CHAPTER ONE

1. INTRODUCTION

1.0 DEFINITION OF TRADITIONAL MEDICINE, HERBAL PREPARATION AND FINISHED HERBAL PRODUCTS

The World Health Organization (WHO) defines traditional medicine as the sum total of knowledge, skills and practices based on the theories, beliefs and experiences indigenous to different cultures. Traditional medicine is used in the maintenance of health the prevention, diagnosis, improvement or treatment of physical and mental illness, whether explicable or not and is passed on from generation to generation.
Herbal Preparations contain plant parts or plant material in the crude or processed state as active ingredients and may contain excipients. (WHO, 1996a; Busse, 1999). Combinations with chemically defined active substances or isolated constituents are not considered herbal preparations (Busse, 2000; GNDP, 2004).
According to the European Medicine Evaluation Agency (EMEA), herbal preparations are medicinal products containing exclusively herbal drugs or herbal drug preparations as active substances (WHO, 1996b; Busse, 2000). Several chemical constituents with different pharmacological targets are involved in the therapeutic action of herbal preparations. This may be an advantage compared to single isolated compounds, especially when the underlying disease has a multifactorial etiology which is the case in many chronic illnesses.
Herbal preparations may include comminuted or powdered plant material, extracts, tinctures, fatty or essential oils of herbal materials. Herbal preparations are made from different plant parts such as roots, bark, stems, leaves, and fruits whose production involves a fractional, purification, or concentration process (Evans, 1989; Evans, 1996). They also include preparations made by steeping or heating herbal materials in alcoholic beverages and/or honey, or in other materials. Based on the European Medicine Evaluation Agency (EMEA), Ghana Food and Drugs Authority and WHO quality guidelines, the herbal drug or preparation in its entirety must be considered as the active ingredient. It can be deduced from this and from the fact that herbal medicines are complex mixtures of substances that great effort must be made to ensure quality, safety and efficacy.
Finished herbal products consist of herbal preparations made from one or more herbs. If more than one herb is used, the term mixture herbal product can also be used. However, finished products or mixture products to which chemically defined active substances have been added, including synthetic compounds and/or isolated constituents from herbal materials, are not considered to be herbal (WHO/EDM/TRM/2000.1)

1.2 Difference between Herbal Preparation and the Orthodox Drugs

The classical pharmaceutical development concept is targeted at isolating a single component from a medicinal plant which can later be manufactured synthetically or extracted on a large scale (e.g. atropine from atropa belladona). Such a product does not fall under herbal preparation. It is a fact that complex herbal extracts have been shown to be therapeutically active and safe (Linde et al., 1996; Oken et al., 1998).

1.3 Uses of Herbal Preparations

In Ghana, herbal preparations have been used in the treatment of many diseases including malaria, jaundice, menstrual pain, waist pain, piles, erectile dysfunction, hypertension, rheumatism, and many others. Herbal preparations are not only used in Ghana but also in developed countries such as UK, China, United States and India, for various disease conditions (CSIR, 1992; Gulla et al., 2001).
Examples of these herbal preparations in include; Ark Uroplus, Ark Prostacare, Herbaquin, Angel Natural capsules, Angel Cream, Engel Mighty power, Capital O2 Living bitters, Ark Bactima, Ark Virex, Angel Fatwikεkε, Top tonic, Ahuodzen ancient Herbal Brew, Yafo Man Capsules, Taabea Herbal mixture. Other preparations are St. John Wort, Dan Shen, Kava, Comfrey, Dong Quai, Siberian, Ginkgo Biloba, Valerian, Saw Palmetto, Feverfew, Garlic, Ginger, Cranberry, Aloe, Chan Su, Cat’s claw, Asian ginseng, Soy milk, Ephedra, Calamus, Chaparral, Licorice, Shankhapushi, Borage oil, Senna, Pokeweed, Hawthorn (Wahed and Dasgupta, 2001; Dasgupta et al., 2002).

