An Investigation Into the Anti-Bacterial Efficacy of Zingiber Officinale on Staphylococcus Aureus and Escherichia Coli

An Investigation into the Anti-Bacterial Eﬃcacy of Zingiber oﬃcinale on Staphylococcus aureus and Escherichia coli

By Linda S. Dhliwayo

Supervisor: Dr. Zvidzai

A Laboratory Based Project submitted in Partial Fulﬁlment of the Requirements for the Degree of Bachelor of Science Education in the faculty of Science Education

Bindura University of Science Faculty of Science Education Department of Biological Sciences September 2013 Dedicated to my loving mum, father, sister, brothers, husband and son.

iii Table of Contents

iv List of Tables

3.1 Zone Size Interpretive Chart ...... 14

4.1 Mean Zone Diameters and Standard Errors ...... 17 4.2 Mean minimum inhibition concentrations in mg/ml ...... 18 4.3 Mean Minimum Bactericidal Concentrations in mg/ml ...... 19

5.1 Minimum Inhibiton Concentrations in mg/ml of Z. officinale . . . . 27 5.2 Minimum Inhibiton Concentrations in mg/ml of Penicillin ...... 28 5.3 Minimum Bactericidal Concentrations in mg/ml of Z. officinale . . 28

v List of Figures

vi Abstract

The research was basically on finding out the effectiveness of Zingiber officinale extract as an antibacterial agent against two bacterial strains; Escherichia coli and Staphy- lococcus aureus. The experiments were done in comparison with the effects of the antibiotic Benzyl penicillin against these two bacteria. The Kirby Bauer method was used to determine the susceptibility of bacteria to antibacterial agents by measuring the size of inhibition zones they formed under standardized conditions.The Tube Dilu- tion method was used to determine the minimum inhibitory concentration(MIC) and the minimum bactericidal concentration(MBC).Thirteen different concentraions were used, each experiment being run three times on each bacterial strain and the mean values were calculated. The MIC of Z. officinale was 28.13mg/ml against E. coli and 14.06mg/ml against S.aureus. The MBC values were 112.5mg/ml and 56.25mg/ml respectively. These were being compared to the MIC of penicillin, which was the same as its MBC and the value was less than 0.22 for both bacterial strains. These results show that indeed Z.offinale has antibacterial effects, which are stronger on S. aureus than on E.coli. Therefore it can be used to treat conditions arising from infection by these bacteria, probably with a few modifications such as synergism.

ii Acknowledgements

I am greatly indebted to my supervisor Dr. Zvidzai who made sure this project became a success. Also to the chairman of the Biology Department, Mr Matekaire for his support throughout the period I was working on this research. I acknowledge the greatest support that came from my family; my daughter Juanita for being a good girl during the long hours I was away from home; my son Shammah for being patient with me. Last, but most importantly, my deepest gratitude goes to my endeared husband Lawrence for his tireless encouragement and support, especially with the typing of this project. Thank you.

iii Chapter 1

Introduction

1.1 Background to the Study

Rising costs of living have not excluded the health care department. Infact, health care costs are ranked among the top ﬁve departments that receive huge sums of money in almost every national annual budget of Zimbabwe. However, despite large sums of money allocated to the Ministry of Health, Zimbabwe, there are always reports of shortages of drugs in hospitals and clinics, especially those in rural areas. This is due to high microbial and other infectious diseases outbreaks which increase the demand on the limited drug resources, which are very expensive since the bulk of them are imported. The shortage and high prices of drugs has therefore necessitated the use of herbs to come up with alternative ways of treating various ilnesses and diseases. According to the proceedings of the First International IOCD-Symposium held at Victoria Falls, Zimbabwe (1996), African medicinal plants have long provided important sources of drugs to local population, but due to the decreasing number of traditional healers the cumulation of their valuable knowledge is progressively diminishing. It is therefore important to exploit much of this data as possible before it is too late. Zingiber oﬃcinale , commonly known as ginger, or tsangamidzi in Shona, is among the

1 commonly used medicinal plants with potential for development in medicine. Ginger is known to treat such pathologies as diarrhoea, hypertension, nausea and many other disorders resulting from microbial infections. Producing medicine from ginger will offer several significant advantages which include ease of availability at reasonable prices. It will be also a way of utilising the available resources in a profitable way.

