Peptidoglycan Lipopolysaccharide Teichoic Acid Lipoprotein Lipoteichoic Acid Peptidoglycan Overall Thickness Thicker (20-80 nanometres) Thinner (8-11 nanometres) Outer Membrane No Yes Periplasmic Space Absent Present in all Porin Proteins No Yes Permeability to Molecules More permeable Less permeable (Taken from Talaro and Talaro, 1996)

2.2.3 Mycology Mycology is the study of fungi; their genetic and biochemical properties, their taxonomy, their use to humans as a source of medicines like penicillin, food as well as their dangers such as poisoning and infection. Pathogenic fungi have the ability to actively attack and invade tissues (Bauman, 2007; Hawksworth, 1974). Fungi are ubiquitous in the environment, and infection due to fungal pathogens has become more frequent (Walsh and Groll, 1999; Fleming et al., 2002). Although, fungal-related disease may not be as common as other bacterial infections, when they are present, they could be difficult to eradicate, especially in immunosuppressive situations (Bryce, 1992). Mycotic infections have been observed to be the primary cause of mortality in patients with severely impaired immune mechanisms (Kelbergh, 1997).

Research indicated that all over the world, 58-81% of all patients contract a fungal infection at some time during the primordial stage or after developing AIDS and 10-20% have died as a direct consequence of fungal infections (Drouhent and Dupont, 1989). Opportunistic fungal pathogens have become a common cause of morbidity and mortality with the rise in HIV (Garbino et al., 2001). In HIV patients, the presence of oral candidiasis is the earliest opportunistic infection (Fan-Havard et al., 1991). Clinically, the fungal infection is identified as creamy-white, curd-like patches on the tongue or other oral mucosal surfaces which are removed by scrapings. If left untreated, this leads to difficulty in chewing and swallowing and is

sometimes associated with severe diarrhoea (Drouhent and Dupont, 1989; Dube and Mutloane, 2001). Hence, those who suffer from oral candidiasis often lose substantial weight because of a sore throat, which prevents them from eating (Sanne, 2001). Candida albicans was included in this study because it is a pathogenic microorganism causing oral thrush particularly in immunocompromised individuals (Samaranayake, 2000).

Furthermore, since the first human kidney was transplanted in 1954, the frequency of organ transplantation has increased and the number of immuno-compromised hosts has grown steadily with the introduction of novel immuno-suppressive agents and improved rates of survival. Thus the incidence of opportunistic infections, including fungal infections, has also increased (Rubin et al., 1981; Winston et al., 1995; Fishman and Rubin, 1998). As a result, antifungal therapy is playing a greater role in health care and the screening of traditional plants in search of novel antifungals is now more frequently performed (McCutcheon et al., 1994; Jones et al., 2000; Motsei et al., 2003).

The search for novel antifungal agents relies mainly on ethnobotanical information and ethnopharmacologic exploration. The medicinal knowledge of North American First Nations peoples has been shown to be a valid resource. Studies have revealed a high degree of correlation between traditional medicinal uses and laboratory analysis (McCutcheon et al., 1994; Bergeron et al., 1996; Jones et al., 2000). The traditional preparations of these medicines often involve a broth or tea. However, much of the antifungal research conducted to date has assessed ethanol or methanol extracts while few studies have utilized aqueous extracts, a closer approximation of the traditional medicine.

Alcohol extracts provide a more complete extraction, including less polar compounds, and many of these extracts have been found to possess antifungal properties. Yet success with aqueous extracts has also been observed (Ali-Shtayeh and Abu Ghdeib, 1999).

2.2.4 Prevention, diagnosis and treatment

There is no specific laboratory test for early diagnosis of septicaemia (Longe and Frey, 2006). Nevertheless, in an effort to diagnose sepsis, the following laboratory tests are performed: a complete blood cell count (CBC), blood culture, urinalysis (UA), urine culture, and cerebrospinal fluid (CSF). The tests are performed on all patients presenting with possible sepsis (American

College of Emergency Physicians, 2003). In neonates, 1-2 ml of blood is acceptable for a blood culture. The urine culture should be obtained via sterile urethral catheterization or suprapubic aspiration in infants, and using an aseptic technique in adults. A urine analysis showing 10 or greater white blood cells (WBCs)/high-power field (HPF) and/or bacteria on Gram stain suggest a urinary tract infection. Accepted normal range values for preterm and term neonates respectively are 0-25, 0-22 cells/mm3 (WBCs); 65-150, 20-170 mg/dl (protein); 24-63, 34-119 mg/dl (glucose) (McMillan et al., 1999).

Africans in the Eastern Cape have a long tradition of using plants in treating both human (Hutchings, 1989a) and animal (Masika, et al., 1997) ailments. However, the effectiveness of the various herbal remedies remains controversial. Historically, cases where herbal remedies were effective in treating livestock diseases in the Eastern Cape have been reported. Smith wrote in the late 19th century that, whenever herds sickened and died, the herbal preparations administered by African herdsmen produced results far in advance of European remedies of that time. A documented case involved the treatment of a horse with a cancer-like growth by the latex of a plant Euphorbia. Samples that were sent to Germany revealed it to be a new species of Asclepiad which was later used in the treatment of cancer patients (Johannes, 1915).

It is said that treatment of bacterial infections is a frequent problem due to the emergence of bacterial strains resistant to numerous antibiotics (Keasah et al., 1998; Marimoto and Fujimoto, 1999). Only recently, activated protein C (APC) (drotrecogin alfa [activated]) has been the first biologic agent approved in the United States for the treatment of severe sepsis though with controversies (Tetta et al., 2003a).

Infectious diseases are usually characterised by clear symptoms, so it is likely that traditional healers have been able to recognise such diseases and have developed effective therapies. Moreover, as antibiotics mostly have clear effects, the chance of finding antimicrobially active traditional medicine is considered to be high (Sofowora, 1984; Elmi et al., 1986).

2.2.5 Antibiotic therapy Natural products from microorganisms have been the primary source of antibiotics, but with the increasing acceptance of herbal medicine as an alternative form of health care, the screening of medicinal plants for active compounds has become very important because these may serve as promising sources of novel antibiotic prototypes (Meurer-Grimes et al., 1996; Rabe and Van 9

Staden, 1997; Koduru et al., 2006). In older studies, the green parts of the plant, Harpephyllum caffrum, have given negative tests for the presence of antibiotics (Osborn, 1943). Yet, with improved methods it was found to have antimicrobial properties against microorganisms like Candida albicans.

2.2.6 Antibiotic resistance Bacteria have evolved numerous defences against antimicrobial agents, and drug-resistant pathogens are on the rise. This resistance is conferred by multi-drug resistance pumps, membrane translocases that extrude structurally unrelated toxins from the cell. These protect microbial cells from both synthetic and natural antimicrobials (Stermitz et al., 2000).

The growing problem of resistance in pathogenic microorganisms against many of the antibiotics in routine use, combined with the existing problem of the adverse effects of many antibiotic treatments, has recently increased research on the antimicrobial activities of various extracts and compounds isolated from the plant species used in herbal medicine (Nostro et al., 2000; Kokoska et al., 2002).

Currently, the emergence of resistant pathogens to many of the commonly used antibiotics has provided an impetus for further attempts to search for new antimicrobial agents to combat infections and overcome the problems of resistance to currently available antimicrobial agents (Balandrin et al., 1985; Xu and Lee, 2001). The use of plant extracts and phytochemicals can be of great significance in therapeutic treatments and could help curb the problem of these multidrug resistant organisms. In a study done with Pseudomonas aeruginosa, which is resistant to different antibiotics, its growth was inhibited by extracts from clove, jambolan, pomegranate and thyme (Nascimento et al., 2000).

The pattern of antimicrobial resistance varies widely. It is highest in countries where effective therapy is either unavailable or too expensive and facilities for diagnosis are inadequate. The pattern of antimicrobial resistance is often associated with a high prevalence of HIV infection (Ison et al., 1998).

2.3 Bacteria and fungi selected for investigation

Gram-positive and Gram-negative bacterial strains were included in this study together with some fungi. The bacterial strains include specific ATCC strains bought from Mast QC Sticks but supplied by Davis Diagnostics, and clinical isolates obtained from National Health Laboratory Service, Port Elizabeth, South Africa.

2.4 Traditional Herbal Medicines

Traditional medicine is the sum total of knowledge, skills and practices based on the theories, beliefs and experiences indigenous to different cultures. It is used to maintain health, as well as to prevent, diagnose, improve or treat physical and mental illness (WHO, 2008). The art of healing with herbal remedies is empirical, and it is usually transferred directly by oral teaching from master (father) to apprentice (son). Written documentation of the principles and practices of herbal therapy has been sporadic at best. In some areas, documenting the art and practice of herbalists is urgent because the knowledge is threatened with extinction (Masika et al., 2000).

In the World Health Report of 1995, ‘Bridging the gap’, the Director General of the WHO stated that ‘poverty is the world’s deadliest disease’ (Anon, 1995). Due to this, some 80 per cent of the world’s inhabitants rely mainly on traditional medicines for their primary health-care needs and utilize medicinal plants (Akerele, 1993). Reliance of human communities on plant-based remedies has a long-standing history and remains a vibrant culture interfacing with modern healthcare (Makunga et al., 2008). A list of 21,000 species of medicinal plants used world wide has been prepared (Penso, 1983), but others believe that this is a conservative estimate with the number of species used being between 35,000 to 70,000 (Farnsworth and Soejarto, 1991). Dr X. Zhang, a Medical Officer of Traditional Medicine for WHO, has stated that the majority of the population in developing countries make use of their local medicinal plants because of the lack of medical doctors, pharmaceutical products and money (Zhang, 1996).

Today, most of the population in urban South Africa, as well as smaller rural communities, is reliant on herbal medicines for their health care needs. Apart from their cultural significance, this is because herbal medicines are generally more accessible and affordable (Mander, 1998), easily available and cheaper than modern medicine (Otshudi et al., 2000). As a consequence, there is an increasing trend, world wide, to integrate traditional medicine with primary health care. Dual

health systems and the regulation of medicines exist simultaneously for both complementary/traditional medicines and pharmaceuticals in most developing countries. According to WHO, 65 out of 193 of its member states have regulatory systems dealing with traditional medicines.

In countries such as Vietnam and China, the modern and traditional health systems are integrated at the level of medical education and practice (Bodeker, 2001). In India, there are more than 200,000 registered traditional medicine practitioners, the majority having received training in degree granting colleges of Ayurvedic or Unani medicine.

As a result of a telephone survey conducted in the USA, it was estimated that some 42.5 million visits were made to herbalists in 1990, and that 425 million visits would have been made to unconventional therapists contrasting with the 388 million actual visits made to primary healthcare physicians (Eisenberg et al., 1993). In South Africa there has been interest in integrating the different health systems, such as the 48-bed hospital in Kwa-Mhlanga founded by a traditional African healer that combines the different aspects of traditional African, western and other complementary healing practices (Helwig, 2000).

Although traditional remedies were regarded as of little relevance to modern drug discovery in earlier years, they are now regarded as an important source of potentially therapeutic drugs (Cox and Balick, 1994).

The global demand for natural, healthy living conditions during the last century led to a greater awareness of traditional medicine (Koo and Wright, 1999). From an economic point of view, the high cost of imported conventional drugs and/or inaccessibility to western health care facilities implies that traditional mode of health care is the only form of health care that is affordable and available to the rural people. On the other hand, even when western health facilities are available, traditional medicine is viewed as an efficient and an acceptable system from a cultural perspective (Munguti, 1997) and as a result, traditional medicine usually exists side by side with western forms of health care (Sindiga, 1994). As First World countries became more aware of the positive correlations between traditional remedies and scientific proof of their healing effect, pharmaceutical formulations were developed on the basis of natural biochemical compounds. The identity of various bioactive substances were determined and synthesized by pharmaceutical companies for uses against a wide range of diseases (Walsh, 1998). 12

Recently, biologically active compounds and extracts isolated from such plant species used in herbal medicine have been the centre of interest (Nishino et al., 1987; Abdulla et al., 1989; Brantner et al., 1996; Anonymous, 1997). Numerous kinds of metabolites have been isolated from various plants and its chemical structures have been elucidated (Ghazal et al., 1992; Brantner et al., 1996).

2.5 Medicinal plants and their importance

Plants were once a primary source of all the medicines in the world and they still continue to provide mankind with new remedies. Natural products and their derivatives represent more than 50% and higher plants contribute no less than 25% to the total of all drugs in clinical use in the world (Kinghorn and Balandrin, 1993). There is a growing interest in natural and traditional medicines as a source of new commercial products (Van Wyk et al., 1997).

It is believed that there are about 27 million consumers of medicinal plants in South Africa (Mander, 1998). These medicinal plants form an important foundation in various ethnobotanical studies and phytochemical analysis in the different traditions worldwide. Medicinal plants and their derivatives contribute to more than fifty percent of all drugs used worldwide (Van Wyk et al., 1997; Kong et al., 2003).

Despite well-documented ethnobotanical literature, very little scientific information (e.g. efficacy, phytochemistry) has become available on indigenous medicinally used plants. It is only recently (1997-2008) that a number of findings have emerged on the chemistry and biological activity of plants used in traditional healing. This recent emergence in the scientific validation of South African medicinal plants can possibly be attributed to public awareness, method advancements and a number of citations in local books confirming the need for such studies (Hutchings et al., 1996; Van Wyk et al., 1997). Available reports tend to show that indigenous folk-medicinal plant preservation and study is vital because such plants are fully adapted to local environments and to conditions compared to introduced species (Qin and Xu, 1998). Pharmacologically active compounds and phytochemicals isolated from such endemic and indigenous plants used in folk medicine have been the center of interest during the past few decades (Farnsworth, 1994; Benzi and Ceci, 1997).

For some of the old medicinal plant extracts, purification and isolation of a single active ingredient has enabled standardized doses to be given in tablet or capsule form. From the view point of the patient, it matters little as to whether or not the capsule or tablet contains a synthetic or a natural medicinal drug. Attempts to synthesize these active compounds have largely been successful, but in most cases this has proved to be uneconomic in comparison to isolation from plant material. Thus today, drugs such as morphine (the potent pain-killer from the opium poppy) and digoxin (for treatment of congestive heart failure, from foxglove) are isolated from their plant sources. Despite considerable efforts to produce synthetic analogues with safer profiles for use in humans, both of these drugs continue to play prominent roles in medicine. It is claimed that natural products isolated from higher plants (including their derivatives and analogues) account for 25 per cent of the number of drugs in clinical use today (Balandrin et al., 1993).

Over the years, various alternative uses for plants were discovered and developed, such as plant derivatives used in the formulation of health and functional foods (Coghlan, 1996; Mozersky, 1999; Mukhtar and Ahmad, 2000). Having access mostly to their immediate surroundings, the natural environment provided indigenous people with remedies for the relief of pain and disease. A broad range of medicinal plants is used against microbial related illnesses, such as infections, diarrhoea or intestinal problems, asthma, psoriasis, articular rheumatism and skin problems (Hutchings et al., 1996; McGaw et al., 2000).

The regulation of traditional medicinal plant use embodies three fundamental aspects: quality, safety and efficacy. Unfortunately, comprehensive safety and efficacy data on traditional medicines are lacking (Springfield et al., 2005). The shortage of safety and quality controls of South African Medicinal plants is further compromised by the fact that there is currently no pharmacopoeia that documents indigenous medicinal plants of South Africa (Fennell et al., 2004).

In other countries, pharmacopoeias or medicinal manuals of useful available plants were compiled, later called medicinal plants (Van Wyk and Gericke, 2000; Lall, 2000). Today, these natural resources are still used. They make a significant contribution to primary health care especially in developing countries (Grierson and Afolayan, 1999), but also in the western world. Already an estimated 122 drugs from 94 plants species have been discovered through ethnobotanical leads (Fabricant and Farnsworth, 2001).

Of the 250,000 species of higher plants on earth, the majority have not been examined in any detailed way for their pharmacological properties. One of the largest investigations into potential new drugs from plants has been undertaken by the National Cancer Institute in the USA. Between 1960 and 1982, some 35,000 samples of higher plants were collected and provided 114,000 extracts which were screened for anticancer activity (Cragg et al., 1993). Recently, important new anticancer drugs such as taxol (a highly effective drug against breast and ovarian cancers, extracted from the bark of the Pacific yew [Taxus brevifolius]) and vincristine (a binary indole alkaloid used in breast and uterine cancer chemotherapy (Bruneton, 1995) extracted from Catharanthus roseus), have been developed (Van Wyk et al., 1997).

Aloe plants have been used in folk medicine for the treatment of various conditions including gastrointestinal disorders, insect bites, skin burns and other skin injuries. There are many products ranging from aloe gels, powders, capsules and creams that have recently appeared on the commercial market prepared from different aloe species (El Sohly et al., 2004). Products such as ‘Aloe 4 U’ commercially sold in South Africa consists of many different aloe species mixed together to reportedly give a stronger effect (Ndhlala et al., 2009).