1.4 STATEMENT OF THE PROBLEM

The use of traditional medicine has expanded globally and has gained popularity. It has not only continued to be used for primary health care of the poor in developing countries, but has also been used in countries where conventional medicine is predominant in the national health care system. With the tremendous expansion in the use of traditional medicine worldwide, safety and efficacy as well as quality control of herbal medicines and traditional procedure-based therapies have become important concerns for both health authorities and the public (Zhang Xiaorui, 2000). In recent years, there has been growing interest in alternative therapies and the therapeutic use of natural products, especially those derived from plants (Goldfrank et al., 1982; Vulto and Smet, 1988; Mentz and Schenkel, 1989). WHO has developed and issued a series of technical guidelines such as the General Guidelines for Methodologies on Research and Evaluation of Traditional Medicine, Regulation and Registration of Herbal Medicine, Guidelines for the assessment of herbal medicines; Research guidelines for evaluating the safety and efficacy of herbal medicines and The WHO Traditional Medicine Program. These programs and guidelines however are still not sufficient to cover the many challenging issues in the research and evaluation of traditional medicines and its practice. European countries such as Germany, France, Sweden, Denmark and Switzerland have established specific national regulations concerning the evaluation of the quality parameters of herbal products. Other countries such as the Netherlands, the United Kingdom and Portugal evaluate them in the same way as pharmaceuticals. In the dietary supplement market in the United States, herbal products are easily available to the general public (Busse, 2000; Ang-Lee et al., 2001). A major problem with this is that products which contain potentially toxic contaminants or products which do not contain the accurate amount of constituents may be on the market.
In Ghana, however, even though herbal products are on the market, not much research has been done in relation to its quality, safety and efficacy. The manufacturers of the herbal preparations in Ghana do not have the required laboratories or expertise to perform quality control on the preparations they produce. This brings the problem of inconsistency on the quality, safety and efficacy of the herbal preparations in the country.
This project therefore seeks to evaluate the activity and efficacy of a selected herbal preparation on the Ghanaian market using harmonized procedures.

1.5 AIMS AND OBJECTIVES * To determine antibacterial activity of Ark Uroplus against selected organisms that cause urinary tract infections. (Escherichia Coli and Staphylococcus Aureus)

1.6 JUSTIFICATION

WHO’s policy of encouraging the development and utilization of traditional medicine in the Primary Health Care delivery system, particularly, in the third world countries is based on the sound recognition of the role that traditional medicine is already contributing to the health care program in most developing countries such as Ghana. According to UNDP (2007), it is estimated that about 80% of the Ghanaian populace rely on herbal preparations. The Food and Drugs Law in Ghana mandates the Ghana Food and Drugs Authority (FDA) to implement regulatory measures that aim at achieving high standards of quality of food and drugs, including herbal preparations (GNDP, 2004).
It was reported that some herbal practitioners started packaging “Tampico” orange juice manufactured by Fan Milk Ghana Limited and Turpentine, a toxic-colorless liquid mostly used by carpenters and painters, deceitfully claiming they are herbal concoctions for treatment of HIV/AIDS, severe headache and other infections. (The Herald Newspaper, 2010). Also a survey conducted by (M.J. Newman, 2000) at the Korle Bu teaching hospital, showed that urinary tract infections represented 13.1% and 4.2% of hospital-acquired and community-acquired infections respectively. This highlights the importance of ascertaining the activity and efficacy of herbal preparations and the need for more research into herbal remedies for urinary tract infections.

CHAPTER TWO

2.0 LITEREATURE REVIEW

2.1 EFFICACY REQUIREMENTS

It is important for herbal medicines, and particularly for those made from mixture herbal products, that the requirements for proof of efficacy and activity, including the documentation required to support the indicated claims. For the treatment of minor disorders, for non- specific indications, or for prophylactic uses, less stringent requirements (e.g. observational studies) may be adequate to prove efficacy. However, the extent of traditional use and the experience with a particular herbal medicine and supportive pharmacological data are taken into account. The level of the evidence and the grading of recommendations must correspond to the nature of the illness to be treated. (WHO/EDM/TRM/2000.1)

2.1.1 Assessment of activity and efficacy

Ark Uroplus herbal preparation is used to treat urinary tract infections; cystitis, urethritis, pyelonephritis.