1.2 Statement of the Problem

An investigation into the antibacterial efficacy of Zingiber officinale againstEscherichia coli and Staphylococcus aureus as compared to the antibiotic penicillin. There is a great shortage of drugs in Zimbabwe and those available are not affordable to the or- dinary citizen. Zingiber officinale is one of the plants available in this country which is a potential source of medicine.

1.3 Justiﬁcation of Study

Plants are a rich potential source of drugs as they produce a vast array of novel bioactive molecules, many of which probably serve as chemical defences against infection. There are well over 265 000 flowering species alone on earth, of which less than 1% have thus far been screened for the presence of any bioactive molecules of potential therapeutic use (Walsh, 1998). The bioactive compounds contained in ginger can be directly extracted from the plant material and standardized in a simple way that can be done by local people and also some ginger-derived drugs can be manufactured in part by direct chemical synthesis (Peterson and Shanholter, 1992). The findings of this study will provide the socio-technical support needed to exploit the medicinal effects of ginger on the micro-organisms Escherichia coli and Staphylo- coccus aureus. The purpose of this is that it will be used in place of some antibiotics

2 which are expensive and not always available.

1.4 Research Aim

The principal aim of this study is to determine the potential medical usage of Zingiber oﬃcinale extract by testing its eﬀects on Escherichia coli and Staphylococcus aureus.

1.5 Objectives

1. To determine the size of inhibition zones formed by Z. oﬃcinale extract on the two bacterial cultures, as compared to those formed by benzyl penicillin.

2. To determine the minimum inhibition concentration (MIC) of the extract.

3. To determine the minimum bactericidal concentration (MBC) of the extract.

3 Chapter 2

Literature Review

2.1 Zingiber oﬃcinale Plant Structure and its Uses

2.1.1 Botanical Classiﬁcation of the Plant 2.1.2 Origin and Plant Physiology

The Zingiber oﬃcinale plant, commonly known as ginger, cooking ginger, tsangamidzi in Zimbabwean Shona language, originated in tropical Asia,but is now grown as a commercial crop for the ginger root in Latin America, South East Asia and Africa (http://www.friedli.com/herbs/ginger.html). Ginger is a herbaceous perennial with upright stems and narrow medium green leaves arranged in two ranks on each stem. The plant gets to a height of 1.2m with leaves about 1.9cm wide and 17.8cm long. It grows from an aromatic tuber-like rhizome which is warty and branched. The rhizomes are thickened and have secretory cells with ethereal oils; vessel elements in

Kingdom Plantae Phylum Magnoliophyta Class Liliopsida Order Zingiberales Family Zingiberaceae Genus Zingiber Species oﬃcinale

4 roots. The inﬂorescence grows on a separate stem from the foliage stem, and forms a dense spike. The bracts are green with translucent margins and the small ﬂowers are yellow green with purple lips and cream coloured blotches.

2.1.3 General Uses

Ginger root is widely used around the world as a spice or food additive. It is used fresh (green ginger), preserved in syrup, crystallised (especially in Hong Kong) or dried and powdered. Ginger is also used to ﬂavour biscuits, cakes, sweets, ginger beer, ginger wine, and brandy; as well as shampoos and cosmetics.