Ndukwe et al. (2004) conducted a study investigating the antibacterial activity of chewing sticks, his study confirms that chewing sticks have a potential to prevent oral ailments. A majority of the plants tested in his study reveals that chewing sticks are capable of inhibiting Gram-positive and Gram-negative bacteria such as Bacillus subtilis, Porphyromonus gingivalis and Fusobacterium nucleatum. Several African tribes use the common traditional chewing sticks called ‘Muthala’, scientifically known as Diospyros lysioides DESF (Khan, 1978) and/or Euclea natalensis A.D.C. (Lall and Meyer, 2000).

Plant remedies are becoming popular in modern-day western countries, where natural products are now often preferred to synthetic chemicals (Elvin-Lewis, 2001). Not only the natives used plants for their healing properties, but settlers and farmers were also dependent on these resources, especially before the development of and access to pharmacies or commercial products (Moolman, 1984).

Natural remedies are known to be used predominantly by Latin American, Asian and African countries, which include India (Jain, 1994; Valsaraj et al., 1997; Srinivasan et al., 2001); Turkey (Sokmen et al., 1999); Israel (Alkofahi et al., 1997; Mahasneh and El-oqlah, 1999; Khafagi and 15

Dewedar, 2000); Mexico (Navarro et al., 1996; Rojas et al., 2001); Rwanda (Boily and Van Puyvelde, 1986; Sindambiwe et al., 1999) and South Africa (Cunningham, 1991; Grierson and Afolayan, 1999; Lin et al., 1999). Third world countries are often subject to shortage of funds, medical facilities, qualified personnel and newly developed medicine, which make them more dependent on their natural resources (Mammem and Cloete, 1996; Shale et al., 1999).

Sadly, consequences posed on plant biodiversity by commercial harvesters have become more evident, especially over the last century (Cunningham, 1991). Therefore, organizations initiated programs to conserve and manage endangered flora. More attention has been focused on biodiversity conservation and protection of natural products, especially in the northern hemisphere. Still, the greatest natural variety, and subsequently also the most rapid loss of biodiversity in the world, is currently found in Southern countries (Koo and Wright, 1999).

The high demand of medicinal plants in South Africa has led to over 10% of more than 20,000 plant species being threatened with a decreased availability and listed in the South African Red Data books (Goldring, 1996; George and Van Staden, 2000). The Durban municipality in Kwa Zulu-Natal has a medicinal plant nursery, Silverglen, which cultivates about 120 at risk plant species. Despite the fact that certain more conventional traditional healers believe that plants grown as agricultural crops will not have the same medicinal properties as those harvested from the wild (Cunningham, 1993), 82% of urban-based healers and 69% of the clinic patients in the Eastern Cape reported that they would make use of cultivated plants for medicinal purposes (Dold and Cocks, 2002).

2.5.1 Economic importance of traditional medicine

Herbal treatments are the most popular form of traditional medicine, and are highly lucrative in the international marketplace. Annual revenues in Western Europe reached US$5 billion in 2003-2004. Herbal medicine revenue in Brazil was US$160 million in 2007 (WHO, 2008). However, the use of herbal medicine is increasingly becoming mainstream with retail sales of herbal products in Australia estimated to be A$200 million (Wohlmuth et al., 2003).

The worldwide use of herbal medicinals has soared in the past decade reaching annual sales in excess of 60 billion U.S. dollars and is expected to reach 55 trillion U.S. dollars by 2050. Although most herbal remedies are consumed by adults, a growing proportion is consumed by

children of all ages. Two recent surveys report that up to 20% of children who are scheduled for elective surgery consume herbal medicine (Lerman, 2005). This has happened over the past years.

According to a survey by the International Trade Centre, trade in medicinal plants and their derivatives in pharmacy has declined in many industrialized countries, owing to the volume of competitive synthetic products currently marketed. Overall, the trade in botanicals has increased, through their use in the health food and cosmetic industries. Imports, however, represent only a minute percentage of the value of internal trade in medicinal plants. For example, in the United States, imports in 1980 were valued at US$ 44.6 million compared to an internal trade in medicinals and botanicals in 1981 estimated at US$ 3900 million. Over-the-counter sales of herbal medicines in the USA and Canada during 1990 reached US$860 million with an annual growth rate of 15% (Zhang, 1996). In Europe, the sales of herbal products have been referred to as ‘Europe’s growth market’ which amounted to US$1.4 billion during 1992 (Anon, 1992).

De Smet (2005) reported the market estimates of 2003. He stated that European countries spent almost $5 billion on over-the-counter herbal medicines. He continued saying that, all European countries have not accepted herbal treatments with equal interest. While Germany and France are leading on the over-the-counter sales, physicians in Great Britain prescribe herbal medicines, which are not covered by the National Health Service.

A survey of herbal products sold in UK pharmacies indicated that about 150 medicinal herbs are in common use (Newall et al., 1996). The majority of herbal products sold in the UK, whether from pharmacies, health-food shops, supermarkets or mail order outlets, are not licensed as medicines. Such products are controlled under food legislation and are referred to as food supplements, or dietary integrators or nutraceuticals.

In a report by Akerele and collegues, the annual production of traditional plant remedies in China was valued at US$ 571 million and the countrywide sales of crude plant drugs at US$ 1,400 million (Li Chaojin, 1987). Traditional medicines in China were reported to account for 30% - 50% of medicines consumed and the total sales of their herbal medicines amounted to US$2.5 billion in 1993. In addition, China exported medicinal herbs in 1993 with an estimated value of US$40 million (Bodeker, 1994). An article by WHO (2008), states that in China, sales of herbal products totalled to US$14 billion in 2005. 17

One Italian company (Indena of Milan) specializes in the preparation of medicinal plant extracts and utilizes some 12,000 metric tones of dried plant material annually. The company prepares some 1,139 plant extracts for medicinal use together with 202 pure compounds, which are isolated directly from plant material and used as such or as semi-synthetic derivatives. The main markets for these products are Europe, USA, Japan and South Korea. Among the most popular extracts used in Europe are garlic (Allium sativum, antimicrobial, blood cholesterol lowering), ginkgo (Ginkgo biloba, circulatory insufficiency), saw palmetto (Serenoa repens, diuretic, reduction of enlarged prostate), milk thistle (Silybum marianum, treatment of liver disorders) bilberry (Vaccinium myrtillus, inflammation of mucous membranes and diarrhoea), and grape seeds (Vitis vinifera, antioxidant and cardiovascular disease treatment) (Bombardelli, 1996).

Inappropriate methods of collection, processing and storage with undesirable contaminants in the products, have all contributed to the negative impact with regards to African natural plant products competing in international markets (Tadmor et al., 2002). The bulk of the medicinal plant trade takes place at informal street markets and involves the sale of unprocessed or semi- processed products. Raw plant material undergoes very little processing (e.g. grinding or boiling) before being administered to the patient (Mander and Le Breton, 2006).

A recent study on South African medicinal plants recommended for the treatment of HIV/AIDS revealed that many plants had high bacterial and fungal numbers due to low environmental sanitation and a low standard of processing during preparation (Govender et al., 2006). A study on African herbal teas showed samples containing a high microbial count, unacceptable in modern food and food supplement markets (Tadmor et al., 2002).

The global market for traditional therapies stands at US$ 60 billion a year and is steadily growing. In addition to the patient safety issue and the threat to knowledge and biodiversity, there is also the risk that further commercialization through unregulated use will make these therapies unaffordable to many who rely on them as their primary source of health care. For this reason, policies on the protection of indigenous or traditional knowledge are necessary (WHO, 2009).

2.6 Medicinal plants under investigation

Medicinal plants contain molecular structures and a built-in ability to produce substances (secondary metabolites) to protect itself from injury and disease. Plants are able to produce a wide array of secondary metabolites through intricate metabolic pathways. Lovkova et al. (2001), reports that the pharmacological activities of saponin-containing medicinal plants result in their marked therapeutic effects. Secondary metabolites synthesized by plants serve as defence mechanisms against predation by microorganisms (Cowan, 1999). These defence chemicals or secondary metabolites of plants can serve several types of functions and are found in abundance in plant species (Tsao et al., 2002). The ability of plants to manufacture secondary metabolites has been widely exploited by man. Plant biotechnology can make important contributions to the natural products sector even though, use of this technology in Africa is limited (Makunga et al., 2008).

Medicinal plants contain the ability to initiate the production or release of specific compounds. These substances are able to inhibit the growth of particular microorganisms and suppress their associated infections. The use of higher plants and preparations made from the medicinal plants to treat infections is an age-old practice in a large part of the world population, especially in developing countries, where there is dependence on traditional medicine for a variety of diseases (Ahmad et al., 1998). Bioassay-guided fractionation of plant species may lead to the discovery of new antibacterial agents and a better understanding of how ethnomedicine can treat infections. The larger part of the current research in ethnopharmacology is focused on understanding the molecular mechanisms whereby plants construct chemical defense systems (Trankina, 1998). Another key area that was relevant to this project was: Phytotherapy (healing using plants). The protocols and principles of these two disciplines were intertwined to determine the medicinal value of selected plant species as well as their possible implementation as scientifically validated health care alternatives in less advantaged communities.

Due to genetic, ecological and environmental differences, plants harvested from wild generally vary in quality and consistency of active compounds (Bopana and Saxena, 2007). Medicinal plant gatherers collect their materials throughout the year to supply the persistent demand for medicinal plants. If mature trees or plants cannot be found, then younger ones suffice, which results in availability of inconsistent plant material of the same species (Von Ahlefeldt et al.,

2003). Plant age, seasonal variation and geographical deviation in harvest site are contributing factors towards variation in biological activity (Taylor and Van Staden, 2001; Shale et al., 2005; Buwa and Van Staden, 2007).

Collections of Harpephyllum caffrum Bernh. (Anacardiaceae) bark from the same female tree at different times of the year showed the highest antibacterial activity in summer months (Buwa and Van Staden, 2007). Buwa and Van Staden, (2007) studied the effects of collection time on the antimicrobial activities of Harpephyllum caffrum bark. Three collections were made from a female tree in the Eastern Cape during 2003/2004 and tested for antibacterial and antifungal activity. The highest antibacterial activity was detected with plant material collected in June 2003 and December 2003, with a decline in activity in extracts collected in September 2004. Highest antifungal activity was detected in plant material collected in December 2003. TLC analysis revealed some variation in chemical composition of each of the H.caffrum bark extracts tested.

Plant-based antimicrobials and antibacterials represent a vast untapped source for medicines and hence have enormous therapeutic potential (Phillipson, 1994). They are effective in the treatment of infections while mitigating many of the side effects associated with synthetic antimicrobial and antibacterials (Mathews et al., 1999; Bagghi, 2000). Many medicinal herbs are rare and difficult to identify. To the untrained eye, especially when found in cut or chopped form, one plant may look just like another. One species can however be the source of a life-saving drug while a very similar plant may be a deadly poison (McKenna, 1996).

2.6.1 Chironia baccifera Chironia baccifera is a shrub with widespread distribution in the Western and Eastern Cape Provinces of South Africa (Springfield et al., 2005). It is also known as aambeibossie, bitterbossie (Afrikaans); Christmas berry, Wild gentian, Piles bush, Toothache berry (English). It belongs to the family of Gentianaceae. This is an important medicine in South Africa, traditionally used by the Khoi as a purgative for haemorrhoids and to treat boils. A decoction of the whole plant is taken as a blood purifier to treat acne and sores. An infusion may be used as a remedy for diarrhoea, or for leprosy (Van Wyk and Gericke, 2000). A tea or decoction made of the whole plant is drunk as a cleanser for skin problems and haemorrhoids {1/4 cup of fresh herb to 1 cup of boiling water, draws for 3 minutes, then strain}(Roberts, 1990). It has shown that 45g

20 of dried plant is fatal to a rabbit and half a pound fatal to a sheep. Confirmation of toxicity in the sheep and the rabbit discloses cyanosis of the mucosae, degenerative changes in heart muscle and liver, much blood in and oedema of the lungs and catarrhal gastroenteritis (Watt and Breyer- Brandwijk, 1962).

Figure 2.1: Chironia baccifera (Taken from http://www.plantzafrica.com/plantcd/chironbac.htm)

For labour, it is one of the most important Cape herbs. An infusion of fresh leaves and stems, and fruits if present, is very bitter and is taken post-partum to expel a retained placenta (Dawid Bester, pers. comm.). Furthermore, the plant is used to treat stomach ulcers, syphilis, kidney and bladder infections and diabetes. Side-effects that are known include a slightly loose stool, but never to the extent of causing diarrhoea (Dawid Bester, pers. comm.). The plant has been reported to be diaphoretic (Van Wyk and Gericke, 2000), cause sleepiness and increase perspiration (Roberts, 1990).

2.6.1.1 Active ingredients C.baccifera roots contain various secoiridoids, of which gentiopicroside is the main component, together with smaller amount of swertiamarine, chironioside and others (Wolfender, 1993). Other species of the family Gentianaceae, such as Gentiana lutea (yellow gentian) and C.krebsii are known to contain gentiopicroside (Dictionary of Natural Products, 1996) and chironioside (Wolfender, 1993), respectively. These are bitter substances, traditionally used in the liquor industry (Bruneton, 1995).

2.6.1.2 Pharmacological effects The bitter iridoids are known to stimulate appetite, but the compounds responsible for the healing properties of Chironia appear to be unknown (Van Wyk et al., 1997).

2.6.2 Harpephyllum caffrum This plant is also known as umgwenya (Xhosa, Zulu); mothekele (Northen Sotho); wild plum (English); wildepruim (Afrikaans). It belongs to the family of Anacardiaceae (mango family), which is the fourth largest tree family in Southern Africa. The natural distribution is restricted to Southern Africa (Palmer and Pitman, 1972). H.caffrum grows from the Eastern Cape northwards through KwaZulu-Natal, Swaziland, Southern Mozambique, Limpopo Province and into Zimbabwe. The generic name Harpephyllum is of Greek derivation, meaning sickle-like leaves, referring to the shape of the falcate leaflets. The specific name caffrum is derived from its place of origin, Kaffraria, now part of the Eastern Cape. This word means ‘indigenous’.

Figure 2.2: Harpephyllum caffrum (Taken from www.plantzafrica.com/planthij/harpephylcaf.htm).

The edible fruits are commonly used for eating and for making jams and jellies. They contain citric and malic acids (Watt and Breyer-Brandwijk, 1962). The tree has some potential as a commercial fruit crop but preliminary trial plantings in Israel were rather disappointing. The bark is a popular traditional medicine and cosmetic for facial saunas and skin washes (Van Wyk and Gericke, 2000). Williams (1930) claimed that the bark yields 18.2 per cent of tanning matter and the leaf and twig 11.4%.

It is also used to treat acne and eczema (Pujol, 1990) and decoctions of the bark are used as blood purifiers or emetics (Watt and Breyer-Brandwijk, 1962). According to Mr Mutwa (pers.com), the bark is primarily used to purify the blood in a person with “bad blood,” which manifests as pimples on the face.

2.6.2.1 Active ingredients The chemical constituents of Harpephyllum caffrum are poorly known (Dictionary of Natural products, 1996), but the presence of polyphenolic compounds and flavonoids have been reported

(El Sherbeiny and El Ansari, 1976). These include organic acids, such as protocatechuic acid, and flavonols, such as kaempferol.

2.6.2.2 Pharmacological effects The benefits derived from the use of this traditional medicine may be related to the phenolic compounds (El Sherbeiny and El Ansari, 1976).

2.6.3 Hermannia cuneifolia This is also known as agtdaegeneesbossie, broodbos, geneesbossie, pleisterbos, geelpleisterbos, vet-en-broodbos (Afrikaans) and belongs to the family, Sterculiaceae. It is a sturdy, much- branched shrublet, up to 500 mm high. Leaves occur in tufts and coarsely toothed, both surfaces are covered with star-like hairs. It has yellow flowers that become red-brown with age and is sweetly scented. The plant is very palatable and drought resistant. This plant is used medicinally to heal wounds, therefore the name, geneesbos. The Europeans apply an infusion or a decoction of this plant to sores and take the preparation internally (Watt and Breyer-Brandwijk, 1962). Distribution: widespread in fynbos, karoo and grassland, from the Western Cape, Free State, to Lesotho.

Figure 2.3: Hermannia cuneifolia (Taken from www.plantzafrica.com/planthij/hermannia.htm).

The genus was named after Prof. Paul Herman (1640-1695), a German professor of botany at Leyden and one of the first travelers and collectors at the Cape. Hermannias possess strong, thick, rootstocks and underground stems, which enable them to withstand fires in the highveld and overcome times of severe drought in arid areas. They are mostly very palatable to stock and small game, and are generally heavily grazed.