2.2 URINARY TRACT INFECTIONS - UTIs

Infections in the urinary tract are relatively common (JJustad, 2010). These infections are often referred to as “bladder infections”. They are also known as urinary tract infections or UTIs. When an infection is confined to the bladder, the correct term to use is cystitis. The kidneys can also become infected. This is referred to as pyelonephritis. UTIs are believed to be the most common bacterial infection with an estimated 150 million cases globally (Hooton TM et al., 1996).
The urinary system (also called the excretory system) basically produces, stores and eliminates urine. It includes two kidneys, two ureters, the bladder, and the urethra as well as two sphincter muscles. * Kidneys: Located in the upper abdomen close to the spine, at the level of or just below the ribcage. Kidneys filter water soluble waste products from the blood and form urine. * Ureters: Thin tubes through which urine travels from each kidney to the bladder. Small muscles that are present in the walls of these tubes constantly tighten and relax to force urine away from the kidney. * Bladder: Hollow organ shaped like a balloon. It is located in the pelvis and held in place by ligaments that attach to other organs and the pelvic bones. Its function is to store urine. It can swell to be quite large but generally holds about two cups of urine comfortably for 2 to 5 hours.

* Urethra: Tube that allows urine to pass from the bladder to outside the body. * Sphincter Muscles: Circular muscles that keep urine from leaking out of the bladder. The urinary system is designed to minimize the risk of serious infection in the kidneys. It does this by preventing the urine from flowing back up into the kidneys from the bladder. The majority of urinary infections are confined to the bladder. Nerves in the bladder signal the brain when it is time to urinate. The sensation to urinate becomes stronger as the bladder continues to fill and reaches its limit. When a person urinates, the brain signals the bladder muscles to tighten and squeeze the urine out of the bladder. It also signals the sphincter muscles to relax so that urine can exit through the urethra. Urine is normally clear, and has a yellow/amber color. 2.2.1 CAUSES OF URINARY TRACT INFECTIONS

The following are possible ways by which an individual can develop a UTI aside pathogenic causes:

2.2.1.1 Changes in the Acid-Alkaline Balance of the Urinary Tract.

Changes in the amount or type of acid within the genital and urinary tracts are major contributors to lowering the resistance to infection. For example, beneficial organisms called lactobacilli increase the acidic environment in the urinary tract. Reductions in their number, increases pH and therefore the risk of infection (The HPCSA and the Med-Tech Society).

2.2.1.2 Biofilm

One theory, called the biofilm mode of growth, suggests that sometimes bacteria form capsules that adhere to the urinary tract and this protects them from many of the body's normal defenses (The HPCSA and the Med-Tech Society).

2.2.1.3 Medical devices

Another common source of infection is through the use of catheters, or tubes, placed in the urethra and bladder. A person who cannot void or who is unconscious or critically ill often needs a catheter that stays in place for a long time. Some people, especially the elderly or those with nervous system disorders who lose bladder control, may need a catheter for life. Bacteria on the catheter can infect the bladder (The HPCSA and the Med-Tech Society).
According to several studies, women who use a diaphragm are more likely to develop a UTI than women who use other forms of birth control. Recently, researchers found that women whose partners use a condom with spermicidal foam also tend to have growth of Escherichia coli bacteria in the vagina (Foxman, 2002).

2.2.2 TYPES OF URINARY TRACT INFECTIONS

The following types of urinary tract infections have been identified (JJustad, 2010).

* Urethritis: Infection of the urethra * Cystitis: Infection of the bladder * Pyelonephritis: Infection of the kidneys

2.2.3 RISK FACTORS

* Gender: Females are more prone to urinary tract infections than males. This is because the urethra is shorter which cuts down the distance that bacteria have to travel to reach the bladder. The urethral opening is also much closer to the anus and can come into contact with bacteria more readily. Females also lack the prostatic secretions which are present in males. Prostatic secretions are bacteriostatic which means that they keep the bacteria from growing and multiplying as readily. As males age, they often have enlargement of the prostate gland. An enlarged prostate gland also can slow the flow of urine, thus raising the risk of infection. When the bladder does not completely empty, bacteria are not fully flushed out and can multiply and cause an infection. In males who are not circumcised, there are more bacteria living closer to the opening of the urethra which increases their risk for developing an infection. After menopause, females are also more prone to infections due to lack of estrogen (JJustad, 2010).

* Sexual activity: Increased sexual activity leads to more frequent urinary tract infections in women especially when a spermicide is used and also when there is more than one sexual partner. (JJustad, 2010).