2.1.4 Medicinal Uses

Zingiber officinale has a broad spectrum of medicinal purposes worldwide. It is used as an excellent remedy for digestive problems such as flatulence, nausea, indigestion, intestinal infections and certain types of food poisoning(Huang, et al , 1990 ). The combination of sweat and circulatory stimulation allows ginger to move blood to the periphery. This makes it a good remedy for high blood pressure and fever. According to Srivastava, et al (1964), ginger inhibits platelet aggregation and therefore should be the ideal condiment for people predisposed to clotting which may lead to either heart attack or stroke. The Lancet, a highly respected British medical journal, reported excellent results in scientific tests using ginger to treat nausea and morning sickness. In Zimbabwe and other African countries, ginger is normally dried and ground into a powder then dissolved in hot or cold water to form a decoction which is then used to treat some of the conditions mentioned above. According to most herb users, use of hot water quickens the dissolution process and is encouraged when the decoction is for infants (Cunningham, 1993). Elderly people normally chew the ginger raw as they believe that its effects are most effective in this state. However due to its hot and biting

5 ﬂavour, most people would rather take it in other forms than when it is raw.

2.1.5 Chemical Components of Ginger

The chemical components of ginger include volatile oils such as zingiberol, zingiberene and camphene; oleoresine (gingerol, shogaol), phenol, proteolytic enzyme (zingibain), vitamin B6, vitamin C, calcium, magnesium, phosphorous, potasium and linoleic acid. According to Lee and Ahn (1985), ginger has a high content of antioxidants which means it has antimutagenic and antiinﬂammatory properties. Inevitably, ginger is believed to have antibacterial properties which is one of the objectives of this research to ﬁnd out.

2.2 Bacterial Strains and their Eﬀects

Diarrhoea which is quite common in infants and adults in Zimbabwe is the net loss of ﬂuid from the human body or the expression of watery or bloody stools resulting from the malabsorption of salt and water (Eley, 1992). It occurs when intestinal mucosa is stimulated to secrete salt and water; when mucosal permeability is altered followimg gross destruction of mucosal cells, or when there are disturbances of gastrointestinal motility and transit (Eley, 1992). The bacterium mostly implicated in infantile diarrhoea is Escherichia coli and this together with Staphylococcus aureus are implicated in stomach upsets leading to adult diarrhoea (Stanier et al., 1992). According to Sussman (1985), E.coli and S.aureus colonize the umbilical stump, perineum, skin and sometimes the gastrointestinal tract soon after birth and their sources are found to be mother and the inanimate environment.

2.2.1 Staphylococcus aureus

Staphylococcus aureus is a gram positive, salt tolerant, non-motile and non-sporeforming cocci which belongs to the Micrococcus family (Norton,1986). The bacteria divides

6 randomly giving rise to irregular clusters of cells. It is a facultative anaerobe which grows rapidly on many types of media with individual colonies sharply defined as smooth and convex. The species name aureus comes from the tendency of those organisms to have a yellow pigmentation as a result of mannitol fermentation, a phe- nomenon which occurs only when cultured in mannitol salt agar media. Infections caused by S. aureus cause significant morbidity and mortality, despite the availability of potent antimicrobial drugs. The constituents of its cell wall are important determinants of virulence (Norton, 1986). Peptidoglycan is the main component of the cell wall and it represents 60-90% of the cell wall weight. It is a polysaccharide polymer eliciting production of endogenous pyrogen and has potent endotoxin like activity. Important enzymes like catalases, coagulases, hyaluronidases and beta-lactamases are produced by S. aureus and have been implicated in the pathogenesis of its infections. Coagulase initiates formation of blood clots that can protect bacteria from phagocyto- sis (Stanier et al., 1992). Beta-lactamase confers penicillin resistance to the bacteria. This means that when this enzyme is present the bacteria is protected from the ef- fect of penicillin. Cytotoxins are also produced; leukocidins which kill leucocytes and hemolysins which lyse red blood cells in vitro and are toxic to leucocytes. S.aureus occurs as a commensal on the skin and nasal passages of healthy humans and animals. It produces five distinct serologically distinct enterotoxins which cause food poisoning. The bacterium proliferates and produces toxins in poorly refrigerated and undercooked food. Ingestion of the contaminated food causes a sudden onset of nausea, vomiting, abdominal cramps and diarrhoea after two to six hours (Barnwart, 1989). Most adults recover sooner from this kind of diarrhoea than youths, but loss of fluid from the body can be fatal in both the young and the old. Most S. aureus strains are susceptible to penicillin and methicillin,but some have

7 developed resistance against them. Some beta-lactamase mediated and intrinsic methicillin resistances have been developed by S. aureus against penicillin and methicillin respectively (Bryan, 1982). Due to the rapid increase in resistance by microbes to modern medicine, this project was aimed at investigating the antibacterial activities of Zingiber oﬃcinale on the bacterium so that people can use it more eﬀectively to treat some of the above mentioned ailments.