2.6.3.1 Economic and cultural value Many members of the genus are used medicinally, for anything ranging from respiratory diseases, coughs and internal aches, as stimulants or purgatives, to soothing wounds and cuts.

Figure 2.4: Hermannia cuneifolia leaves and seeds

The common name pleisterbos (Hermannia cuneifolia) refers to the use of the leaves as plasters. In some places the leaves are infused in a tea, and used to clean the blood. A root infusion was used by the early European colonial settlers against epilepsy. A lotion of the leaf was used for eczema and shingles. Certain species have magical significance and are used to drive out spirits and to wash the divining bones (www.plantzafrica.com/planthij/hermannia.htm).

2.6.4 Pentzia incana (Karoo bush) The plant belongs to the family Asteraceae. It is a small shrub in the sunflower family. Its yellow ball-like flowers appear during the winter. Near Middelburg C.P., an infusion of the leaf of Pentzia incana is commonly used by the Africans for the relief of stomach troubles (Kling, 1923).

Figure 2.5: Pentzia incana (Taken from www.fs.fed.us/.../weeds/iw_karoo_bush.shtml).

For erysipelas and for pain after eating, the woody parts of Pentzia incana and of Antizoma capensis are chewed and the saliva swallowed (Kling, 1923). It is said to be of value as pasture (Botha, 1939) and is reported to be much eaten by all types of domestic stock (Burtt, 1912; Marloth, 1913-32; Sim, 1907). Henrici (1952) has studied the starch and sugar content of the plant.

2.6.5 Rhigozum obovatum It is an ideal shrub or small tree to bring life to gardens in dry areas where water is in short supply and garden maintenance is low. Although the yellow pomegranate looks drab during a large part of the year, it turns into a spectacular showy sight after the first rains in spring or early summer when it is covered in a mass of bright yellow flowers. When rain is sporadic it may have several flushes of bloom.

Figure 2.6: Rhigozum obovatum (Pictured by Mrs E. Baxter)

2.6.5.1 Description This is a much-branched, spiny shrub or small tree 1.0 - 4.5 m high. The leaves are opposite or in a cluster, trifoliolate (compound leaf with three leaflets) or rarely simple, and they are borne on short, spine-tipped side shoots. The leaflets are obovate (egg-shaped and widest at the tips), 5-13 x 2-5 mm, greyish green, often finely notched, the margin is entire, rolled under, and the petiole is 2-6 mm long. The flowers are produced singly along the branches where the leaves emerge. They are showy, bright yellow, tubular or funnel-shaped with 5 lobes. Flowers are followed by fruits which are pendulous, flattened, pod-like capsules, up to 80 mm long, white brownish in colour, and split along the flat surface when mature to release buff-coloured seeds with papery wings.

2.6.5.2 Distribution This beautiful plant grows in the central to eastern Karoo, extending into south-eastern Lesotho and the Southern Free State. It often grows on rocky outcrops (koppies) in this summer rainfall, but semi-arid region. While winters are cold, the plant is frost tolerant. It can be grown on the highveld provided it is not over-watered and it will flower there and produce viable seed.

2.6.5.3 Derivation of name and historical aspects Rhigozum is derived from the Greek rhigios meaning stiff and ozos, a branch, a reference to the plant's rigid branches and obovatum is derived from the Latin word meaning egg-shaped with the widest point near the tip, referring to the leaf shape.

2.6.5.4 Uses and cultural aspects It is heavily browsed by game and stock.

2.6.6 Felicia muricata Felicia muricata belongs to the family Asteraceae. It is locally known as Ubosisi in the Eastern Cape of South Africa. This is a small drought resistant perenial herb growing up to 20cm in height. The leaves are simple and are arranged in alternate fashion. Its name was derived from muricate (rough, with sharp tubercles or protuberances). The species is regarded as an indicator of desertification, becoming increasingly invasive in grassland regions (Jordan and Kruger, 1993).

Figure 2.7: Felicia muricata (Taken from www.zimbabweflora.co.zw/speciesdata/species.php?)

F.muricata is used by the traditional healers in the management of headaches, stomach catarrh, pains and inflammation (Hutchings, 1989; Hutchings and Van Staden, 1994; McGaw et al., 1997). Extracts from the plant have been reported to show 80-90% inhibitory activity against cyclooxygenase, an important enzyme in the prostaglandin biosynthesis pathway (McGaw et al., 1997; Okoli and Akah, 2004).

2.7 Traditional healers’ beliefs and practices

Traditional healers in South Africa are most commonly known as “inyanga” and “isangoma”. These terms are used to refer exclusively to herbalist and diviner respectively, but in modern

times the distinction has become blurred, with some healers practising both arts. In addition to the herbalists and diviners who are believed to be spiritually empowered, there are traditional birth attendants, prophets, spiritual healers (Zulu: “abathandazi”), spirit mediums, intuitives and dreamers. Most elderly folk in rural areas have knowledge of herbal lore, and function as first- aid healers with a family repertoire of herbal remedies or “kruierate” (Van Wyk et al., 1997).

Van Wyk et al. (1997) suggest that there is the magical, ritual, spiritual and symbolic aspects of the use of indigenous medicinal plants which have not been much recorded. The informal oral- tradition medical systems of the Khoi-San people, the Nguni and the Sotho-speaking peoples have not yet been systematised, and are passed on by word of mouth from one generation to the next. These medical systems and their herbal, animal and mineral materia medica have ancient origins which may date back to palaeolithic times.

Traditional healers often unknowingly conserved their natural remedies by selective harvesting. The bark of some trees could only be removed at one side of the trunk to “ensure healing of the patient,” hereby preventing ring barking of the tree. Furthermore, bark that is used for treating kidney diseases are sometimes only harvested from the eastern and western sides of the tree, traditionally symbolizing the kidneys, thereby preventing ring-barking (Van Wyk et al., 1997). Similarly, when a piece of bulb was to be cut from a bulbous plant, the new hole in the soil had to be left open, in this way ensuring healing of the fresh wound in the plant tissue (Reyneke, 1971). In Gazankulu, a Shangaan taboo dictates that if the remaining root system of Elephantorrhiza elephantine is not covered after a portion has been removed, the patients treated with it will not get better.

Herbalists claim to use underground plant parts more frequently, believing that they contain the highest concentration of potent healing agents (Shale et al., 1999). These slow growing storage organs can normally be kept for longer periods, retaining their medicinal compounds for later use.

In general, natives rely on wild plants instead of cultivated varieties, and traditional healers normally prefer to work with freshly harvested material (Zschocke and Van Staden, 2000). More collections of medicinal plants are made in areas with higher altitude, also called the ‘highlands’ (Shale et al., 1999). Traditional healing practices are usually specific regarding the plant parts that should be used in disease treatment, as well as their appropriate usage. Almost any part of 28

the plant e.g. roots, bark, leaves, bulbs, rhizomes, tubers, stems, flowers, fruits, seeds, gums, exudates and nectar, can be used for medicinal purposes. The choice of usage may depend on the nature of disease, characteristics of certain plants and the recommended method of administration (Van Wyk et al., 1997). The most common methods of administering plant medicines are outlined as orally, sublingually, rectally, topically, nasally, smoking, steaming and bathing (Van Wyk et al., 1997).

It might be interesting to study the traditional combination of one plant part added to another substance. This combination is used to treat women of various ailments. Examples of the plants used are: Achyranthes aspera, Cassia auriculata, Dichrostachys cinerea and Sida acuta, all of which are mixed with onions, Blepharis madaraspatensis with limestone, Leucas aspera with garlic, and Phyllanthus amarus with goat’s milk. The reason as to why the plant parts are mixed with other substances and administered by local/indigenous people is unclear (Bhanumanthi et al., 1999). When Chironia baccifera is combined with Peucedanum galbanum (wildeselery), it makes a well-known Cape remedy for arthritis (Pvt. Daniels, pers. comm.). Chironia baccifera and C. palustris have been used for colic and diarrhoea in children. While under the name Pentzia virgata, Pentzia incana has been used in the Western Cape districts, with Tarchonanthus camphorates and possibly also with Pentzia globosa, as a poultice on the chest for the relief of bronchitis (Kling, 1923). A number of interesting outcomes have been found with the use of a mixture of natural products to treat diseases, most notably the synergistic effects and polypharmacological application of plant extracts (Gibbons, 2003). Scientific investigation should be conducted in this direction (Bhanumanthi et al., 1999).

2.8 Poisonous plants

The subject of the toxicity of medicinal plants is very topical and at the center of passionate debate. Because of this concern, many healers have been treated like poisoners or witches. Today, to protect its citizens, governments have prepared a list, sometimes arbitrary, of the plants they consider to be dangerous, prohibiting their cultivation, processing and sale. Although herbs appear to be harmless and beneficial to everybody, numerous toxic compounds were found in remedies such as deadly nightshade and jimson weed (Dantra). Medicinal plants collected from the wild may be contaminated by other species or plant parts through misidentification (WHO, 2003). Poisoning from traditional medicines is frequently a consequence of misidentification

(Stewart et al., 1998). Poisonings by traditional plant-based remedies in South Africa is not uncommon, particularly in children (Stewart et al., 1999; Steenkamp et al., 2002). Nonetheless, medicinal personnel tend not to ask about the use of traditional medicines administered to young children, especially not babies (Van Wyk and Els, 2008). A recent case study reported a single case of neonatal organo-phosphate-like poisoning, presumed to be caused by traditional medicine. The traditional healer was contacted and a sample of the traditional medicine administered to the child was sent for analysis. Unfortunately, the specimen was misplaced during transfer and no analysis could be performed (Van Wyk and Els, 2008).

Some medicinal herbs contain toxic compounds such as pyrrolizidine alkaloids (e.g. species of Crotalaria, Heliotropium, Senecio, Symphytum), which may cause liver toxicity and cancer or venocclusive disease. Other toxic compounds present in some herbal plants include lectins, toxins, lignans, saponins, diterpenes, and cyanogenetic glycosides. Contrary to popular belief, herbs may cause adverse reactions including allergic, cardiac, endocrine, and irritant effects. There is also the possibility that herbal treatment may interact with other medicines, for example if a herb lowers blood pressure or increases blood coagulation time or is a sedative, then it may interact with concurrent antihypertensive, anticoagulant or sedative therapy (Newall et al., 1996).

The deliberate addition of biologically active material to a plant preparation (known as adulteration) is a common method of malpractice used to enhance the efficacy of the products (Yee et al., 2005). There had been no reports of deliberate adulteration of traditional African herbal remedies until recently with reports of two separate incidences in which South African traditional remedies were adulterated with Western pharmaceuticals causing severe toxicity (Snyman et al., 2005).

It is certainly useful to clear the most dangerous plants and to avoid contact with them or to avoid using them, but it is important to remember that they merit our respect and the right to live. Indeed, they often contain highly concentrated active principles that are sometimes useful in small doses for an expert in pharmacognosy (Schneider, 2002).

More recently, mainly in the last century, the Western World, with its new inventions and technology, succeeded to distinguish between toxic or ineffective plant remedies, and those that proved to be pharmacologically useful (Lall, 2000). Results from these investigations provided significant scientific support for the traditional usage of plants, leading to an increasing trust in 30

the rich heritage of knowledge left behind by native folklore (Hutchings et al., 1996; Lin et al., 1999; Shale et al., 1999; Kelmanson et al., 2000).

The prescription and use of traditional medicine in South Africa is currently not regulated, with the result that there is always the danger of misadministration especially of toxic plants. The potential genotoxic effects that follow prolonged use of some of the more popular herbal remedies, are also cause for alarm (SATMERG, 1999).

Many South African medicinal plants are harvested from the wild. This not only threatens medicinal plant biodiversity and population stability but also leads to speculations with regard to safety as industrial encroachment has led to contamination of water sources and natural habitats. The deposition of processed and unprocessed mining and industrial waste materials (Naicker et al., 2003; Roychoudhury and Starke, 2006) have led to questions of safety for South African medicinal plants that are harvested near to these resources. According to Verster et al. (1992) large quantities of polluted water and tones of dry sewage sludge is being disposed on South African soils, much of this on agricultural land. Numerous reports have revealed heavy metal contamination of South African rivers and soils (Abbu et al., 2000; Binning and Baird, 2001; Okonkwo and Mothiba, 2005). As a result of polluted harvest sites or poor farming practices, medicinal plant products may be contaminated with pesticides, microbial contaminants, heavy metals, toxic substances and adulterants (Chan, 2003).

2.9 Mutagenic effect

Mutagenesis is observed as reversion to the histidine prototroph, i.e. mutated organisms are capable of growing in the absence of exogenous histidine (Maron and Ames, 1983; Wall et al., 1988). Salmonella typhimurium tester strains contain mutations in the histidine operon, thus making them dependent on histidine for growth. In addition, the tester strains contain mutations in certain repair systems, and this greatly increases their ability to detect mutagens (Maron and Ames, 1983). Strain TA98, which detects compounds causing various frameshift mutations, and strain TA100, which detects mutagens causing base-pair substitutions, are most often used (Maron and Ames, 1983; Wall et al., 1988).

There is a continuous search for new medical preparations against a great number of ailments, including cancer. This is because existing medication often has unwanted side effects or because

31 of loss of efficiency in the long run. Epidemiological studies indicate that many cancers are dependent on multiple mutational etiologies, as well as on inherited mutator phenotype. The search for inhibitors of mutagenesis may therefore be useful as a tool to discover anticarcinogenic agents. The approximately 25,000 plant species of South Africa form a potential pool for the screening of new anti-tumour compounds. Such screening may include the screening for inhibitors of mutagenesis. Screening traditional medicinal plants that have been used for ages against several illnesses, including cancer, and that proved to be efficient in curing people may be one way to narrow the search (Van Wyk et al., 1997).

Recently, there has been considerable interest in mutagenicity (Elgorashi et al., 2002-2003) and antimutagenicity (Verschaeve et al., 2004) of medicinal plants used by traditional herbalists. Previously, the possible mutagenic effects of 51 South African species frequently used in traditional medicine, were reported (Elgorashi et al., 2003). Frequently used plants in traditional medicine are assumed safe, due to their long-term use (Elgorashi et al., 2002) and are considered to have no side effects because they are “natural” (Popat et al., 2001). This concept is largely circumstantial and it is important to determine toxicology of plant extracts, especially those that are used frequently over long periods. As new natural compounds come on the market in drug form as industrial and agricultural inputs, their toxic or genotoxic action on man or on genetic material must increasingly be evaluated to prevent the use of substances that could interact and cause severe cellular and molecular organization disorders. Genetic assays used in these studies, especially the Salmonella/microsome test, allow faster screening of these mutagenic and probably carcinogenic substances at a lower cost (Mortelmans and Zeiger, 2000). The Ames test (Maron and Ames, 1983) was used in this study as it is widely used in the determination of possible gene mutations caused by plant extracts. A positive response in any single bacterial strain either with or without metabolic activation is sufficient to designate a substance as a mutagen (Zeiger, 2001).

It is important to note that most of the traditional medicinal plants have never been the subject of exhaustive toxicological tests such as is required for modern pharmaceutical compounds. Based on their traditional use for long periods of time they are often assumed to be safe. However, research has shown that a lot of plants which are used as food ingredients or in traditional medicine have in vitro mutagenic (Cardoso et al., 2006; Déciga-Campos et al., 2007) or toxic and carcinogenic (De Sá Ferreira and Ferrão Vargas, 1999) properties. 32

Plants sometimes synthesize toxic substances, which in nature act as a defence against infections, insects and herbivores, but also may affect the organisms that feed on them. An assessment of cytotoxic and mutagenic potential of extracts used in traditional medicine is necessary to ensure their safe usage (Cavalcanti et al., 2006).

2.10 The concept of Western medicine versus Traditional medicine

Herbs and plants were used as the very first medicinal therapies known to man. With time, the original inhabitants of an area gained knowledge as to which plants could be used for certain disease or states of illness. In addition, they also gained knowledge of which plants were hazardous or poisonous. The very first original pharmacy, in fact, was Mother Nature, with her expensive variety of medicinal plants (White and Foster, 2000). Knowledge of the plants used in the preparation of remedies was not in the public domain. Many treated it as a secret to be guarded closely (Masika et al., 2000).

Literature by Felhaber (1999) discusses Southern African traditional healers and their belief that healing rests on the assumption that if the mind is healed, the body takes care of itself. Whereas in Western medicine, the opposite is often considered true: If the body is healed, the mind takes care of itself. According to research by Hewson (1998), a growing number of articles show that physicians can improve their clinical effectiveness by adopting the traditional approach of embracing both physical and mental aspects of illness.

Felhaber (1999) also explains that the main method of alternative healing is to work WITH the body instead of working AGAINST the disease, as is the case with Western therapies. Rather than fighting against bacteria, an attempt is made to enhance the body’s own built-in defence mechanism.