* Urinary tract abnormalities: Neurological abnormalities such as a neurogenic bladder can lead to recurrent urinary tract infections as the bladder does not empty correctly. Other anatomical variations that block the flow of urine and emptying of the bladder cause an increase in infections. Having to use a catheter to empty the bladder leads to infections mostly due to introduction of bacteria to the inside of the bladder when the catheter is inserted. When a catheter is left in the bladder for more than a couple of days, the bladder becomes colonized with bacteria and is prone to more infections (JJustad, 2010).

* Suppressed immune system: Diabetes and other diseases can impair the immune system and increase the risk of infection. The immune system is the body’s defense against bacteria and developing infections (JJustad, 2010).

2.2.4 SYMPTOMS OF INFECTION

Most infections involve the lower urinary tract; the bladder and urethra. Infections in the urinary tract do not always cause symptoms. Frequent symptoms of an infection include: * Strong, persistent urge to urinate * Burning sensation when urinating * Passing urine frequently in small amounts * Cloudy, pink, or brown urine * Strong-smelling urine * Pelvic or rectal pain More specific symptoms may depend on which part of the urinary system is infected (JJustad, 2010). * Kidneys (pyelonephritis) * Upper back and side (flank) pain * High fever * Shaking and chills * Nausea and/or vomiting * Bladder (cystitis) * Pelvic pressure * Lower abdomen discomfort * Frequent, painful urination * Bloody or discolored urine * Urethra (urethritis) * Burning with urination

Sometimes no symptoms are present. Elderly individuals may not have discomfort of any type. However, a new onset of confusion or worsening of confusion can frequently be seen when an infection is present in the elderly (JJustad, 2010).

2.2.5 INVESTIGATION AND DIAGNOSIS OF INFECTION

* FBC * Mid-stream specimen of urine for microscopy, culture and sensitivity (re-culture urine after treatment) * Abdominal ultrasound scan in children if indicated * Urinalysis * Imaging of urinary tract in recurrent or persistent cases to exclude anatomical abnormalities, lower urinary tract obstruction etc. * Fasting Blood glucose * Urethrocystoscopy in selected cases
(Standard Treatment Guidelines, 2010)

2.2.6 TREATMENT

PHARMACOLOGICAL TREATMENT

ANTIBIOTICS
In mild/moderate cases
Ciprofloxacin oral
Adults
500 mg 12 hourly for 7 days
Co-amoxiclav oral
Children
12 years and above; one 500/125 tablet 12 hourly for 7 days
6-12 years; 5 ml of 400/57 suspension 12 hourly for 7 days
1- 6years; 2.5 ml of 400/57 suspension 12 hourly for 7 days
1month-1 year; 0.25ml/kg body weight of 125/31 suspension 8 hourly for 7 days
Neonates; 0.25 ml/kg body weight of 125/31 suspension 8 hourly for 7 day
OR
Cefuroxime oral
Children
12-18 years; 250 mg 12 hourly for 5-7 days 2-12 years; 15 mg/ kg 12 hourly (maximum 250 mg) for 5-7 days
3 months-2 years; 10 mg/kg 12 hourly (maximum 125 mg) for 5-7 days
In severe cases
Ciprofloxacin IV
Adults
200 mg 12 hourly for 7 days
OR
Gentamicin IV
Adults
40-80mg for 7 days
OR
Ceftriaxone IV
Adults
1-2g daily for 7 days

Amoxicillin IV
Children
1 month-18years; 20-30 mg/kg 8 hourly (maximum 500 mg) for 5-7days
Neonates (dose doubled in severe infection) 7-28 days; 30 mg/kg 8 hourly for 5-7days < 7 days; 30 mg/kg 12 hourly for 5-7days plus Gentamicin, IV, (slow intravenous injection over at least 3 minutes)
Children
12-18years; 2 mg /kg 8 hourly
1month-12years; 2.5 mg/kg 8 hourly
OR
Cefuroxime IV
Children
1 month-18 years; 20 mg/kg 8 hourly maximum 750mg, (increase to 40-50 mg/kg maximum 1.5g 6-8 hourly in severe infections)
Neonates (double the dose in severe infections, IV route only) 21-28 days; 25 mg/kg 6 hourly 7-12 days; 25 mg/kg 8 hourly < 7days; 25 mg/kg 12 hourly
Treatment is dependent on severity of infection as well as the age of the patient (Standard Treatment Guidelines, 2010).