2.2.2 Escherichia coli

Escherichia coli are a gram negative, motile, peritrichous, fimbriate and non-spore forming bacillus belonging to the Enterobacteriaceae family (Sussman, 1985). It is a facultative anaerobe that grows readily on simple culture and synthetic media with glycerol or glucose as the only source of carbon and energy. In addition, it ferments lactose in MacConkey agar to lactic acid and gas giving rise to pink, convex and glossy colonies (Harigan, 1998). E.coli have a thin cell wall with relatively porous peptidoglycan comprising 5-20% of cell wall weight. Unlike S.aureus which has lysine, it has diaminopimelic acid in its peptidoglycan and it contains an outer wall layer madeup of lipopolysaccharide. A lipoprotein layer is found on the inner side of the outer membrane which lacks teichoic acids and has poris which are transmembrane channels (Brock and Madigan, 1991). The principal reservoir of E. coli is the human intestinal flora. It gets to the exter- nal environment through excretion in faeces (Jay, 1986). The enterotoxigenic E.coli strains are implicated in causing traveller’s diarrhoea in adults. They do this by al- tering the balance of sodium and chloride ions entering and leaving the enterocytes which directly affects water absorption and secretion resulting in net efflux of these ions with more water being secreted than is absorbed (Eley, 1992; Campbell, 1996). Onset of symptoms of infection which include abdominal pains occurs seven hours to several days and the infection also lasts several days. Poor hygienic practices have

8 led to contaminatioon of baby food and even adults’ food with faecal E. coli leading to severe outbreaks of diarrhoea in Zimbabwe.

2.3 Antibacterial Agents

2.3.1 Characteristics of Antibacterial Agents

Antibacterial agents are among the most valuable therapeutic agents in modern health care. According to Prescott and Dunn (1959), those used in the medical field should not be relatively toxic, precipitant to serum proteins, haemolytic to blood cells or adversely affect phagocytes. They should have a long shelf life and long in vivo life. They should be free from pyrogenicity and must not cause histamine-like responses (Hawkey and Lewis, 1989). Moreover, the agents must be effective against pathogens under the conditions of use, being able to reach site of infection and excretion and also be well tolerated by the individual in the doses required. Side effects must be very few if any. Hawkey and Lewis (1989) asserts that the outcome of antibacterial therapy of infection is affected by pharmacokinetic and pharmacodynamic factors which are the interactions between the agent, the pathogen and the patient. The effects of antibacterial agents on organisms can either be bactericidal or bacteriostatic , or both but at different concentrations. Bactericidal agents kill the bacteria, while bacteriostatic agents only stop the action of the bacteria without killing it (Prescott and Dunn, 1959). Antibacterial agents have several sites and modes of action on bacteria. The cell wall, cell membrane, ribosomes and nucleus are the most targeted sites. The structures and functions of these sites are altered such as replication of genetic information and the translation process (Norton, 1986).

9 2.3.2 Methods used to Determine Susceptibilities of Bacteria to Antibacterial Agents

Bioassays which involve the in vitro methods like agar diffusion, macrotube dilution, microtube dilution and serum bactericidal determinations are used to determine bacterial susceptibilities to antibacterial agents (Wang and Peterson, 1984). According to Linton and Hawker, (1988) bioassay is a method of assessing activity of sample of unknown potency where the activity is compared with that of a standard preparation against a test organism known to be sensitive to the antibacterial agent under test. Quality control is very important during bioassay of natural products because it ensures that all methods employed are of internationally recommended standards. Zingiber officinale root extract was bioassayed in this project, using the Kirby Bauer method (agar diffusion) and the tube dilution method. These two method have been used succuessfully to study the antibacterial activity of 7-O-beta glucopyranosyl- nutanocoumarin from Chaptala nutans against S. aureus, Pseudomonas aureginosa and E. coli (http://www.antibacterial activities of the members of the Asteracea family).