Health is defined by the WHO as a complete state of physical and mental well-being, and not merely the absence of disease or infirmity. This definition is in line with the practice of traditional healers who look at the whole body (physical, mental, spiritual), whereas biomedicine heals only the affected parts of the body and is forever looking for germs (Kubukeli, 2000).

Western medicine may diagnose a disease in terms of a bacterial infection for example, and treat that infection effectively with antibiotics. An African traditional healer will seek to understand why the patient became ill in the first place, and the treatment administered will address the 33 perceived cause, usually in addition to specific therapies to alleviate the signs and symptoms of the condition (Van Wyk et al., 1997).

Although traditional and Western therapies are usually seen as opposing entities, they actually have quite a lot in common: an approximate amount of pharmaceutical products sprout directly from medicinal plants; these plants and pharmaceutical products, contain healing agents that can change body processes. That is the reason why these compounds are used as medicine in order to transform the body from a state of illness and disorder to a state of emotional and physical health (White and Foster, 2000).

Felhaber (1999) has formed the opinion that there are few areas for collaboration between traditional healers and modern medicine. This is an unfortunate situation, because traditional healers perform an important role in the delivery of health care, especially in rural areas where access to Western medical care is limited. In contrast, Taylor and Van Staden, (2001) explains that the evaluation and authentication of traditional medicine can contribute towards the formulation of an integrated health care system combining both traditional and Western practices.

2.11 Methods of diagnosis and treatment of disease

Table 2.2: Western versus Traditional Diagnosis and Treatment

Western Diagnosis Traditional Diagnosis Diagnosis of illness in a patient is based The diagnostic process investigates how upon: the disease originated (in relation to natural • The patient’s medical history; causes), and why the disease is manifesting • A physical examination of the patient or itself (the deeper meaning or motivation • Any special investigations or tests that behind the causes). are thought to be necessary by the Diagnosis is made after combining medical practitioner to assist in the information gathered from observing the successful treatment of the patient. patient, patient self-diagnosis and divination or spiritual guidance. Western Medicine Traditional Medicine Antibiotics are used to treat infectious Traditional treatment has elements of being

diseases. They must be taken for the entire curative, protective and preventive, and can length of time for which they have been either be natural or ritual, or both. prescribed. Stopping the drug before the Formulas are prepared from various natural prescribed time can cause bacteria that are substances (animal, mineral and vegetable) resistant to the drug to grow out of control, and can be applied in various ways which makes treating the illness even more according to the body site and severity of difficult the second time. the illness.

(Compiled from: Felhaber, 1999; Gould & Mackenzie, 2002; Maher, 1999)

In the context of Nguni people’s perception, illness is a condition of internal corruption (accumulation of toxins) and ‘uncleanliness’ (Hutchings, 1989b). As a result, many herbal medicines were found to seek the cleansing of the inner system of the patient by inducing purging and vomiting (Masika et al., 2000).

2.12 Methods of preparation and administration of traditional remedies

The methods of preparation are critical, as it includes the amount of fresh or dry plant material to be used, the addition of appropriate volumes of solvents such as water or alcohol, and additional activities such as boiling for a specified length of time, or partial burning to achieve a desired colour. The common forms and methods of preparation are enemas, extracts, infusions, decoctions or teas, inhalations, snuffs, linctuses, tinctures, lotions, liniments, mixtures, ointments and nasal drops. For internal use, the preparation is mostly prepared as a tincture, decoction or infusion. For external use, the powder or the juice of the plant can be directly applied on the wound or introduced in incisions (Van Wyk et al., 1997).

The most common methods of administering plant medicines are orally (mainly infusions, decoctions, syrups and tinctures, powdered herbs, partially burned herbs or the ash of burned herbs taken directly by mouth, usually followed by a mouthful of water), sublingually (under the tongue), rectally (infusions and decoctions are commonly administered as enemas), topically, nasally, smoking, steaming and bathing (Van Wyk et al., 1997). In administering Harpephyllum caffrum by traditional healers, it is first given as a facial steaming, then as a phalaza, and then orally as an imbiza daily. The bark is used fresh and can be collected in any season (Van Wyk and Gericke, 2000).

Modern pharmaceuticals are typically oral dosage forms containing single synthetic chemicals that have potent clinical activity. However, natural products from higher plants continue to be used in pharmaceutical preparations either pure or as extracts (Gogtay et al., 2002).

2.13 The study area

South Africa boasts with a unique and diverse botanical heritage with over 30,000 plant species of which 3,000 species are used therapeutically (Van Wyk et al., 1997). Not only is the South African flora rich in diversity but it is also mostly endemic (Mulholland, 2005). In addition to this unique botanical heritage, South Africa has a cultural diversity with traditional healing being integral to each ethnic group. African traditional medicine is the oldest medicinal system, often culturally referred to as the Cradle of Mankind (Gurib-Fakim, 2006).

The Eastern Cape Province in South Africa, falls within the latitudes 30o00'- 34o15'S and longitudes 22o45' – 30o15'E. It is bounded by the sea in the East and the drier Karoo (semi-desert vegetation) in the west. The elevation ranges from sea-level to approximately 2,200m in the north of the province (Grierson and Afolayan, 1999). To the north-west the province borders on KwaZulu-Natal and meets the southern tip of the Drakensberg range; further south, mountains and hills predominate, the northern section in the dry karoo being flatter. The long curve of coastline, large area (at nearly 170,000 square kilometers covering 13.9% of the country) and the considerable east-west and north-south distances it covers, and the vegetation is veld type number 7, known as the Eastern Cape thorn veld (Acocks, 1975). The coastal areas receive good summer rainfall and have a moderate climate, becoming more sub-tropical to the north-west.

According to a study by Hostettmann et al. (2001), plant species growing in a hostile environment (such as warm and humid conditions) will attempt to protect itself by synthesizing insecticidal, fungicidal, antibacterial or virucidal constituents, e.g. roots often synthesize antifungal substances as soil is rich in pathogenic fungi.

CHAPTER 3: RESEARCH METHODOLOGY

3.1 Introduction

The current chapter discusses the materials and methods used in the sampling of Harpephyllum caffrum, Chironia baccifera, Hermannia cuneifolia, Pentzia incana, Rhigozum obovatum and Felicia muricata plant materials, as well as performance of antimicrobial activity screening of the respective extracts of plants using four testing methods. The agar diffusion method using the Mast multipoint inoculator, the determination of minimum inhibitory concentration using microtitre plates, thin layer chromatography fingerprints used to detect the inhibition of bacterial growth by active compounds separated from plant extracts, and the Ames test required to assess the possibility of bacterial mutagenesis upon exposure to plant extracts which can lead to carcinogenicity, are also discussed.

3.2 Sampling technique

Renewed interest in traditional pharmacopoeias has meant that researchers are concerned not only with determining the scientific rationale for the plant’s usage, but also with the discovery of novel compounds of pharmaceutical value. Instead of relying on trial and error, as in random screening procedures, traditional knowledge helps scientists to target plants that may be medicinally useful (Cox and Balick, 1994). Although there are other approaches of selecting plant species for biological investigation, selecting species that are used in traditional medicine is reported to be more valuable and many drugs that are of plant origin were discovered from plants used in traditional medicine (Cotton, 1996). The investigated plants were selected on the basis of their reported ethnobotanical use in South African traditional medicine and on availability (Hutchings et al., 1996; Van Wyk et al., 1997). The materials were collected in the Eastern Cape Province during the summer months. In their work, Mitscher et al. (1972) found that extracts are generally richest in antibacterial agents after the flowering (sexual) stage of their growth is complete, and that plants taken from stressful environments were particularly active.

In this study the following plants were chosen, Harpephyllum caffrum, Chironia baccifera, Hermannia cuneifolia, Pentzia incana, Rhigozum obovatum and Felicia muricata, because of their traditional medicinal use as well as pasture for livestock. In this part of the world, people do not only use traditional medicine for their own welfare, but also for that of their animals. A

previous study has shown that almost 75% of resource-limited livestock owners use traditional medicine to treat animal ailments (Masika et al., 2000).

3.3 Plant preparation, extraction and methods.

3.3.1 Preparation

Herbalists use various methods to prepare herbal remedies. One method is preparation of infusions, where the plant parts are soaked in water, and left for some time to allow the active ingredients to infuse into the water. Another method involves the preparation of decoctions whereby the plant material is boiled in water. Other remedies are prepared in a powdered form by grinding dried or roasted plant material. Pastes are made by mashing fresh plant parts. Other medicines consisted of juices obtained by pressing the sap out of fresh leaves, stems, flowers and other plant parts (Masika et al., 2000).

Proper drying conditions are largely overlooked by the collectors, growers and traders (Ramakrishnappa, 2002). Inadequate drying may result in mould growth (Whitten, 1997), which can lead to deterioration of the plant product (Ramakrishnappa, 2002). Yeasts and moulds can cause opportunistic infections in humans and are more significant in HIV patients (Govender et al., 2006). Hence great care was taken during the drying process.

Plant materials were dried in an oven at 40oC and weighed. Dried material is preferred in cases where there is a delay between collection and processing. Unlike fresh material in which the interfering water content can pose problems in subsequent processing by liquid-liquid extraction, dry material has fewer problems with large-scale extraction (George et al., 2001). Mahanom et al. (1999) evaluated three different drying methods that are routinely used in the detection of physiologically active substances (PAS). These are: • Oven drying at 70oC for 5 hours, • Oven drying at 50oC for 9 hours and • Freeze drying The research concluded that drying treatments caused significant reduction in the levels of all phytochemicals analysed. Oven drying caused greater reduction compared to freeze-drying. Freeze-drying resulted in the lowest percentage losses of all the phytochemicals analysed, followed by oven drying at 70oC and oven drying at 50oC.

Harborne (1998) advises that the drying procedure should be carried out under controlled conditions to avoid too many chemical changes occurring. Plant materials should be dried as quickly as possible, without using high temperatures, preferably in a good air draft. Once thoroughly dried, plants can be stored for long periods of time.

Figure 3.1: Powdered leaves of Rhigozum obovatum

The dried plant parts were powdered using a pestle and mortar. Powdering of the plant material increases the surface area accessible to the solvents, which results in better extraction (Harborne, 1998; George et al., 2001).

3.3.2 Plant Extraction Extraction methods used pharmaceutically, involve separation of medicinally active portions of plant tissues from the inactive/inert components by using selective solvents with appropriate extraction technology. During extraction, solvents diffuse into the solid plant material and solubilise compounds with similar polarity (Green, 2004).

An extraction method adapted from Eloff (2000) was employed using approximately 2g of dried plant leaves, bark or stem for the six plants. Choosing a solvent for extraction depends on the aim of the screening. For instance antibacterial screening of plants requires no interferences by the solvent, in the bioassay procedure (George et al., 2001). A publication by Eloff (1998b) identified acetone as the most favourable solvent for plant extraction in antimicrobial studies. Plant extracts tested with methanol have proved valuable, as a high percentage of antibacterial activity is present (Rabe and Van Staden, 1997). The solvents chosen for this study were acetone, methanol because of low toxicity, ease of evaporation at low heat, promotion of rapid 39 physiologic absorption of the extract, preservative action and inability to cause the extract to complex or dissociate (Hughes, 2002) and sterile distilled water.

The extraction procedure was performed as follows with the dried plant material: • Weighed 2g of plant material and 30ml of each solvent, were placed in screw cap tubes, respectively and shaken vigorously for 5 min.

Figure 3.2: Centrifuged extracts in the respective solvents

• Centrifugation of different extracts in tubes at 4000rpm for 5 min using the Eppendorf centrifuge 5810 separated the supernatant from the plant material for each extract. • Supernatant of each plant extract was transferred into pre-weighed beakers. • Procedure was repeated twice more for re-extracting the remaining plant material with additions of 30ml solvent, respectively. • The pre-weighed beakers containing the plant extract supernatants were allowed to dry completely to obtain a solvent-free dried extract residue.

Figure 3.3: Centrifuged extracts in evaporating beakers 40

• Methanol and acetone plant extract supernatants were subjected to overnight drying under airflow in a fume cupboard. • Incomplete drying of the aqueous plant extract supernatants prompted alternative drying at 37oC overnight. • Dried beakers were re-weighed and calculated extract residues resuspended in Dimethyl Sulphoxide (DMSO). • Plant extract concentrations were standardized between the different plants and solvents used for extraction. According to Mathekaga and Meyer (1998), in vitro screening methods could provide the needed preliminary observations necessary to select crude plant extracts with potential useful properties for further chemical and pharmacological investigations.

3.3.3 Assays The antimicrobial susceptibility test (AST) is an essential technique in many disciplines of science. It is used in pathology to determine resistance of microbial strains to antimicrobials, and in ethnopharmacology research, it is used to determine the efficacy of novel antimicrobials against microorganisms, essentially those of medical importance. The test is the first step towards new anti-infective drug development. There are various AST methods that are employed by researchers and these could lead to variations in results obtained (Lampinen, 2005). Other factors impacting on methods have been studied which include storage of plant material and its effect on microbial efficacy (Eloff, 1999; Stafford et al., 2005).

Figure 3.4 below represents the proportion of studies and the methods used to assess antimicrobial activity mostly for the period 1997 – 2008. The two most common methods used to investigate the antimicrobial activity of South African medicinal plants are disc diffusion and minimum inhibitory concentration (MIC) assays. Initially disc diffusion studies were the first method of choice, possibly due to their simplicity and capacity to analyse a large number of test samples. Many earlier publications (65.0% before 2004) used this method as a means of determining activity (Van Vuuren, 2008).

Figure 3.4: Methods used for antimicrobial investigation versus No. of Publications

(Compiled by Van Vuuren, 2008)

Essential oils possess biological activity including antibacterial, antiviral, antifungal and anti- inflammatory effects. Oils from Cinnamomum osmophloeum have been shown to possess antibacterial activity against Escherichia coli, Enterococcus faecalis, Staphylococcus aureus (including methicillin resistant S.aureus) and Vibrio parahaemolyticus, with cinnamaldehyde being the main antibacterial component isolated. This compound has also been widely used in antiseptic mouthwashes because of its activity against oral bacteria (Wallace, 2004).

The bacteria and fungi selected for investigation in this study were isolated from patients diagnosed with septicaemia. The following ATCC strains and microorganisms from clinical isolates were used: Escherichia coli (ATCC 35218); Bacillus cereus (ATCC 10876); Staphylococcus aureus (ATCC 43300); Candida albicans (ATCC 66027); Pseudomonas aeruginosa (ATCC 27853); Enterococus faecalis (ATCC 29212); The clinical isolates included one strain of the following bacteria: Streptococcus bovis; Enterococcus faecalis; S. group b; Methicillin Resistant Staphylococcus epidermidis; Staphylococcus hominis; Staphylococcus Coagulase Negative; Providentia stuartii; Leuconostoc pseudomesenteroides; Enterobacter agglomerans; Acinetobacter baumanii; Acinetobacter haemolyticus; Acinetobacter lwoffii; Candida krusei; Candida lusitaniae; Staphylococcus haemolyticus and Aeromonas hydrophila; plus 7 strains of Methicillin Resistant Staphylococcus aureus (MRSA); 2 strains of Klebsiella pneumoniae; 5 strains of Klebsiella oxytoca; 5 strains of Salmonella species; 2 strains of Enterobacter cloacae and 7 strains of Candida albicans. Each organism together with the ATCC

strains were stored in cryotubes (microbank™) supplied by Davies Diagnostics and were subcultured onto specific culture media and incubated at 37oC overnight before testing.

3.3.3.1 Agar diffusion method Mueller-Hinton (MH) agar was prepared according to the manufacturer’s directions and autoclaved, allowed to cool to about 50oC before the addition of the extracts. The extracts were prepared by dissolving in a known amount of DMSO per plate (0.4% V/V). Each plant extract was carefully added into 25ml Mueller-Hinton agar to obtain the final agar plate concentration of 10mg/ml. These were poured into Petri dishes, swirled carefully until the agar begun to set and left overnight for the solvents to evaporate (Afolayan and Meyer, 1997). The ATCC strains and the clinical isolates were recovered for testing by growth in nutrient broth overnight at 37oC. Nutrient broth was subcultured onto solid agar. A standardized inoculum was prepared by suspending colonies in normal saline and adjusting the density to that of a 0.5 McFarland standard solution (McFarland, 1907) which is equivalent to 106-108 CFU/ml. The final volumes of 30μl prepared inoculums were transferred into each well of the sterile inoculum pots. Antimicrobial activity of the tested microorganisms was carried out by the use of Mast multipoint inoculator (Mast systems, London, UK) based on the principles of agar dilution (Andrews, 2001) and plates were incubated at 37oC for 24 to 48 hrs.

Figure 3.5: Mast Multipoint Inoculator

Agar plates containing 0.5ml of acetone, methanol or DMSO were used as controls respectively. Tests were performed in triplicate. Complete suppression of growth was required for an extract to be declared active.