TRADITIONAL MEDICINE TREATMENT

* Ark Uroplus Mixture (Dried Guava leaves + Dried Mango Stem Bark): 10ml three times daily (Ark Clinic and Laboratory).

NON PHARMACOLOGICAL TREATMENT

* Liberal oral fluids to encourage good urinary output * Personal hygiene and proper cleaning after defecation * Pre-coital and post-coital emptying of the bladder
(Standard Treatment Guidelines, 2010).

2.2.7 PREVENTION

The following steps can be taken to reduce the risk of urinary tract infections. * Drinking of water helps to dilute urine and causes increased frequency of urination which flushes out bacteria (JJustad, 2010). * Carbonated beverages (soda), caffeine (coffee, tea, soda), and alcohol are the most common irritants of the bladder. Tomato based products, sugar, chocolate, and highly spiced foods can also cause irritation in the bladder. Artificial sweeteners, especially aspartame should also be avoided (JJustad, 2010). * After urinating or bowel movements, wiping from the front to the back helps prevent bacteria in the anal region from spreading to the vagina and urethra (JJustad, 2010). * Flushing the bacteria out of the bladder and urethra is important to prevent infections. This can be achieved by regular emptying of the bladder (JJustad, 2010). * Cranberry products: Cranberry juice or tablets are promoted as a way to help prevent infections. Reviews of literature have found evidence for this in women but not in everyone (JJustad, 2010).

2.2.8 CAUSATIVE MICROORGANISMS OF URINARY TRACT INFECTIONS (UTIs)

Some of the causative microorganisms for UTIs are the following:

2.2.8.1 Pathogenic bacteria Escherichia coli accounts for 85% of community acquired and 50% of hospital acquired urinary tract infections. Within the Escherichia coli species a number of subgroups are frequently isolated from patients with urinary tract infections (Brooks et al., 1981; Gruenberg, 1969; Roberts and Phillips 1979; Vosti et al., 1964). Gram negative bacteria such as Klebsiella and Proteus; and Gram positive Enterococcus faecalis and Staphylococcus aureus are causative agents for the remainder of community acquired infections (Kennedy et al., 1965). The remainder of hospital acquired infections usually occur after colonization with Klebsiella, Enterobacter, Citrobacter, Serratia, Pseudomonas aeruginosa, Providencia (Kennedy et al. 1965). Notably, the patient’s age may influence the type of infective organism present with Staphylococcus aureus now accounting for 10% of urinary tract infections in young females compared to less than 1% in elderly female patients (Kennedy et al., 1965).
According to the WHO, Escherichia coli is a bacterium that is found in the gut of humans and animals. Most strains of Escherichia coli are harmless. Most human beings have a significant concentration of such "good" Escherichia coli in their gut (typically up to 1,000,000 per gram of faeces).

Escherichia coli is responsible for 75 - 90% of uncomplicated cystitis cases. In most cases of UTIs, Escherichia coli, which originates as a harmless microorganism in the intestines, spreads to the vaginal passage, where it invades and colonizes the urinary tract. Some bacteria may be able to invade into deeper tissue in the bladder, where they survive to re-infect the patient after resolution of the previous infection. In addition, a considerable number of Escherichia coli UTI cases results in bacteremia and consequently death (The HPCSA and the Med Tech Society).

Staphylococcus aureus is a gram-positive, spherical bacterium with a diameter of 1–1.3 µm. When viewed microscopically, it appears in clusters, like bunches of grapes. Growing in food, some strains can produce toxins which cause acute gastro-intestinal diseases if ingested. They also cause infections of the urinary tract. (The Food Safety File, Edition 2008)

2.3 HERBAL PREPARATIONS – ARK UROPLUS

HERBAL PREPARATIONS | INGREDIENTS | PHARMACOLOGIC ACTIVITY | Ark Uroplus | Psidium guajava leavesMagnifera Indica Stem bark | Antibacterial | | | |
ARK UROPLUS (HP/AU/01 - HP/AU/05)
Table 1. Ingredients and pharmacological action of Ark Uroplus herbal preparation.