2.3.3 Kirby Bauer method

The susceptibility of bacteria to antibacterial agents is determined by the size of inhibition zones they form under standardized conditions. The pH and composition of media,size of innoculum, antibiotic disc concentration, incubation temperature and time must all be standardized. The incubation temperatures are suggested to be 37 degreesCelsius for 18-24 hours. If incubation occurs at lower temperatures, the growth of the organism or the herbal activity could be hindered and longer incubation period would cause overgrowth (Hawkey and Lewis, 1989). Several factors inﬂuence the zones of inhibition. These include concentration of bacteria spread on the susceptibility of the pathogen the antibacterial agent, antibiotic

10 diffusion effects, agar depth, growth temperatures, nutrient availability and drug an- tagonists. The diffusion of the antibacterial agent is influenced by its concentration, molecular weight, solubility in water, pH and ionization (Wang and Peterson, 1984). Diameters of zones produced are proportional to amount of antibacterial agent on each disc as well as the solubility, diffusion coefficient and overall effectiveness of the agent. The size of the inhibition zone is used to determine whether bacteria are sensitive or resistant to the antibacterial agent, and if sensitive, the antibacterial agent will either be bacteriostatic or bactericidal and this is determined by the tube dilution method.

2.3.4 Tube Dilution method

This is a quantitative method used to determine the minimum eﬀective concentrations, that is the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). MIC is the in vitro minimum concentration of antibacterial agent required to halt microbial growth for as long as the inhibitory substance is present, but if removed growth resumes (Norton, 1986). The MIC is correlated with the concentration of the antibiotic achievable in blood. Several studies have shown that most antibacterial agents are active at two to seven times the MIC in vivo (An- halt and Washington, 1985). Knowledge of the MIC can provide the physician with precise information regarding the organism’s degree of susceptibility. MBC is the minimum concentration showing no growth or 99% kill on subculture of each MIC tube. According to Peterson and Shanholter, (1992) MBC data for which cidal agents are recommended. The tube dilution method also requires a standard innoculum and the same incubation conditions as the Kirby Bauer method. One advantage of using the tube dilution method is that it has readily observed endpoints. Susceptibility of slowly growing organisms demanding unusual growth conditions can be best determined using the MIC and susceptibility can be related more accurately

11 to drug levels achievable in body ﬂuids other than serum (Bryan, 1982).

12 Chapter 3

Materials and Methods

The crude extract of Zingiber oﬃcinale root was tested for antibacterial activity against strains of E. coli and S. aureus.

3.1 Preparation of the Zingiber oﬃcinale crude extract

Fresh root tubers of Z. officinale were washed to remove soil particles and other debris. A sterile knife was used to chop 90g of the tubers into small pieces which were then ground into a paste using sterile piston. The paste was soaked in 100ml of sterile distilled water and left to stand for 4 days. During this period the mixture was swirled occasionaly to maximise dissolution of active compounds into the water. At the end of this period the mixture was filtered using filter paper and the filtrate was then used in the experiments.

3.2 Antibacterial Susceptibility Tests

MacConkey agar was used as the culture media. The susceptibilities of the bacteria cultures to the Z. oﬃcinale extract were determined using the Kirby Bauer method.