3.3.3.2 Determination of minimum inhibitory concentration The most prominent South African publication (Eloff, 1998a), dedicated to antimicrobial methods, is the quick, sensitive microtitre plate minimum inhibitory concentration determination method, which has been extensively cited. Although the results of the agar diffusion assay cannot always compare to the MIC data (Njenga et al., 2005), three extracts of plants namely Harpephyllum caffrum, Hermannia cuneifolia and Chironia baccifera, which showed positive antimicrobial activity against some of the microorganisms tested in the agar diffusion bioassay were further tested for the determination of MIC. Dried plant extracts were dissolved at 50mg/ml with the extracting solvents. All extracts were initially tested at 25mg/ml in 96-well microtitre plates and serially diluted two-fold to 0.049mg/ml, after which 50µl bacterial culture was added to each well. Each extract was tested against Escherichia coli (ATCC 35218), Bacillus cereus (ATCC 10876), Staphylococcus aureus (ATCC 43300), Candida albicans (ATCC 66027), Pseudomonas aeruginosa (ATCC 27853), Enterococcus faecalis (ATCC 29212) and 73 microorganisms from clinical isolates.

Bacterial culture strains that were subcultured on nutrient agar and placed in an incubator overnight at 37oC, were prepared by suspending colonies in MH broth and adjusting the density to that of a 0.5 McFarland standard prior to use in the bioassay. Extract-free solution was used as a blank control. The microtitre plates were incubated overnight at 37oC. As an indicator of bacterial growth, 40 µl p-iodonitrotetrazolium violet (INT) dissolved in water was added to the wells and incubated at 37oC for 30 minutes. MIC values were recorded as the lowest concentration of the extract that completely inhibited bacterial growth. The colourless tetrazolium salt acts as an electron acceptor and is reduced to a red-coloured formazan product by biologically active organisms (Eloff, 1998a). Where bacterial growth was inhibited, the solution in the well remained clear after incubation with INT.

Table 3.1: Microtitre plate layout with plant extracts

Rows Columns • A: Undiluted extract • 1-3 Methanol extract • B: 1 in 2 dilution • 4-6 Acetone extract • C: 1 in 4 dilution • 7-9 Aqueous extract • D: 1 in 8 dilution • 10-12 Sterility controls

• E: 1 in 16 dilution • F: 1 in 32 dilution • G: 1 in 64 dilution • H: Positive Growth Controls

3.3.3.3 Thin Layer Chromatography (TLC) fingerprints TLC is a type of liquid chromatography in which the stationary phase is a thin (~0.25mm) uniform coating of a solid fine material spread, on a glass plate, aluminium foil, or plastic sheet (Sherma and Fried, 2005) is utilized in a manner similar to that of the paper in paper chromatography (Voet and Voet, 2004). It is recommended for the quality control of Traditional Chinese Medicine (TCM) in the Chinese Pharmacopoeia (Committee of National Pharmacopoeia, 2005). It is quick, convenient, and cost effective technique widely used for pharmaceutical analyses. TLC has the special ability to assay many samples at the same time on a single plate (Li, et al., 2004). It is recommended as an effective method for identification of plant derivatives by Chinese, American and European Pharmacopoeias (Fuzzati, 2004). TLC enables reliable separation and analysis of compounds from a wide variety of classes in many types of biological samples (Sherma and Fried, 2005).

The plant extracts and herbal medicinal products are usually highly complex samples, and in most cases it is difficult to identify all compounds by means of common approaches (e.g. one- dimensional liquid chromatography). However, there are many methods, in which traditional techniques are used in the analysis of complex samples. These methods are usually focussed only on some components of which the identity is known, regarding them as “marker compounds” (Chen et al., 2006). Contrary to synthetic drugs and isolated natural products the entire plant extracts should be regarded as active components, so the aforementioned method may sometimes appear insufficient in the quality control of highly complex natural samples (Cieśla and Waksmundzka-Hajnos, 2009).

3.3.3.3.1 Materials and Techniques The TLC fingerprints of different extracts were prepared by spotting 0.5 mg of methanol, acetone and water extracts as a 1cm band onto a large TLC plate (silica gel 60 F254, 0.25 mm, 20cm×20cm, Merck). 45

The plates were developed in petroleum ether: ethyl acetate: chloroform: formic acid (8:7:5:1), over a distance of 17 cm. Once the extracts were separated by TLC and the solvent evaporated,

the separated components were visualised under visible and ultraviolet light UV300 using the 300 series Foto UV transilluminators (Gibbons and Gray, 1998).

BIOAUTOGRAPHY ASSAY: During the extraction and purification procedure, bioautography assays (Slusarenko et al., 1989) were performed on TLC plates using the ATCC strains. An inoculated layer of Mueller-Hinton broth was sprayed with fresh culture of bacteria over a developed TLC plate and incubated for 24 to 48hrs at 37oC in a moist environment. As an indicator of bacterial growth, 0.2mg/ml INT solution was sprayed over the plate and incubated at 37oC for 30 min. The inhibition of bacterial growth by compounds separated on the TLC plate was visible as white spots against a grey background. The white spots on the front of the mobile

phase were used to calculate the Rf values (= distance of travel of the zone divided by the distance of the mobile phase front) (Sherma and Fried, 2005).

3.3.3.4 The Ames test The Ames test assay is a rapid and effective bacterial assay for carcinogenicity that is based on the high correlation between carcinogenesis and mutagenesis (Voet and Voet, 1995). The Ames test (Maron and Ames, 1983; Mortelmans and Zeiger, 2000) is a well-known bacterial mutagenicity test. In this test reverse His- → His+ mutations are visualised by plating Salmonella typhimurium bacteria in a histidine poor growth medium. In this medium only His+ mutants are able to form visible colonies. Different bacterial strains are available to identify different types of mutations. Strain TA98 gives an indication of frame-shift mutations, while a positive response from strain TA100 indicates base-pair substitution. For a substance to be considered genotoxic in the Ames test, the number of revertant colonies on the plates containing the test compounds must be more than twice the number of colonies produced on the solvent control plates (i.e., a ratio above 2.0). In addition, a dose-response should be evident for the various concentrations of the mutagen tested.

The procedure of the Ames test as performed is described in Elgorashi et al. (2003), Verschaeve et al. (2004) and Reid et al. (2006). Stock solution of Salmonella typhimurium bacteria (100µl/ml) in 20ml Oxoid nutrient broth were incubated for 16hrs at 37oC. The bacterial cultures (500μl) were added to 500µl of plant extract in 1000μl phosphate buffer and 10ml of agar containing histidine (0.5mM). The mixture was poured on a Davis minimal agar plate and 46

incubated at 37oC for 48hrs. Samples were tested in triplicate. Two dilutions (200 and 600µg/ml) of histidine were used per sample. 4-Nitroquinoline-N-oxide (4-NQO) was used as a positive control. Bacterial colonies were counted at the end of 48hrs. Results are expressed as means of three independent experiments.

The Salmonella typhimurium strain TA100 (ATCC 49416) was bought from MicroBiologics. The strain was subcultured on nutrient agar prior to the assay at Nelson Mandela Metropolitan University (NMMU) Microbiology laboratory and it was confirmed using the Analytical Profile Index (API) method inorder to rule out possible contamination.

Figure 3.6: Salmonella typhimurium reaction on API Kit

A disadvantage of the Ames test is that any one strain is not able to detect all classes of mutagens. Therefore, it may be necessary to use several strains in a single assay, and this might not be practical for certain screening programs (Skopek et al., 1978). In this study, Harpephyllum caffrum and Hermannia cuinefolia were the plants that were selected for Ames test because of their marked inhibitory activity in the agar difusion and serial dilution methods.

CHAPTER 4: RESULTS

4.1 Introduction

In chapter 3, the author discussed the materials and methods used in sampling, preparation, extraction and antimicrobial testing. This chapter will look at the outcomes of the assays performed.

4.2 Agar diffusion method

An extract is considered having antimicrobial activity with a specific solvent if it inhibits a particular microorganism in all the three runs performed at 10mg/ml concentration. The results of water, methanol and acetone extracts are presented in Tables 4.1 to 4.7 below. Generally, Gram-negative bacteria have been reported to be more resistant to plant extracts than the Gram- positive strains (Rabe and Van Staden, 1997; Grierson and Afolayan, 1999; Afolayan, 2003). Harpephyllum caffrum, Chironia baccifera and Hermannia cuinefolia are the only plants whose extracts inhibited some bacteria in the tables below.

Figure 4.1: Agar plate containing extracts of Pentzia incana showing slight inhibition of bacteria

The comparison of the plate with acetone extract of P.incana (left) to the growth control (right) shows slight inhibition of the inoculated microorganisms. The left plate: observing the diameter of colonies from the far left vertical line where there are four colonies to the third vertical line

48 where there are six colonies, the top most colony (B.cereus) has been markedly reduced in diameter (slightly inhibited) due to the addition of the P.incana acetone extract.

Figure 4.2: Agar plates containing extracts of P.incana, F.muricata & H.cuneifolia

In figure 4.2 above, the top two plates contained acetone and methanol extracts of P.incana at a concentration of 10mg/ml. The activity of the extracts showed slight inhibition as compared to the two middle plates which contained methanol and acetone extracts of F.muricata that showed no activity after 48 hours incubation. In contrast, methanol and acetone extracts of H.cuneifolia

inhibited four strains of B.cereus, one strain of Candida albicans, three Staphylococcus species and three strains of Staphylococcus aureus as shown by the last two plates at the bottom of figure 4.2.

Figure 4.3: Agar plate containing methanol extracts of H.cuneifolia compared with the control plate

In figure 4.3 above, the plate at the right with large colonies is the growth control while the plate at the left contained methanol extracts of H.cuneifolia which inhibited some microorganisms.

Figure 4.4: Agar plate containing acetone extracts of H.cuneifolia compared with the control plate

Figure 4.4 above, is a comparison of the growth control to acetone extracts of H.cuneifolia. It shows inhibition of microorganisms as in figure 4.3 of methanol extracts.

Figure 4.5: Agar plate containing aqueous extracts of H.cuneifolia compared with the control plate

Lack of antimicrobial activity in aqueous extracts of H.cuneifolia has been shown by the two plates in figure 4.5. All the microorganisms grew almost like the growth control after 48 hours incubation.

Table 4.1: Antimicrobial results of Hermannia cuneifolia extracts

H.cuneifolia 1st run H.cuneifolia 2nd run H.cuneifolia 3rd run Number Strain Bacteria Name {10mg/ml} {10mg/ml} {10 mg/ml} W M A W M A W M A 1 973723 A. baumannii + + + + + + + + + 2 984736 A. haemolyticus + + + + + + + +/- +/- 3 987221 A. lwoffi + + + + + + + +/- +/- 4 10876 B.cereus ATCC + - - + - - + - - 5 3 B. cereus + - - - - - + - - 6 1 B. cereus + - - + - - + - - 7 2 B. cereus + - - + - - + - - 8 66027 C. albicans ATCC + + + + + - + + + 9 66027 C. albicans ATCC + - - + - - + - - 10 892996 C. albicans ++ + + + - - + + + 11 3814 C. albicans + + + - - + + + +

12 8102 C. albicans + + + + - - + + + 13 1801 C. albicans + + + + + + + + + 14 2996 C. albicans + + + + + + + + + 15 3305 C. albicans + + + + + + + + + 16 3271 C. albicans + + + + + + + + + 17 991374 C. lusitaniae 18 972711 C. krusei 19 973420 CNS + + + + + + + + - 20 981534 E. agglomerans + + + + ++ ++ + + + 21 977418 E. cloacae + + + + ++ + + + + 22 989694 E. cloacae + + + + + + + + + 23 984997 E. cloacae + + + + + + + + + 24 980654 E. faecalis + +/- +/- + + + + +/- +/- 25 864936 E. faecalis + + + + + + + + + 26 893080 E. faecalis ++ + + + + + + + + 27 29212 E. faecalis ATCC + + + + + + + + + 28 893314 E. faecalis + + + + + + + +/- +/- 29 895614 E. faecalis + + + + + + + + + 30 896074 E. faecalis + + + + + + + + + 31 987039 ESBL E. coli + + + + + + + +/- +/- 32 980232 E. coli + + + + ++ + + + + 33 973732 E. coli + + + + ++ ++ + + + 34 979562 E. coli + + + + ++ ++ + + + 35 893080 E. coli + + + + + + + + + 36 891784 E. coli + + + + + + + +/- +/- 37 891838 E. coli + ++ ++ + + + + + + 38 892894 E. coli + ++ ++ + + + + + + 39 890897 E. coli + + + + + + + + + 40 35218 E. coli ATCC + + + + + + + +/- +/- 41 980244 K. oxytoca + +/- + + + + + +/- +/- 42 984997 K. oxytoca + + + + + + + + + 43 983633 K. oxytoca + + + + + + + +/- +/- 44 980659 K. pneumoniae + + + + + - + + + 45 984400 K. pneumoniae + + + + ++ ++ + + + L. 46 978882 pseudomesenteroides + + + + - - + + - 47 977070 MRSE + - - + - - + - -

48 978024 MRSE + + + + + + + + - 49 976687 MRSE 50 976802 MRSA + - - - - - + - - 51 867949 MRSA + + - + + - + + + 52 976672 MRSA + + - + + + - - - 53 990866 P. stuartii + + + + + + + + + 54 984374 P. aeruginosa + + + + + + - + + 55 1825 P. aeruginosa + + + + + + + + + P. aeruginosa 56 27853 ATCC + +/- + + + + + + + 57 891825 P. aeruginosa + + + + + + + + + 58 892026 P. aeruginosa + + + + + + + + + 59 864263 P. aeruginosa + + +/- + + + + +/- +/- 60 867357 P. aeruginosa + + + + + + + + + 61 8128 P. aeruginosa + + + + ++ ++ + + + 62 977403 P. orizihabitans 63 973004 P. stutzeri 64 897 P. aeruginosa 65 974563 Salmonella species + + + + + + + + + 66 979403 Salmonella siecies + + + + + + + +/- +/- 67 985626 Salmonella species + + + + + + + + + 68 980342 Salmonella species + + + + + + + + 69 867716 S. aureus + + - + + - + + - 70 978523 S. aureus + + + + +/- +/- + - - 71 980218 S. aureus + + - + - - - - - 72 867181 S. aureus + + - + + - + + + 73 891564 S. aureus + + + + +/- - + +/- +/- 74 4330 S. aureus ATCC + - - + - - + + - 75 868060 S. aureus + + + + + + + + + 76 984375 S. haemolyticus + + + + - - + - - 77 984253 S. hominis + - - + - + + - - 78 984254 Streptococcus bovis 79 991504 Strep. Group b + +/- +/- + - - + - -

Note: H.cuneifolia: Hermannia cuneifolia; W: Water; M: Methanol; A: Acetone; MRSA: Methicilin-resistant S.aureus; MRSE: Methicillin-resistant S.epidermidis; CNS: Coagulase Negative Staphylococci; (-): No growth (+/-): Slight inhibition (very weak activity); (+): Normal

growth (no activity); (++): Stimulated growth; Blank box: Organism did not grow on the control plates.

Some strains of Staphylococcus and Bacillus have been successfully inhibited by methanol and acetone extracts of H.cuneifolia. The growth of Pseudomonas species was observed to be affected by the addition of DMSO. The colonies were slightly smaller and flat on the DMSO control as compared to the plain growth control. Seven strains of Escherichia coli were stimulated on exposure to the H.cuneifolia extracts.

Table 4.1: Summary of microorganisms inhibited by extracts of Hermannia cuneifolia

Bacteria Strain Solvent (10mg/ml) WATER METHANOL ACETONE Bacillus cereus ATCC 10876 + - - Bacillus cereus 1 + - - Bacillus cereus 2 + - - Bacillus cereus 3 + - - Candida albicans ATCC 66027 + - - MRSE 1 + - - MRSA 1 + - - Staphylococcus aureus ATCC 4330 + + - Staphylococcus aureus 1 + + - Staphylococcus aureus 2 + + - Staphylococcus hominis 1 + - +

Note: (-): Inhibited; (+): Not inhibited; MRSA: Methicilin-resistant S.aureus; MRSE: Methicillin-resistant S.epidermidis

Table 4.1 is a summary of all consistent results as shown by the activity of H.cuneifolia extracts in the respective solvents. Four strains of Bacillus cereus, three Staphylococcus species, three strains of Staphylococcus aureus and one strain of Candida albicans are microorganisms that were inhibited by the extracts of H.cuneifolia at 10mg/ml concentration.