2.4 PHYTOCHEMISTRY OF PLANTS USED IN ARK UROPLUS HERBAL PREPARATION

The main chemical constituents of magnifera indica are the following: flavonoids, citric acid, reducing sugars, terpinolene, mycrene, careen, terpenoid, sabinene and limonene. The chemical constituents of psidium guajava are tannins (hydrolysable), volatile oils (sesquiterpenes), saponins and sapogenins, reducing sugars, flavonoids, calcium oxalate crystals, starch, vitamin A, vitamin B group (niacin, nicotinic acid, thiamine) and vitamin C; carotenoids, fiber and fatty acids (Begum, et al., 2002; Arima and Danno, 2002).
Complex mixtures such as fats, fixed oils, volatile oils, and resins had been prepared and used, although, virtually nothing was known about their composition. Not all the chemical compounds elaborated by plants are of equal interest. The active principles are frequently alkaloids or glycosides and flavonoids and these, therefore deserve special attention. Other groups such as carbohydrates, fats and proteins are of dietetic importance.

2.4.1 Flavonoids

Flavonoids (Kawal et al., 2009) are a major class of oxygen-containing heterocyclic natural products that are widespread in green plants. Generally, they are found as plant pigments in a broad range of fruits and vegetables. These are C15 compounds composed of two aromatic rings linked through a three-carbon bridge with a carbonyl functional group located at one end of the bridge. Flavonoids have been recognized as having a protective effect in plants against microbial invasion by plant pathogens. Flavonoid-rich plant extracts have been used for centuries to treat human disease. Isolated flavonoids have been shown to possess a host of important biological activities, including antifungal and antibacterial activities. The potential of naturally occurring flavonoids as anti-infective agents has been recognized. However, reports of activity in the field of antibacterial flavonoid research are widely conflicting, probably owing to inter and intra assay variations in the susceptibility testing.

2.4.2 Alkaloids Alkaloids (Omprakash, 2013) have been defined in various ways, but one definition comes fairly close to actuality. An alkaloid is a plant-derived compound that is toxic or physiologically active, contains nitrogen in a heterocyclic ring, is basic, has a complex structure, and is of limited distribution in the plant kingdom. It can also be described as group of mildly alkaline compounds containing nitrogen, mostly of plant origin and of moderate molecular complexity, which produce various physiological effects on the human body. Nearly 3,000 alkaloids have been recorded; the first to be prepared synthetically in 1886 was one of the simplest, called coniine, or 2-propyl piperidine, (C5H10NC3H7). It is highly poisonous; less than 0.2g is fatal. Coniine, obtained from seeds of the hemlock, was the poison used in the execution of Socrates. Some 30 of the known alkaloids are used in medicine. For example, atropine, obtained from deadly nightshade, causes dilation of the pupils; morphine is a painkiller; quinine is a specific remedy for malaria; nicotine is a potent insecticide; and reserpine is a valuable tranquilizer (Robbers, 1988). In an experimental study conducted by the Institute of Chemistry and Institute of Mycology and Plant Pathology, University of the Punjab, Pakistan, it was found that the magnifera indica contained five flavonoids which possessed antibacterial activity against bacterial species. Namely, Lactobacillus sp., Escherichia coli, Azospirillium lipoferum and Bacillus sp. (Kawal et al., 2009). All the tested concentrations of the five flavonoids significantly reduced the growth of all the five tested bacterial species. These flavonoids were; * epicatechin-3-O- β-glucopyranoside * 5-hydroxy-3-(4-hydroxylphenyl)pyrano[3,2-g]chromene-4(8H)-one * 6-(p-hydroxybenzyl)taxifolin-7-O-β-D-glucoside (tricuspid) * quercetin-3-O-α-glucopyranosyl-(1→2)- β -glucopyranoside * epicatechin(2-(3,4-dihydroxyphenyl)-3,4-dihydro-2H-chromene-3,5,7-triol)