13 3.2.1 Kirby Bauer Method (Disk Diﬀusion Test)

Four agar plates were warmed up to room temperature to avoid excess moisture build up after innoculation. The water that built up during the warming period was decantd because it would leach antibiotic material from the discs. The agar was innoculated with the bacterial cultures, two with E. coli and the other two with S. aureus, by streaking the entire surface in four different directions. This was done aseptically and the plates left to stand for 10 minutes to facilitate absorption of innoculum into the agar. Sterile filter paper discs of 8mm in diameter were aseptically dispensed directly onto the innoculated agar. Two filter paper discs were placed on each plate and they were at the same distances from the edges of the plate and from each other to avoid overlapping of the inhibition zones. A sterile micropipette was used to collect 0.011ml of Z.officinale and drop it on each disc giving a disc concentration of 9.9mg/ml. Each disc was gently tapped with sterile forceps to ensure contact with the agar surface then the plates were incubated at 37oC for 24 hours, in an aerobic atmosphere (Oberhofer, 1985). At the end of the incubation period the plates were recorded for any growth inhibition and the zones of inhibition were measured with a metric rule in mm (see Appendix 1).The mean zone diameters produced by each bacteria and the standard errors were determined and the results statistically analysed in comparison to the National Com- mittee for Clinical Standards (1999) shown in Table (??).

Table 3.1: Zone Size Interpretive Chart

Zone size produced (mm) Bacteria Disc content Resistant Intermediate Susceptible S. aureus 10 units ≤ 20 21-28 >29 E. coli 10 units ≤11 12-21 >22

14 The minimum eﬀective concentrations of Z. oﬃcinale were then determined.

3.3 Determination of the Minimum Inhibition Con- centration

1. 26 universal bottles, divided into two sets were labelled 1 to 13. 10ml of nutrient broth were aseptically pipetted to each bottle.

2. 10ml of Z. oﬃcinale extract were aseptically pipetted into the ﬁrst bottles of the two sets, the serial two-fold dilutions were carried out upto a concentration of 0.22mg/ml.

3. 1ml of innoculum was added to each bottle, the ﬁrst set for S. aureus and the second for E. coli. All the bottles were gently shaken and incubated at 37oC for 24 hours.

4. Tubes showing growth were determined visually.The results were recorded as (+) where there was turbid growth, and (-) where there was no detectable growth. The MIC was taken to be the concentration of the last bottle showing no growth.

3.4 Determination of Minimum Bactericidal Con- centration

A loopful of culture was aseptically removed from each tube that showed no growth and innoculated on MacConkey agar. The plates were divided into quadrants and the loopfuls from each bottle were innoculated onto their respective sectors. The diﬀerent quadranrs were labelled according to the concentrations in their source bottles. The plates were inverted and incubated at 37oC for 24 hours and then checked for growth.

15 The last quadrants showing no growth were taken as the MBCs (Paul and Wolf, 1979).

3.5 Controls for the Antibacterial Tests

Benzyl penicillin, an antibiotic with established activities against E.coli and S. aureus strains was used as the control for the antibacteial tests. The disk diﬀusion test was done as described above using a disk concentration of 10mg/ml. The minimum eﬀective concentrations procedures were done as those described above.

3.6 Statistical analysis of the results t-tests for two independent small samples were used for comparing the mean inhibition zone sizes, the MICs and MBCs of the Z. oﬃcinale crude extract with those of the benzyl penicillin.

16 Chapter 4

Presentation of Results

4.1 Antibacterial Susceptibility Tests

The raw data for the sizes of inhibition zones formed by E. coli and S. aureus, the minimum inhibitory concentrations and the minimum bactericidal concentrations are given in the Appendices 1, 2 and 3.

4.2 Disk Diﬀusion Tests

The largest inhibition zones were produced by S. aureus in both plant extract of Z. oﬃcinale and the antibiotic penicilin, as compared to those produced by E.coli, as depicted in Table (??).

Table 4.1: Mean Zone Diameters and Standard Errors Bacteria Antibacterial agent Zingiber oﬃcinale Penicillin Staphylococcus aureus 25.8 ± 0.28 29.1 ± 0.28 Escherichia coli 21.2 ± 0 23.4 ± 0.42

17 4.3 Minimum Inhibitory Concentration(MIC) De- termination

Lower concentrations of Z. oﬃcinale and of penicillin were required to inhibit growth of S. aureus strains as compared to the E. coli strains, as presented in Table (??).