Table 4.2: Antimicrobial results of Pentzia incana extracts

P.incana 1st run P.incana 2nd run P.incana 3rd run {10mg/ml} {10mg/ml} {10mg/ml} Number Strain Bacteria Name W M A W M A W M A 1 973723 A. baumannii + ++ ++ + ++ ++ + ++ ++ 2 984736 A. haemolyticus + + + + + + + + + 3 987221 A. lwoffi + ++ + + + + + + + 4 10876 B. cereus ATCC + - - + +/- +/- + +/- +/- 5 3 B. cereus + - - + +/- +/- 6 1 B. cereus + + - + - +/- + +/- +/- 7 2 B. cereus + + + + +/- +/- + +/- +/- 8 66027 C. albicans ATCC + + + + - - + + + 9 66027 C. albicans ATCC + + + + + + + + + 10 892996 C. albicans + + + + - + + + + 11 3814 C. albicans + + + + + + 12 8102 C. albicans + + + + - - + + + 13 1801 C. albicans + + + + + + + + + 14 2996 C. albicans + + + + + + + + + 15 3305 C. albicans + + + + + + + + + 16 3271 C. albicans + + + + + + + + + 17 991374 C. lusitaniae 18 972711 C. krusei 19 973420 CNS + ++ ++ + ++ ++ + ++ ++ 20 981534 E. agglomerans + + + + + +/- + +/- + 21 977418 E. cloacae + ++ ++ + ++ ++ + ++ ++ 22 989694 E. cloacae + ++ ++ + ++ ++ + ++ ++ 23 984997 E. cloacae + ++ ++ + + + + + + 24 980654 E. faecalis + + + + + + + + + 25 864936 E. faecalis + + + + + + + + + 26 893080 E. faecalis + + + + + + + + + 27 29212 E. faecalis ATCC + + + + + + + + + 28 893314 E. faecalis + + + + + + + + + 29 895614 E. faecalis + + + + +/- +/- + +/- +/- 30 896074 E. faecalis + + + + +/- +/- + +/- +/- 31 987039 ESBL E.coli + ++ ++ + ++ ++ + ++ ++ 32 980232 E. coli + ++ ++ + + + + + + 33 973732 E. coli + ++ ++ + + + + + +

34 979562 E. coli + ++ ++ + + + + + + 35 893080 E. coli + ++ ++ + ++ ++ + ++ ++ 36 891784 E. coli + ++ ++ + ++ ++ + ++ ++ 37 891838 E. coli + ++ ++ + ++ ++ + ++ ++ 38 892894 E. coli + + + + + + + + + 39 890897 E. coli + ++ ++ + + + + + + 40 35218 E. coli ATCC + ++ ++ + ++ ++ + ++ ++ 41 980244 K. oxytoca + ++ ++ + ++ ++ + ++ ++ 42 984997 K. oxytoca + ++ ++ + ++ ++ + ++ ++ 43 983633 K. oxytoca + + + + + + + + + 44 980659 K. pneumoniae + ++ ++ + + + + + + 45 984400 K. pneumoniae + ++ ++ + ++ ++ + ++ ++ L. 46 978882 pseudomesenteroides + + + + - + + + + 47 977070 MRSE + + + + + + + + + 48 978024 MRSE + + + + + + + + + 49 976687 MRSE 50 867949 MRSA + + + + - +/- + +/- +/- 51 976672 MRSA + + + + +/- +/- + + + 52 976802 MRSA + + + + - - + + + 53 990866 P. stuartii + ++ ++ + + + + ++ ++ 54 984374 P. aeruginosa + + + + + + + + + 55 1825 P. aeruginosa + + + + + + + + + P. aeruginosa 56 27853 ATCC + + + + +/- +/- + + + 57 891825 P. aeruginosa + + + + + + + + + 58 892026 P. aeruginosa + + + + + + + + + 59 864263 P. aeruginosa + + + + + + + + + 60 867357 P. aeruginosa + + + + + + + + + 61 8128 P. aeruginosa + + + + + + + + + 62 977403 P. orizihabitans 63 973004 P. stutzeri 64 897 P. aeruginosa 65 974563 Salmonella species + ++ ++ + ++ ++ + ++ ++ 66 979403 Salmonella species + ++ ++ + ++ ++ + ++ ++ 67 985626 Salmonella species + ++ ++ + + + + + + 68 980342 Salmonella species + + + + +/- +/-

69 867716 S. aureus + + + + - +/- + + + 70 978523 S. aureus + + + + + + + +/- +/- 71 980218 S. aureus + - + + + + + +/- +/- 72 867181 S. aureus + + + + - +/- + +/- +/- 73 891564 S. aureus + + + + + + + + +/- 74 4330 S. aureus ATCC + + + + + + + + + 75 868060 S. aureus + + + + ++ ++ + + + 76 984375 S. haemolyticus + + + + - + + + + 77 984253 S. hominis + - + + - + + + + 78 984254 Streptococcus bovis 79 991504 Strep. group b + + + + - + + + +

Note: P.incana: Pentzia incana; W: Water; M: Methanol; A: Acetone; MRSA: Methicilin- resistant S.aureus; MRSE: Methicillin-resistant S.epidermidis; CNS: Coagulase Negative Staphylococci; (-): No growth (+/-): Slight inhibition (very weak activity); (+): Normal growth (no activity); (++): Stimulated growth; Blank box: Organism did not grow on the control plates.

Four strains of Bacillus cereus, one strain of Enterobacter agglomerans, two strains of Enterococcus faecalis, two strains of Methicillin Resistant Staphylococcus aureus and five strains of Staphylococcus aureus species showed slight inhibition by extracts of P.incana at 10mg/ml concentration. The two strains of Klebsiella pneumoniae and nine strains of Escherichia coli were stimulated by extracts of P.incana.

Table 4.3: Antimicrobial results of Rhigozum obovatum extracts

R.obovatum 1st run R.obovatum 2nd run R.obovatum 3rd run Number Strain Bacteria Name {10mg/ml} {10mg/ml} {10mg/ml} W M A W M A W M A 1 973723 A. baumannii + - + + - + + + + 2 984736 A. haemolyticus + - - + - + + + + 3 987221 A. lwoffi + - + + - + + + + 4 10876 B. cereus ATCC ++ ++ ++ + ++ ++ + ++ ++ 5 3 B. cereus ++ ++ ++ + ++ ++ + ++ ++ 6 1 B. cereus ++ ++ ++ + ++ ++ + ++ ++ 7 2 B. cereus ++ ++ ++ + ++ ++ + ++ ++ 8 66027 C. albicans ATCC + + + + + + + + + 9 66027 C. albicans ATCC + + - + - + + + +

10 2996 C. albicans + + + + + + + + + 11 3814 C. albicans + + + - - + + + + 12 8102 C. albicans + + + + + + + + + 13 1801 C. albicans + + + + + + + + + 14 892996 C. albicans + + + + + + + + + 15 3305 C. albicans + + + + + + + + + 16 3271 C. albicans + + + + + + + + + 17 991374 C. lusitaniae 18 972711 C. krusei 19 973420 CNS + - + + - + + + + 20 981534 E. agglomerans + + + + + + + + + 21 977418 E. cloacae + + + + + + + + + 22 989694 E. cloacae + + + + + + + + + 23 984997 E. cloacae + + + + + + + + - 24 980654 E. faecalis + + + + - + + + + 25 864936 E. faecalis + + + + + + + + + 26 893080 E. faecalis + + + + + + + + + 27 29212 E. faecalis ATCC + + + + + + + + + 28 893314 E. faecalis + + + + + + ++ + + 29 895614 E. faecalis + ++ ++ + + ++ + + + 30 896074 E. faecalis + + + + + + + + + 31 987039 ESBL E. coli + + + + + + + + + 32 980232 E. coli + + + + - + + + + 33 973732 E. coli + + + + + + + + + 34 979562 E. coli + + + + + + + + + 35 893080 E. coli + ++ + + + + ++ + + 36 891784 E. coli ++ ++ ++ + + ++ ++ + ++ 37 891838 E. coli ++ ++ ++ + + ++ ++ + ++ 38 892894 E. coli ++ ++ ++ + + + + + + 39 890897 E. coli ++ ++ + + + + + + + 40 35218 E. coli ATCC + ++ + + + + ++ + + 41 980244 K. oxytoca + + + + - + + + + 42 984997 K. oxytoca + + + + - + + + + 43 983633 K. oxytoca + + + + ++ + + + + 44 980659 K. pneumoniae + + + + - - + + + 45 984400 K. pneumoniae + + + + - + + + + 46 978882 L. + - + - - - + + +

pseudomesenteroides 47 977070 MRSE + - + - - - + + + 48 978024 MRSE + - + + - - + + + 49 976687 MRSE 50 867949 MRSA + - - + - - + + + 51 976672 MRSA + - + + - - + + + 52 976802 MRSA + - + - - - + + + 53 990866 P. stuartii + - + + - - + + + 54 984374 P. aeruginosa + + + + + + + + + 55 1825 P. aeruginosa + + + + ++ + + + + P. aeruginosa 56 27853 ATCC + + + + + + + + + 57 891825 P. aeruginosa + + + + + + + + + 58 892026 P. aeruginosa + + + + + ++ ++ + + 59 864263 P. aeruginosa + + + + + ++ ++ + + 60 867357 P. aeruginosa + + + + + ++ ++ + + 61 8128 P. aeruginosa + + + + + ++ ++ + + 62 977403 P. orizihabitans 63 973004 P. stutzeri 64 897 P. aeruginosa 65 974563 Salmonella species + + + + ++ ++ + ++ ++ 66 979403 Salmonella species + + + + ++ ++ + ++ ++ 67 985626 Salmonella species + + + + ++ + + + + 68 980342 Salmonella species + + + + ++ + + + + 69 867716 S. aureus + - + + - - + - + 70 978523 S. aureus + - + + - - + + + 71 980218 S. aureus - - - + - - + + + 72 867181 S. aureus + - + + - - + + + 73 891564 S. aureus + - + + - - + + + 74 4330 S. aureus ATCC + - + + - - + + + 75 868060 S. aureus + - + + - - + + + 76 984375 S. haemolyticus + - + + - - + + + 77 984253 S. hominis ------+ + + 78 984254 Streptococcus bovis 79 991504 Strep. group b + - + + - + + + +

Note: R.obovatum: Rhigozum obovatum; W: Water; M: Methanol; A: Acetone; MRSA: Methicilin-resistant S.aureus; MRSE: Methicillin-resistant S.epidermidis; CNS: Coagulase 59

Negative Staphylococci. (-): No growth (+/-): Slight inhibition (very weak activity); (+): Normal growth (no activity); (++): Stimulated growth; Blank box: Organism did not grow on the control plates.

While the strains of B.cereus in the study were inhibited by extracts of H.cuneifolia, they were also stimulated and grew into extra large colonies on exposure to the extracts of Rhigozum obovatum at 10mg/ml concentration. Six strains of Escherichia coli and two strains of Salmonella species were stimulated by extracts of R.obovatum.

Table 4.4: Antimicrobial results of Felicia muricata extracts

F.muricata 1st run F.muricata 2nd run F.muricata 3rd run Number Strain Bacteria Name {10mg/dl} {10mg/dl} {10mg/dl} W M A W M A W M A 1 973723 A. baumannii + ++ ++ + + + + + + 2 984736 A. haemolyticus + + + + + + + + + 3 987221 A. lwoffi + ++ + + + + + + + 4 10876 B. cereus ATCC + ++ + + + + + ++ + 5 3 B. cereus + ++ + + ++ + 6 1 B. cereus + ++ + + + + + ++ + 7 2 B. cereus + ++ + + + + + ++ + 8 66027 C. albicans ATCC + + + + + + + + + 9 66027 C. albicans ATCC + + + ++ + + + + + 10 2996 C. albicans + + + + + + + + + 11 3814 C. albicans + + + + + + + 12 8102 C. albicans + + + + + + + + + 13 1801 C. albicans + + + + + + + + + 14 892996 C. albicans + + + + + + + + + 15 3305 C. albicans + + + + + + + + + 16 3271 C. albicans + + + + + + + + + 17 991374 C. lusitaniae 18 972711 C. krusei 19 973420 CNS + ++ ++ + + + + + + 20 981534 E. agglomerans + ++ + + + ++ + ++ + 21 977418 E. cloacae + ++ ++ + + + + + + 22 989694 E. cloacae + ++ ++ + + + + + +

23 984997 E. cloacae + ++ + + + + + + + 24 980654 E. faecalis + + + + + + + + + 25 864936 E. faecalis + + + + + + + + + 26 893080 E. faecalis + + + + + + + + + 27 29212 E. faecalis ATCC + + + + + + + + + 28 893314 E. faecalis + + + + + + + + + 29 895614 E. faecalis + + + + + + + + + 30 896074 E. faecalis + + + + + + + + + 31 987039 ESBL E.coli + ++ + + + + + + + 32 980232 E. coli + ++ ++ + + + + + + 33 973732 E. coli + ++ ++ + + + + + + 34 979562 E. coli + ++ ++ + + + + + + 35 893080 E. coli + ++ + + ++ + + ++ + 36 891784 E. coli + ++ ++ + ++ + + ++ + 37 891838 E. coli + ++ ++ + ++ + + ++ + 38 892894 E. coli + ++ ++ + + + + + + 39 890897 E. coli + ++ ++ + ++ + + ++ + 40 35218 E. coli ATCC + ++ ++ + ++ + + ++ + 41 980244 K. oxytoca + ++ ++ + + + + + + 42 984997 K. oxytoca + ++ ++ + + + + + + 43 983633 K. oxytoca + + + + + + + + + 44 980659 K. pneumoniae + ++ ++ + + + + + + 45 984400 K. pneumoniae + ++ + + + + + + + L. 46 978882 pseudomesenteroides + + + + + + + + + 47 977070 MRSE + + + + - + + + + 48 978024 MRSE + + + + + + + + + 49 976687 MRSE 50 867949 MRSA + + + + + + + + + 51 976672 MRSA + + + + + + + + + 52 976802 MRSA + + + - - - + + + 53 990866 P. stuartii + ++ ++ + + + + + + 54 984374 P. aeruginosa + + + + + + + + + 55 1825 P. aeruginosa + + + + + + + + + 56 27853 P. aeruginosa ATCC + + + + + + + ++ + 57 891825 P. aeruginosa + + + + + + + + + 58 892026 P. aeruginosa + + + + + + + + +

59 864263 P. aeruginosa + + + + + + + + + 60 867357 P. aeruginosa + + + + + + + + + 61 8128 P. aeruginosa + ++ ++ + + + + + + 62 977403 P. orizihabitans 63 973004 P. stutzeri 64 897 P. aeruginosa 65 974563 Salmonella species + ++ ++ + + + + + + 66 979403 Salmonella species + ++ ++ + + + + + + 67 985626 Salmonella species + ++ ++ + + + + + + 68 980342 Salmonella species + + + + + + + + + 69 867716 S. aureus + + + + + + + + + 70 978523 S. aureus + ++ + + + + + + + 71 980218 S. aureus + + + + + + + + + 72 867181 S. aureus + + + + + + + ++ + 73 891564 S. aureus + + + + + + + ++ + 74 4330 S. aureus ATCC + + + + + + + + + 75 868060 S. aureus + + + + + + + + + 76 984375 S. haemolyticus + + + + + + + + + 77 984253 S. hominis + + + + + + + + + 78 984254 Streptococcus bovis 79 991504 Strep. group b + + + + + + + + +

Note: F.muricata: Felicia muricata; W: Water; M: Methanol; A: Acetone; MRSA: Methicilin- resistant S.aureus; MRSE: Methicillin-resistant S.epidermidis; CNS: Coagulase Negative Staphylococci; (-): No growth (+/-): Slight inhibition (very weak activity); (+): Normal growth (no activity); (++): Stimulated growth; Blank box: Organism did not grow on the control plates.

The two strains of Klebsiella pneumoniae and ten strains of Escherichia coli were stimulated by extracts of F.muricata. No antimicrobial activity has been observed by the extracts of F.muricata. Out of the seventy-nine microorganisms tested against the four extracts of plants H.cuneifolia, P.incana, R.obovatum and F.muricata, forty-eight microorganisms which were inhibited in most of the runs were selected for further analysis using extracts of Harpephyllum caffrum and Chironia baccifera. The investigation was conducted at 10mg/ml and 20mg/ml concentration because during the preliminary testing the 10mg/ml concentration showed no activity.