Another research study showed that the extracts of the stem bark of magnifera indica had an inhibition zone of 17.75mm (Jasminder Kaur et al., 2010). The crude ethanol extract of mango stem bark at a concentration of 100 mg/mL is found to have potential antimicrobial activity against methicillin-resistant staphylococcus aureus and Escherichia coli (Jasminder Kaur et al., 2010). Furthermore the study conclusively demonstrated the free radical scavenging activity and antibacterial activities of mango stem bark.
A study was designated to evaluate the antibacterial activities of aqueous and ethanol extracts from leaves, roots and stem bark of Psidium guajava (Sanches NR et al., 2005). The antibacterial activities of the extracts against bacteria were tested by using both micro dilution n assay. The aqueous extracts of Psidium guajava leaves, roots and stem bark were active against the gram-positive bacteria Staphylococcus aureus (MICs=500, 125 and 250 µ g/ml, respectively) and Bacillus subtilis (MICs=500 µ g/ml), and virtually inactive against the gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa (MICs >1000 µ g/ml). The ethanol extracts showed higher antimicrobial activity as compared to aqueous extracts. Based on this finding, the ethanol extract of psidium guajava leaves was fractionated on silica gel column chromatography in a bioassay-guided fractionation affording flavonoid mixture, triterpenes (α- and β-amyrin) and sterol ( β -sitosterol). Flavonoid mixture showed good activity on S. aureus with MIC of 25 µ g/ml. β-sitosterol was inactive for all the bacteria tested (Sanches NR et al., 2005).

2.5 SYNERGISM IN HERBAL PREPARATIONS

Natural products including plants offer large structural diversity of chemicals to be used as various class of pharmacological disorders and modern techniques for separation, structure elucidation, screening and combinatorial synthesis have led to revitalization of plant products as sources of new drugs. Synergistic interactions are of vital importance in phytomedicines. Even though several difficulties are faced, isolating the single active compound from the phytomedicine is always targeted. But the efficacy of the whole herb may lie on the low doses of the active constituents present in an herbal product altogether. Until recently there has been little clinical evidences demonstrated which showed that very low dose of the active constituents exert any therapeutically relevant effect. Most of the effective phytomedicines sold on the market are prepared traditionally which are available as whole extracts or individual herb. The alternative medicine practitioners have always believed that synergistic interactions between the components of individual or mixtures of herbs are a vital part of their therapeutic efficacy. In the absence of clinical proof this has led skeptics to dismiss these medicines as placebos, and it is compounded by the fact that there may be result in a measurable efficacy only after continuous administration, which might be due to a cumulative effect (Pulok K Mukherjee et al., 2010).

2.5.1 Mechanisms of Synergy

Two broad types of synergy can be distinguished, based on the nature of the interaction: pharmacodynamic or pharmacokinetic. Pharmacodynamic synergy results from two drugs directed at a similar receptor target or physiological system. For example, combinations of allosteric modifiers at the gamma-amino butyric acid A (GABAA) receptor create potent synergistic interactions (DeLorey TM et al., 1993; van Steveninck AL et al., 1993; Vanover KE et al., 1999). Pharmacokinetic synergy results from the processes of drug absorption, distribution, biotransformation, or elimination. For example, combined administration of drugs which compete for albumin binding will elevate the free drug concentrations, and thus potentiate their actions (Schoener EP, 1986).

CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 General cleansing and sterilization of all materials and apparatus used

All glassware were washed thoroughly in soap solution and rinsed with distilled water and dried before used. The laboratory benches were also cleaned and disinfected before use. All glassware such as pipettes, beakers, stirrers, spatula and test tubes were disinfected with Camel antiseptic solution. Petri dishes, and other equipment were autoclaved to kill all contaminating bacteria. They were then soaked and washed in soapy water also containing disinfectant, after which they were packed into suitable receptacles to dry. Disposable gloves were worn during these operations. The Petri dishes were placed inverted in the canisters to ensure thorough dryness before sterilization. The pipettes and test tubes were also sterilized in an autoclave at 121oC for 15 minutes, after which they were stored in a clean sterile environment.