Table 4.2: Mean minimum inhibition concentrations in mg/ml Bacteria Antibacterial agent Zingiber oﬃcinale Penicillin Staphylococcus aureus 14.06 < 0.22 Escherichia coli 28.13 < 0.22

The Z. officinale crude extract and penicillin were more effective against S. aureus than against E. coli.A higher concentration of the extract was required to inhibit growth of E. coli than was required against S. aureus.The antibiotic penicillin also had reduced action against E. coli strains as compared to S. aureus, but its effects were stronger compared to Z. officinale crude extract as shown by the lower concentrations required to inhibit growth.

4.4 Minimum Bactericidal Concentration Deter- mination

The minimum bactericidal concentrations of penicillin against the two strains were the same as its minimum inhibitory concentrations. The minimum bactericidal concentrations of Z. oﬃcinale against the two strains were higher than that of the antibiotic. Summary statistics of the results are shown in Table (??).

18 Table 4.3: Mean Minimum Bactericidal Concentrations in mg/ml Bacteria Antibacterial agent Zingiber oﬃcinale Penicillin Staphylococcus aureus 56.25 < 0.22 Escherichia coli 112.5 < 0.22

The crude extract of Z. oﬃcinale was less productive as compared to penicillin.The MIC and MBC of penicillin was less than 0.22 for both bacterial strains.

19 Chapter 5

Discussion

The S. aureus and E. coli strains produced larger inhibition zones with penicillin control compared to Z.officinale crude extract. A larger zone does not necessarily mean that one organism is more sensitive than the other but interpretations of the zones depend on the characteristics of the antibacterial agents. The susceptibilities of the two bacterial cultures to Z. officinale crude extract and to penicillin were different and so were the inhibitory and bactericidal activities of the plant extract and the antibiotic against the bacterial strains. This can be attributed to the fact that penicillin, unlike the crude extract of Z.officinale is made up of exclusively the active components which have been concentrated into a drug, which renders it more effective. Probably if the Z. officinale extract had been extracted using a proper solvent and purified by thin layer chromatography, the same results might have been accomplished for the two antibacterial agents,provided their modes of action were the same. However, crude extracts of traditional medicinal plants also have their advantages over modern drugs. They are readily available, considerably easy to prepare and administer. In most African countries including Zimbabwe and South Africa, these crude extracts have been used successfully by herbalists (Kokwaro, 1993). Another advantage is that the pharmacology of the medicine is not known, therefore the crude

20 extract is the best form in which the medicine can be used. Even if the pharmacology is known, it is very expensive to extract the active compounds. For instance, the process of thin layer chromatography requires skilled manpower which is not easy to come by.It is however very important to employ aseptic techniques during extraction, such as washing of hands, the plant material to be used using clean sterile water and equipment. The antibacterial effects of both the Z. officinale and penicillin were stronger on S. aureus which is a gram positive bacteria, than they were on the gram negative E. coli. This suggests a mode of action involving the cell wall. According to Lehninger (1982), penicillin inhibits a late step in the enzymatic synthesis of peptidoglycans in penicillin susceptible organisms, so that the cell wall is incomplete and fails to sustain the normal growth of the cells. It is therefore possible that Z.officinale employs the same mechanism in its action against S. aureus. On the other hand the differences in the cell wall components of the two bacterial strains also contributes to the differences in their susceptibilities to antibacterial agents (Brock and Madigan, 1991). The outer peptidoglycan layer of S. aureus is not an effective permeability barrier compared to E. coli’s outer membrane because it is highly susceptible to antibacterial agents. Hence the MIC of Z. officinale was 28.13mg/ml against E. coli and 14.06mg/ml against S. aureus. For penicillin it was less than 0.22 in both cases showing that it it more effective as an antibacterial agent. Despite having porins which are transmembrane channels, the outer phospholipidic membrane E. coli cell wall provides an apparent barrier to penetration of incoming antibacterial agents.According to Bryan (1982), porins constitute a selective barrier to the hydrophilic solutes with an exclusion limit of about 650 Daltons.It is possible that the Z. officinale cannot dissolve in lipids or it does so slowly, since the cell walls of gram negative bacteria contain lipopolysaccharides, lipoproteins and lipoteichoic acids. In gram positive bacteria all liposubstances are absent and hence Z. officinale