Table 4.5: Antimicrobial results of Harpephyllum caffrum extracts

Number WATER METHANOL ACETONE Strain Bacteria Name {10mg/ml}{20mg/ml} {10mg/ml} {20mg/ml} {10 mg/ml}{20 mg/ml}

1 973723 A. baumannii ------

2 984736 A. haemolyticus ------3 987221 A. lwoffi ------

4 66027 C. albicans ATCC ------

5 66027 C. albicans ATCC ------6 892996 C. albicans ------

7 3814 C. albicans ------

8 8102 C. albicans ------9 1801 C. albicans ------

10 2996 C. albicans ------11 3305 C. albicans ------

12 3271 C. albicans ------

13 991374 C. lusitaniae ------14 972711 C. krusei - - - - -

15 980654 E. faecalis + - + + + -

16 864936 E. faecalis + - + + + + 17 893080 E. faecalis

18 29212 E. faecalis ATCC ------

19 893314 E. faecalis ------20 895614 E. faecalis ------

21 896074 E. faecalis ------

22 973732 E. coli + + + + + + 23 979562 E. coli + + + + + +

24 891784 E. coli + + + + + +

25 890897 E. coli + + + + + + 26 980244 K. oxytoca + + + + + +

27 984997 K. oxytoca + + + + + +

28 983633 K. oxytoca + + + + + +

29 984400 K. pneumoniae + + + + + + 30 L. 978882 pseudomesenteroides

31 976687 MRSE

32 990866 P. stuartii + + + + + + 33 984374 P. aeruginosa + + + + + +

34 1825 P. aeruginosa + + + + + +

35 27853 P. aeruginosa ATCC + + + + + + 36 891825 P. aeruginosa + + + + + +

37 892026 P. aeruginosa + + + + + +

38 864263 P. aeruginosa + + + + + + 39 867357 P. aeruginosa

40 8128 P. aeruginosa + + + + + +

41 977403 P. orizihabitans 42 973004 P. stutzeri

43 897 P. aeruginosa + + + + + +

44 979403 Salmonella species + + + + + + 45 985626 Salmonella species + + + + + +

46 868060 S. aureus ------47 984254 Streptococcus bovis

48 991504 Strep. group b

Note: (+): growth; (-): Inhibition; (BLANK BOX): No growth on control plate; MRSE: Methicillin-resistant S.epidermidis.

Three species of Acinetobacter, nine strains of Candida albicans, 2 other Candida species, four strains of Enterococcus faecalis and one strain of Staphylococcus aureus were inhibited by aqueous, methanol and acetone extracts of H.caffrum.

Table 4.6: Antimicrobial results of Chironia baccifera extracts

Number WATER METHANOL ACETONE Strain Bacteria Name {10mg/ml} {20mg/ml} {10mg/ml} {20mg/ml} {10 mg/ml}{20 mg/ml}

1 973723 A. baumannii + + + + + +

2 984736 A. haemolyticus + + - - - - 3 987221 A. lwoffi + + + + + +

4 66027 C. albicans ATCC + + + + + +

5 66027 C. albicans ATCC + + + + + + 6 892996 C. albicans + + + + + +

7 3814 C. albicans + + + + + +

8 8102 C. albicans + + + + + + 9 1801 C. albicans + + + + + +

10 2996 C. albicans + + + + + + 11 3305 C. albicans + + + + + +

12 3271 C. albicans + + + + + +

13 991374 C. lusitaniae + + + + + + 14 972711 C. krusei + + + + + +

15 980654 E. faecalis + + - - -

16 864936 E. faecalis + + - - - - 17 893080 E. faecalis

18 29212 E. faecalis ATCC + + - - - -

19 893314 E. faecalis + + - - - - 20 895614 E. faecalis + + - - - -

21 896074 E. faecalis + + - - - -

22 973732 E. coli + + + + + + 23 979562 E. coli + + + + + +

24 891784 E. coli + + + + + +

25 890897 E. coli + + + + + + 26 980244 K. oxytoca + + + + + +

27 984997 K. oxytoca + + + + + +

28 983633 K. oxytoca + + + + + +

29 984400 K. pneumoniae + + + + + + 30 L. 978882 pseudomesenteroides

31 976687 MRSE + + - - - -

32 990866 P. stuartii + + + + + + 33 984374 P. aeruginosa + + + + + +

34 1825 P. aeruginosa + + + + + + 35 P. aeruginosa + + 27853 ATCC + + + + 36 891825 P. aeruginosa + + + + + +

37 892026 P. aeruginosa + + + + + +

38 864263 P. aeruginosa + + + + + + 39 867357 P. aeruginosa

40 8128 P. aeruginosa + + + + + +

41 977403 P. orizihabitans 42 973004 P. stutzeri

43 897 P. aeruginosa + + + + + + 44 979403 Salmonella species + + + + + +

45 985626 Salmonella species + + + + + +

46 868060 S. aureus + + - - - - 47 984254 Streptococcus bovis

48 991504 Strep. group b

Note: (+): growth; (-): Inhibition; (BLANK BOX): No growth on control plate MRSE: Methicillin-resistant S. epidermidis.

One strain of Acinetobacter haemolyticus, six strains of Enterococcus faecalis, one strain of Methicillin Resistant Staphylococcus epidermidis and one strain of Staphylococcus aureus were inhibited by methanol and acetone extracts of Chironia baccifera.

Since the MIC method is more sensitive than the agar diffusion method, seven microbes which were inhibited by extracts of H.caffrum, twelve microbes not inhibited by extracts of H.caffrum

66 and other twenty microbes from clinical isolates, totalling to thirty-nine microorganisms, were tested further for MIC determination.

4.3 Minimum inhibitory concentration (MIC)

It was difficult to note other inhibition due to colour similarity; however, the

Figure 4.6: Microtitre plate showing colour intensity

colour intensity had enabled the possibility of differentiation when compared to growth controls. Control wells with growth were much darker than the wells without growth.

Figure 4.7: Microtitre plate showing MIC values

As an organism develops resistance to the plant extract, the MIC will increase, and a greater concentration will be needed to inhibit growth of the bacterial population. The following section contains data table reflecting the MIC values of H.caffrum extracts. These values are an average of the results obtained in triplicate runs.

Table 4.7: Mean MIC results of 39 selected microorganisms tested with extracts of H.caffrum

Number Strain Bacteria Name Harpephyllum caffrum (mg/ml) Water Methanol Acetone 1 1826 P. aeruginosa + + + 2 8128 P. aeruginosa + + + 3 893080 E. coli 0.391 0.049 0.049 4 891838 E. coli 0.391 0.049 0.049 5 892894 E. coli 0.391 0.049 0.195 6 4330 S. aureus 0.195 0.098 0.049 7 867949 MRSA 0.195 0.049 0.049 8 10876 B. cereus ATCC 0.195 0.049 3.125 9 3 B. cereus 0.391 0.195 0.195 10 1 B. cereus 0.391 0.781 0.195 11 2 B. cereus 0.391 0.195 0.195 12 867716 S. aureus 0.195 0.049 0.049 13 891564 S. aureus 0.195 0.049 0.049 14 867181 S. aureus 0.195 0.049 0.049 15 980244 K. oxytoca 0.391 0.049 0.049 16 984997 K. oxytoca 0.391 0.098 0.195 17 977070 MRSE 6.25 0.781 1.563 18 978024 MRSE 6.25 0.781 1.563 19 976672 MRSA 3.125 0.391 0.049 20 976802 MRSA 3.125 0.391 0.195 Salmonella 21 974563 species + + + Salmonella 22 980342 species + + + 23 973723 A. baumannii 12.5 0.781 0.781 24 984736 A. haemolyticus 6.25 3.125 3.125 25 987221 A. lwoffi 6.25 1.563 1.563 26 35218 E. coli ATCC 0.391 0.049 0.049 27 984375 S. haemo lyticus 0.781 0.098 0.195 28 984253 S. hominis 0.391 0.781 0.195 29 980218 S. aureus 0.195 0.049 0.049 30 978523 S. aureus 0.195 0.049 0.049 31 987039 ESBL E. coli 0.391 0.049 0.049 32 977418 E. cloacae + + +

33 989694 E. cloacae + + 34 984997 E. cloacae + + + 35 981534 E. agglomerans + + + 36 973420 CNS 0.195 0.098 0.098 37 980659 K. pneumoniae 0.39 0.049 0.195 38 990866 P. stuartii + + + 39 980232 E. coli 0.391 0.049 0.049

Note: (+): No inhibition even at highest concentration; MRSA: Methicilin-resistant S.aureus; MRSE: Methicillin-resistant S.epidermidis; CNS: Coagulase Negative Staphylococci.

Seventy six percent of the thirty-nine microorganisms selected showed MIC values. Strains of Pseudomonas aeruginosa, Escherichia coli, Klebsiella and Salmonella species which were not inhibited in the agar diffusion assay also showed MIC values ranging from 0.049 to 6.25mg/ml.

Table 4.8: Mean MIC results of 39 selected microorganisms tested with extracts of C.baccifera

Number Strain Bacteria Name Chironia baccifera (mg/ml) Water Methanol Acetone 1 1826 P. aeruginosa + + + 2 8128 P. aeruginosa + + + 3 893080 E. coli + + + 4 891838 E. coli + + + 5 892894 E. coli + + + 6 4330 S. aureus + 0.781 1.563 7 867949 MRSA + 1.563 1.563 8 10876 B. cereus ATCC + + + 9 3 B. cereus + + + 10 1 B. cereus + + + 11 2 B. cereus + + + 12 867716 S. aureus + 0.781 3.125 13 891564 S. aureus + 0.391 1.563 14 867181 S. aureus + 6.25 + 15 980244 K. oxytoca + + + 16 984997 K. oxytoca + + + 17 977070 MRSE + + +

18 978024 MRSE + + + 19 976672 MRSA + + + 20 976802 MRSA + + + Salmonella 21 974563 species + + + Salmonella 22 980342 species + + + 23 973723 A. baumannii + + + 24 984736 A. haemolyticus + 6.25 3.125 25 987221 A. lwoffi + + + 26 35218 E. coli ATCC + + + 27 984375 S. haemolyticus + 3.125 1.563 28 984253 S. hominis + + + 29 980218 S. aureus + 0.781 + 30 978523 S. aureus + 0.781 + 31 987039 ESBL E. coli + + + 32 977418 E. cloacae + + + 33 989694 E. cloacae + + + 34 984997 E. cloacae + + + 35 981534 E. agglomerans + + + 36 973420 CNS + + + 37 980659 K. pneumoniae + + + 38 990866 P. sturtii + + + 39 980232 E. coli + + +

Note: (+): No inhibition even at highest concentration; MRSA: Methicilin-resistant S.aureus; MRSE: Methicillin-resistant S.epidermidis; CNS: Coagulase Negative Staphylococci.

One nonfermentative Gram-negative bacilli, Acinetobacter haemolyticus and eight Gram- positive microorganisms: six strains of Staphylococcus aureus, one strain of Staphylococcus haemolyticus and one strain of Methicillin Resistant Staphylococcus aureus showed MIC values ranging from 0.391 to 6.25mg/ml.

Table 4.9: Mean MIC results of 39 selected microorganisms tested with extracts of H.cuneifolia

Number Strain Bacteria Name Hermannia cuneifolia (mg/ml) Water Methanol Acetone 1 1826 P. aeruginosa + + + 2 8128 P. aeruginosa + 25 12.5 3 893080 E. coli + + + 4 891838 E. coli + + + 5 892894 E. coli + + + 6 4330 S. aureus + 0.19 0.19 7 867949 MRSA + 0.19 0.09 8 10876 B. cereus ATCC + 0.05 0.05 9 3 B. cereus + 0.05 0.05 10 1 B. cereus + 0.05 0.05 11 2 B. cereus + 0.05 0.05 12 867716 S. aureus + 0.09 0.09 13 891564 S. aureus + 1.56 25 14 867181 S. aureus + 3.125 + 15 980244 K. oxytoca + 25 0.78 16 984997 K. oxytoca + 25 + 17 977070 MRSE + + 25 18 978024 MRSE + 3.125 12.5 19 976672 MRSA + 1.56 12.5 20 976802 MRSA + + 6.25 Salmonella 21 974563 species + + 6.25 Salmonella 22 980342 species + + 6.25 23 973723 A. baumannii + + + 24 984736 A. haemolyticus + 25 12.5 25 987221 A. lwoffi + + + 26 35218 E. coli ATCC + 6.25 12.5 27 984375 S. haemolyticus + + 25 28 984253 S. hominis + 12.5 12.5 29 980218 S. aureus + + 25 30 978523 S. aureus + + 25 31 987039 ESBL E. coli + + 12.5 32 977418 E. cloacae + + +

33 989694 E. cloacae + + 12.5 34 984997 E. cloacae + + + 35 981534 E. agglomerans + + + 36 973420 CNS + + + 37 980659 K. pneumoniae + + + 38 990866 P. stuartii + + + 39 980232 E. coli + + +

Note: (+): No inhibition even at highest concentration; MRSA: Methicilin-resistant S.aureus; MRSE: Methicillin-resistant S.epidermidis; CNS: Coagulase Negative Staphylococci.

All the Gram-positive microorganisms which were inhibited by H.cuneifolia in agar diffusion method showed MIC values at concentrations ranging from 0.05 to 12.5mg/ml. Furthermore, some Gram-negative bacteria like Pseudomonas aeruginosa, Klebsiella oxytoca, Salmonella, Escherichia coli, Acinetobacter haemolyticus, and Enterobacter cloacae showed MIC values at concentrations ranging from 0.78 and 25mg/dl. This agrees with the results of Thring et al. (2007) where Candida albicans tested against extracts of Chironia baccifera indicated no inhibition in all solvents used in the disc diffusion assay but showed MIC values of 1.25mg/ml and 5mg/ml in methanol and ethyl-acetate extracts of Chironia baccifera, respectively. This shows that the bioassay (MIC) is more sensitive than the disc or agar diffusion method as more activity was recorded from the MIC bioassay.

4.4 Thin Layer Chromatography

The TLC fingerprints of different extracts were prepared by spotting 0.5 mg of methanol, acetone and water extracts as a 1cm band onto a large TLC plate (silica gel 60 F254, 0.25 mm, 20cm×20cm, Merck).

Figure 4.8: Plant extracts spotted on TLC silica plates

The plates were developed in petroleum ether: ethyl acetate: chloroform: formic acid (8:7:5:1), over a distance of 17 cm.

Figure 4.9: TLC silica plates spotted with extracts developing in air tight glass jar

Once the extracts were separated by TLC and the solvent evaporated, the separated components were visualised under visible and ultraviolet light UV300 (Gibbons and Gray, 1998).

1 2 3

Figure 4.10: Developed TLC silica plates after evaporating and drying

The bioautographic assay showed a number of clear white spots against a grey background. The white spots (inhibition zones) give an indication of antimicrobial activity by compounds present in these areas. Inhibition zones were visible in methanol and acetone extracts of Harpephyllum caffrum tested areas.

F1 F 2 F3

F4 F5 F6

Figure 4.11: F1 to F6 are TLC silica plates after the bioautographic assay viewed under UV light

Plate F1: Staphylococcus aureus growth inhibition visible as white spots. Plate F2: Enterococcus faecalis growth inhibition visible as white spots. Plate F3: Candida albicans growth inhibition visible as white spots. Plate F4: Comparison of F3 and F1 Plate F5: Comparison of F3, F1 and F2 Plate F6: Comparison of F1 and F2. From left to right (F1 towards F2), the extracts added on the spots are: water, methanol and acetone extracts of Harpephyllum caffrum; water, methanol and acetone extracts of Hermannia cuneifolia. Water extracts of H.caffrum did not show any inhibition zones. By separating bioactive extracts on the TLC, it is possible to get information on the compounds present in the plant extract.

In figure 4.11 above, F1 shows four zones of inhibition indicated by the white spots (see arrows) and representing four different compounds that have been separated from methanol and acetone extracts of Harpephyllum caffrum. F2 shows five zones of inhibition representing five compounds that have inhibited Enterococcus faecalis from methanol and acetone extracts of Harpephyllum caffrum. F3 showed one zone of inhibition but after 10 minutes, another zone appeared below it as indicated in F4, signifying that there might be other active compounds in Harpephylum caffrum extracts that can inhibit Candida albicans.

Below is Table 4.11 indicating the Rf values of E.faecalis, S.aureus and C.albicans calculated after visualization of inhibition zones under visible and UV light.

Table 4.10: Rf values of major zones present in chromatograms of methanol and acetone Harpephyllum caffrum extracts.

BAND 1 BAND 2 BAND 3 BAND 4 BAND 5 MICROORGANISM M A M A M A M A M A E.faecalis 0.28 0.28 0.31 0.31 0.45 0.45 0.66 0.66 0.90 0.90 S.aureus 0.25 0.25 0.30 0.30 0.44 0.44 0.95 0.95 C. albicans 0.25 0.28 0.32 0.34 0.48 0.48 Note: M: Methanol; A: Acetone.

E.faecalis showed five zones of inhibition on TLC plates having the same Rf values in methanol

and acetone extracts ranging from 0.28 to 0.90. S.aureus showed four zones of same Rf values in methanol and acetone extracts ranging from 0.25 to 0.95. C.albicans showed three zones, of

which the first and second zones from the bottom had different Rf values in their respective

extract solvent. The third zone had same Rf value in methanol and acetone extracts. The Rf values of C.albicans ranged from 0.25 to 0.48. The different inhibiton zones show differences in the chemical composition of plant extracts (figure 4.11). Methanol and acetone extracts of Hermannia cuneifolia showed a continuous brown and white band appearing faint in other areas.