21 and penicillin molecules enter the cell wall easily. A high volume of antibacterial agent entering freely through the cell wall of gram positive bacteria results in low concentration being built up since concentration is indirectly proportional to volume. Hence gram positive bacteria require low concentrations of antibacterial agent to inhibit their growth. On the contrary, a high concentration of antibacterial agent is needed to inhibit growth or kill E. coli cells. Bacterial susceptibility in vivo is however also affeted by the concentration of the active agent within the infection site, the bacterial phenotype resulting from growth in the host and finally capability of host defences to prevent spread and to eradicate the infective organism (Bryan, 1982). The concentration of agent within the tissue site solely depends on the distribution of the drug in the body. A bacterium in tissue exhibits different phenotypes from those under cultural conditions and in general growth rates are slowed and turnover of target sites may be greatly produced in culture. Bryan (1982) also asserts that new or additional exopolysaccharides may be formed and the structure of the cell envelope may be altered. Above all, inhibiton of isolated bacteria in pure culture require much lower concetration than those necessary for associations of several bacteria in vivo. Synergism can be considered in the development of Z. officinale as an antibacterial agent. This is the combination of two or more agents whose combined action is significantly greater than the sum of each of their effects. Use of Z.officinale with another traditional plant known as Dicoma anomala, would probably lead to decrease in its minimum effective concentrations (Ordy, 1995). Taking the experimental plant’s extract as directed by the elderly for treatment of different infections is therefore centered around reduction of the activity of E. coli by inhibiting its growth when present in the system. The mechanism of action is however still to be discovered. This preliminary study has indicated that the treatment of diarrhoea and stomach upsets with Z.officinale crude extract is due to its antibacterial activities.

22 Use of traditional medicines is being strongly encouraged by the World Health Organ- isation (WHO). However, lack of appropriate documentation and proof of herbalists’ claims on the usefulness, eﬃcacy and non-toxicity of the active compounds in these plants is a major constraint to their use. Furthermore, shortage of resources to use in research is a major limitation to the development of these phytomedicines in almost all of Africa. There is therefore no authenticity and proof of quality of all the materials used in the preparations of most herbal medicines. The shelf life is not usually known and this places a lot of risk of poisoning on the users.

23 References

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26 Appendix

Table 5.1: Minimum Inhibiton Concentrations in mg/ml of Z. officinale Concentration E.coli E.coli E.coli S.aureus S.aureus S.aureus 1 2 3 1 2 3 900 ------450 ------225 ------112.5 ------56.25 ------28.13 ------14.06 + + + - - - 7.03 + + + + + + 3.52 + + + + + + 1.76 + + + + + + 0.88 + + + + + + 0.44 + + + + + + 0.22 + + + + + + MIC 28.13 28.13 28.13 14.06 14.06 14.06

Key: (-) no growth (+) growth evident

27 Table 5.2: Minimum Inhibiton Concentrations in mg/ml of Penicillin Concentration E.coli E.coli E.coli S.aureus S.aureus S.aureus 1 2 3 1 2 3 900 ------450 ------225 ------112.5 ------56.25 ------28.13 ------14.06 ------7.03 ------3.52 ------1.76 ------0.88 ------0.44 ------0.22 ------MIC <0.22 <0.22 <0.22 <0.22 <0.22 <0.22

Table 5.3: Minimum Bactericidal Concentrations in mg/ml of Z. officinale Concentration E.coli E.coli E.coli S.aureus S.aureus S.aureus 1 2 3 1 2 3 900 ------450 ------225 ------112.5 ------56.25 + + + - - - 28.13 + + + + + + 14.06 + + + + + + 7.03 + + + + + + 3.52 + + + + + + 1.76 + + + + + + 0.88 + + + + + + 0.44 + + + + + + 0.22 + + + + + + MIC 112.5 112.5 112.5 56.25 56.25 56.25