4.4 The Ames test

The mutagenicity results shown in Table 4.12 below revealed that all the extracts were non- mutagenic towards Salmonella typhimurium strain TA100. A compound is considered a mutagen if the results show a dose dependent increase in the number of revertants and if the number of revertants is equal to or greater than two times of the negative control (Bulmer et al., 2007).

Table 4.11: Number of histidine+ revertants in Salmonella typhimurium strain TA100 Plant Extract Concentration vs Number of Colonies per plate 600µg/ml 200μg/ml Harpephyllum caffrum Methanol 8 ± 3 10 ± 2 Acetone 12 ± 3 12 ± 3 Water 7 ± 2 9 ± 2 Hermannia cuneifolia Methanol 24 ± 4 22 ± 4 Acetone 23 ± 5 27 ± 4 Water 10 ± 3 10 ± 4 4-nitroquinoline-N-oxide (positive control), 10μg/ml 235 ± 8 colonies Water (negative control) 25 ± 5 colonies

According to the criteria defined in this study, extracts of Harpephyllum caffrum and Hermannia cuneifolia could be classified as having antimutagenic properties. The extracts of the plants presented similar results for the absence of mutagenic induction despite the different solvents used and in the absence of metabolic activation. The average His+ revertants observed ranged between 7 and 27 for all the extracts at concentrations (200µg/ml and 600μg/ml) of histidine while 25 revertant colonies grew on the negative control and 235 revertant colonies on the positive control, 4-nitroquinoline-N-oxide (4-NQO). Therefore, none of the extracts under study could be regarded as mutagenic.

CHAPTER 5: DISCUSSION AND CONCLUSION

The fields of ethnopharmacology, ethnobotany and natural products chemistry, is progressing steadily in South Africa. Ethnopharmacologists, botanists, microbiologists, and natural-product chemists are searching the earth for phytochemicals which could be developed for the treatment of infectious diseases (Tanaka et al., 2006) especially in light of the emergence of drug-resistant microorganisms and the need to produce more effective antimicrobial agents.

It has been shown that in vitro screening methods could provide the needed preliminary observations necessary to select crude plant extracts with potentially useful properties for further chemical and pharmacological investigations (Mathekaga and Meyer, 1998).

This study investigated the antimicrobial activities of Harpephyllum caffrum, Hermannia cuneifolia, Chironia baccifera, Rhigozum obovatum, Felicia muricata and Pentzia incana. The plants were tested against six ATCC strains and seventy-three microorganisms isolated from patients suffering from septicaemia. Three methods were used to determine the antimicrobial activity of the plants. The methods included the agar diffusion method, microtitre plate method to determine MIC, TLC fingerprints with the corresponding bioautographic assays and the Ames test for possible carcinogenicity. Different extracts of the plants were made and it was found that the water extracts did not perform well in this study. Literature supports this finding as reported by Meyer and Afolayan (1995) as well as Masika and Afolayan (2002), stating that the choice of acetone and methanol as solvents for extraction and the exclusion of water extracts in their analysis was based on their previous observation and other reports that water extracts of plants generally showed little or no antimicrobial activities. Cragg et al. (1994) also reported great differences between the activity of aqueous and organic extracts in their anti-HIV screening. It was however still valuable to test water extracts of these plants as traditionally, plant extracts are prepared with water as used in infusions, decoctions and poultices. Thring et al. (2007) is of the opinion that it seems unlikely that the traditional healer will be able to extract those compounds which are responsible for activity in the acetone and methanol extracts by using traditional methods. Observations of very weak (+/-) or no activity (+) of aqueous plant extracts on Gram- negative bacteria are well documented (Eloff, 1998b; Madamombe and Afolayan, 2003; Afolayan, 2003).

In this study, the agar diffusion method showed that the methanol extracts of the plants inhibited a higher percentage of microorganisms when compared to the acetone extracts. Similar observations were reported by Grierson and Afolayan (1999) on water, methanol and acetone extracts from some indigenous plants used for the treatment of wounds in the Eastern Cape, South Africa. They found that the majority of the antibacterial activity observed was in the methanol extracts. Methanol extracts of Hermannia cuneifolia inhibited 16%, acetone extracts inhibited 22% and aqueous extracts inhibited 2% of the microorganisms tested. Slight inhibition was observed with 8% of the methanol extracts, 7% of the acetone extracts and 2% of aqueous extracts. Pentzia incana showed no antimicrobial activity in aqueous extracts. The methanol extracts inhibited 7% of microorganisms while acetone extracts inhibited 2%. Slight inhibition was observed in 8% of the microbes tested in methanol extracts and 10% in acetone extracts. The methanol extracts of Rhigozum obovatum inhibited 21% of the tested microorganisms, acetone extracts inhibited 10%, and aqueous extracts inhibited 3% of the bacteria.

Among all microorganisms that grew, 11% were stimulated (had larger colonies than the control plate) by extracts of Felicia muricata and Rhigozum obovatum. Pentzia incana stimulated 16% and Hermannia cuneifolia stimulated 2% of the bacteria. However, stimulation of microbial growth may facilitate illness in patients treated by such herbal medicine as observed in the plants extracts, while inhibition may suppress the growth and multiplication of the organism hence curing the disease. Hayes (1947) reported in a study entitled “Survey of higher plants for presence of antibacterial substances”, that some plant extracts caused increased bacterial growth.

Another observation in the agar diffusion assay was that none of the Gram-negative bacteria were inhibited by the plants extracts tested. Gram-negative bacteria have an outer phospholipidic membrane with structural lipopolysaccharide components. This composition makes the cell wall impermeable to lipophilic solutes, and the porins in the cell wall form a selective barrier to hydrophilic solutes, with an exclusion limit of about 600 Da (Nikaido and Vaara, 1985; Yao and Moellering, 1995). Four strains of Bacillus cereus, one strain of Candida albicans, and two strains of Staphylococcus aureus were inhibited by methanol and acetone extracts of Hermannia cuneifolia. This may be attributed to the fact that cell wall in Gram-positive bacteria consists of a single outer peptidoglycan layer which is not an effective permeability barrier (Scherrer and Gerhardt, 1971).

Some medicinal plants are named locally after their effects upon consumption by humans. For example, the local name for Hermannia cuneifolia is kwaaiman (Van Wyk and Els, 2008) and it means ‘angry man’ because the infusion that is prepared from a leafy twig burns the throat when used to treat sore throat. Klebsiella pneumoniae is a respiratory pathogen commonly associated with colds and flu (Viljoen et al., 2004). The inhibitory property of extracts from H.cuneifolia on pathogens of respiratory tract might have been the reason for using this plant in the treatment of sore throat and coughs.

The inhibition of strains of Candida albicans by extracts of Harpephyllum caffrum as indicated in Table 4.6 may support the use of this plant as a traditional medicine for treating acne and eczema or as a blood purifier as manifested by pimples on the face. The findings can support the use of this plant in treating fungal skin infections.

Hence, the antibacterial activity of Hermannia cuneifolia and Harpephyllum caffrum extracts on these different clinical isolates of bacteria causing septicaemia strengthen scientific evidence of the effectiveness of medicinal plants in the treatment of various ailments by traditional healers. The ability of the plants extracts to inhibit the growth of several bacteria and fungi species is an indication of the antimicrobial potential which makes these plants likely candidates for bioprospecting for antibiotic drugs.

In this study, when comparing the agar diffusion method to microtitre plate dilution method used for MIC determination, a discrepancy was found with the activity of methanol extracts as compared to acetone extracts. Methanol extracts showed more activity in the agar diffusion method (see Table 4.1 - 4.7) while acetone extracts showed more activity in the microtitre plate dilution method. For example, the MIC results of extracts from Hermannia cuneifolia showed one Staphylococcus aureus and one Klebsiella oxytoca inhibited with values of 3.125 and 25.0 mg/ml, respectively, which were not inhibited in acetone extracts. While one strain of MRSE, one strain of MRSA, two strains of Salmonella species, two strains of Staphylococcus aureus, one strain of Staphylococcus haemolyticus, one strain of E.coli and one strain of Enterobacter cloacae were inhibited at values 25.0, 6.25, 6.25, 25.0, 25.0, 12.5 and 12.5mg/ml respectively, by acetone extracts only (Table 4.10). In contrast, in a study entitled “antimicrobial activity of extracts from Felicia muricata”, Ashafa et al. (2008) reported that methanol extracts were more active than acetone and aqueous extracts using the agar dilution method and the microtitre plate method. This discrepancy may be associated with the presence or absence of soluble phenolic 80

and polyphenolic compounds (Kowalski and Kedzia, 2007). However, it is difficult to compare results obtained, against the published results in literature especially when dealing with plant extracts. The reason is that, several variables influence the results, such as the environmental and climatic conditions under which the plant grew, choice of plant extracts, choice of extraction method, antimicrobial test method and test microorganisms (Nostro et al., 2000; Hammer et al., 1999). Furthermore, Rios and Villar (1988) narrated that the variation, which is often observed in testing crude plant extracts for antibacterial activity can be attributed to a variety of factors. These include the assay technique, culture medium, strain of bacteria used for testing, age of the agar plate, plant source and whether it is used fresh or dried, and the quality of extract tested. In addition, there is no standardized method for expressing the results of antibacterial testing.

In this study, the comparison of methodologies has shown that agar diffusion and MIC methods used to measure antimicrobial activity do not give parallel results, confusing any assessment of antimicrobial activity. Similar results were reported by Njenga et al. (2005) and Youssef and Tawill (1990) who showed that comparison of methodologies has shown that agar diffusion and serial dilution methods to measure antimicrobial activity do not always give parallel results.

The inclusion of TLC fingerprints in modern pharmaceutical herbal monographs is now standard practice (Scott et al., 2004). The results obtained in this study demonstrate the utility of TLC to distinguish with certainty the antimicrobial activity of these herbal plants. Bioautography combines TLC with a bioassay in situ and, therefore allows the researcher to localize the active compounds within a sample (Hamburger and Cordell, 1987; Gibbons and Gray, 1998).

Harpephylum caffrum showed the best separation by the solvent system, petroleum ether: ethyl acetate: chloroform: formic acid (8:7:5:1), which was used. Other researchers have used solvent systems like 1.75M acetic acid: ethyl-acetate: toluene (1:1:1) (Thring et al., 2007); hexane: ethyl acetate (3:1) (Yffa et al., 2002), selected empirically using prior personal experience and literature reports of similar separations as a guide in the mobile phase.

Variation in zone size, zone numbers and colour intensity between the solvent solutions and plant species examined indicate that some quantitative infraspecific variation was present (Scott et al., 2004). Compounds that are naturally coloured, such as chloroplast and bile pigments (Eidam et al., 2001), are viewed directly in daylight or white light with the naked eye. Compounds with native fluorescence are viewed as bright, coloured zones on a dark background

under 366nm or 254nm ultraviolet (UV) light on layers without fluorescent indicator. Compounds that absorb 254nm UV light, particularly those with aromatic rings and conjugated double bonds, can be detected on an “F-layer” containing a fluorescent indicator or phosphor. When irradiated with 254nm UV light, absorbing compounds diminish (quench) the uniform layer fluorescence and can be viewed as dark violet spots on a green or pale-blue background (Sherma and Fried, 2005). Hence, the compounds observed in the study were possibly chloroplasts since the zones were viewed directly in daylight with the naked eye as well as under 300nm UV light.

The Ames test was used because it is reliable, quick and easy. The absence of mutagenic response suggests that Harpephyllum caffrum and Hermannia cuneifolia plant extracts upon exposure to Salmonella typhimurium bacterial strain in the Ames assay are probably safe for use in traditional medicine or is the first step forward in determining the safe use of these plants, though further safety testing may be appropriate. In support of these findings, the Ames test conducted by Elgorashi et al. (2003) on the extracts from the leaves of Harpephyllum caffrum showed negative results in 90% of methanol extracts tested by Salmonella typhimurium strain TA98 and TA100 with and without S9. Hence, the results reported here might be considered sufficient for further studies aimed at isolating and identifying the active compounds.

The determination of toxicity of South African plants is particularly necessary as the use of some traditional medicines has resulted in several cases of acute toxicity, leading to increased morbidity and mortality (Reid et al., 2006). Popat et al. (2001) refers to three case studies in South Africa indicating a high rate of acute toxicity resulting from the use of traditional medicines. Screening is required to identify and eradicate the use of all mutagenic plants as numerous studies have shown that the proportion of carcinogens identified as mutagens by the Ames test ranges from about 50% to 90% (Zeiger, 2001). The occurrence rate of cancer is increasing worldwide and the determination of chemoprevention or chemoprophylaxis compounds is important in an effort to reduce the risk of cancer (Kundu et al., 2004).

Differences in antimicrobial activity of medicinal plants are obviously related to differences in their contents of active compounds (Boakye-Yiadom and Konning, 1975). Available reports tend to show that alkaloids and flavonoids are the responsible compounds for the antimicrobial activities in higher plants (Cordell et al., 2001). Moreover, it is also claimed that secondary metabolites such as tannins and other compounds of phenolic nature are classified as active 82

antimicrobial compounds (Mitscher et al., 1972; Rojas et al., 1992). A study by Springfield et al. (2005) of Chironia baccifera on the microchemical tests in their laboratories indicated the presence of tannins and saponins but the absence for alkaloids and cardiac-, cyanogenic- and anthraquinone glycosides. The presence of these active compound plus those explained by Wolfender (1993) under 2.6.1.1 active ingredients, supports the antimicrobial activities of Chironia baccifera as observed in this study.

Today, there is increased sensitivity towards traditional healers’ issues. Along with the rest of the world, the South African Department of Health is looking at improving conditions for traditional healers’ associations. In addition, the National Indigenous Knowledge Systems Program is working towards registration with the objective that it will lead to the protection of intellectual property rights, equitable compensation, promotion, and the improvement of traditional healing systems (Kubukeli, 2000).

Owing to the fact that millions of South Africans rely on traditional medicines for their primary healthy care needs, there is insufficient pragmatic information regarding the pharmacology and toxicology of commonly used herbal remedies. This information should be available to health care workers such as doctors, pharmacists, nurses, and social workers (Rodriguez-Fragoso et al., 2008).

In the rural communities where there is limited access to medical facilities, people should be empowered through education on proper use of medicinal plants as first aid remedies (Bodeker, 2001; Rabe and Van Staden, 1997). Establishing the antibacterial activity of these plants will contribute to the systematic scientific investigation of indigenous South African medicinal plants.

In the future, integrated research projects among scientists, clinical researchers, and industries will be needed to concentrate all efforts toward innovative therapies for sepsis (Tetta et al., 2003b). As technology offers devices to implement traditional or new modalities, the application of a rigorous analysis derived from basic and clinical sciences in sepsis might lead to major advances provided that randomized, controlled studies are performed. As the sepsis story continues to unravel with the ongoing discovery of more malignant inflammatory mediators, the development and use of extracorporeal blood purification therapies seems a logical, feasible and exciting challenge. Continuous renal replacement therapy (CRRT), plasmafiltration, and coupled

83 plasma filtration adsorption (CPFA) demonstrate exciting potential for improving outcome in critically ill septic subjects (Tetta et al., 2003a).

It is thus concluded that Harpephyllum caffrum, Hermannia cuneifolia and Chironia baccifera do possess antimicrobial properties for at least certain microbes and that this activity is largely dependent on the extract used. However, further studies are required in order to gain more clarity as to the specificity and biochemical mechanisms responsible for the antimicrobial properties of these three plants. The possible loss of volatile components present in the extracts during drying and evaporation might have produced results different from those obtained in studies where other methods of extract drying were employed.

The bioactive compounds responsible for antimicrobial activities of the three plants need to be identified and isolated, and it should be taken into account that it might possibly be the synergistic activity of two or more compounds that is responsible for the medicinal properties of the three plants. It should also be taken into account that results obtained from in vitro assays may not necessarily be reproducible in vivo due to the metabolic processes of the test subjects. It is therefore, suggested that the extracts which showed antimicrobial activity during this study be tested in vivo, and that their toxicity patterns and mutation-inducing capacities be established further.

From the findings of antimicrobial activities of the plants under study, further work needs to be done on the isolation, purification and structural elucidation of the bioactive compounds in these plants. The ethnomedicinal study of plants is important for modern day medicine but its usefulness cannot be over-emphasized if methods are not standardized to obtain comparable and reproducible results. Other pharmacological studies like potentials for combination antimicrobial chemotherapy and mechanisms of action are important additional tests to fully assess antimicrobial activity of plant extracts, and these are subjects of intensive investigation in research laboratories.

Ethnopharmacological research could play an important role in the worldwide HIV/AIDS pandemic and associated opportunistic infections, such as tuberculosis and oral thrush. Successful research in ethnopharmacology encompasses various disciplines which need to be

84 intergrated. Through scientific validation of effective remedies, coupled with training of healthcare practitioners, it may be possible to bring traditional medicine to a level of efficiency and safety where it can be regarded as an acceptable alternative to western healthcare systems.

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