3.1.1 Materials and Apparatus * Incubator capable of maintaining a temperature to within ± 0.5 ºC of 35 and 37 ºC * Autoclave for sterilizing glassware and all equipment and culture media. * Laboratory weighing balance, accuracy ± 0.05 g. * Racks for test tubes and bottles of prepared culture media and dilution water. * Pipettes (10ml), glass beakers (250ml capacity), test tubes (50ml), measuring cylinders, petri dishes, spatula, wash bottles, aluminum foil, filter paper, * Test-tube racks to hold tubes in incubator and during storage. * Bunsen burner * Wire loops for inoculating media * Waste bin. * Culture media: for example Eosin Methylene Blue and Mannitol salt * Disinfectant for cleaning laboratory surfaces and the pipette discard container. * Detergent for cleaning glassware and equipment. * Autoclave tape. 3.2 SAMPLE COLLECTION

The samples were collected in batches from the Ark Clinic and Laboratory at Abeka Lapaz, Accra between November 2012 and April 2013 and analyzed at the Microbiology laboratory of SAS faculty of Central University College. Five different batches of Ark Uroplus herbal preparation were purchased from Ark Herbal Clinic and Laboratory in Accra. The criterion for selection was based on easy accessibility and acceptability of the brand. Coding was done to give each sample a unique identity. The general innovator was AU/HP. All samples were within their shelf lives as at the time of the project. The samples were kept sealed until the time of analysis. For each batch 10ml was taken for the analysis. The freshly opened samples were first analyzed for their organoleptic properties before test for antibacterial activity and efficacy.
3.3 METHODOLOGY

3.3.1 Microbiological Analysis

3.3.2 Preparation of culture media (Microbiological media)

Mannitol Salt (MS) was used to culture Staphylococcus aureus and Eosin Methylene Blue (EMB) for Escherichia coli.

3.3.3 Preparation of Agar Plates
Preparation of EMB media (Ratio is 40g/1L)
7.6g of EMB powder was accurately weighed and dissolved in 100ml of warm deionized water. The resulting solution was stirred with frequent agitation until all the powder was dissolved. After, the solution was sterilized in the autoclave at 121oC for 15 minutes. After sterilization it was then cooled to 45-50oC, and dispersed into plates. The plates were allowed to solidify and placed at an inverted position to avoid excessive moisture on the surface of the medium. Preparation of MS media (Ratio is 76g/1L)
4g of MS powder was accurately weighed and dissolved in 100ml of warm deionized water. The resulting solution was stirred with frequent agitation until all the powder was dissolved. After, the solution was sterilized in the autoclave at 121oC for 15 minutes. After sterilization it was then cooled to 45-50oC, and dispersed into plates. The plates were allowed to solidify and placed at an inverted position to avoid excessive moisture on the surface of the medium.
3.3.4 Inoculation of Culture Plates
An inoculation loop was sterilized by means of heating with a Bunsen burner and then dipped in the 0.5ml microorganism. Holding the petri dish lid at an of 30-45° angle, the inoculating loop was streaked with 0.5ml microorganism culture from the outside of the plate toward the center in a zigzag pattern and that covered approximately 25% of the plate surface. The petri plate was turned 90° to the right, and the inoculation loop was streaked through the last section of the plate, moving from the outside to the inside in a zigzag motion. The process was repeated until the entire plate surface was covered.

3.4 ANTIBACTERIAL SCREENING

3.4.1 Kirby Bauer Disk diffusion method for antimicrobial susceptibility testing.

Filter paper disc diffusion assay:
The Kirby Bauer disk diffusion method was used for screening of antimicrobial activity for each herbal preparation sample. A known volume i.e. 10 ml of each herbal preparation was coated on separate sterile filter paper discs (Whatman No. 1) measuring 5mm in size. These herbal mixture-impregnated discs were made dry under laminar flow cabinet. After streaking of the bacteria with the inoculation loop on each agar plate the coated discs were positioned at four areas of the inoculated agar plate. Sterile distilled water was used as negative control, while broad-spectrum antibiotics i.e. tetracycline, ceftriaxone and ciprofloxacin were used as positive control for obtaining comparative results. All treated and untreated plates were incubated for 48 hours at 37 ºC and size of inhibition zone diameters surrounding filter paper disc was measured. The agar plates were incubated within 15 minutes of disk application. This limits pre-diffusion which may otherwise result in large zone sizes.

CHAPTER FOUR

4.0 RESULTS
The presence of bioactive substances have been reported to confer resistance to plants against bacteria, fungi and pests and therefore explains the demonstration of antibacterial activity (Srinivasan et al., 2001).

Item No. | Herbal Sample | Color | Odor | | Taste | Clarity | 1 | AU/HP/01 | ChocolateBrown | Characteristic | | Bitter | Opaque | 2 | AU/HP/02 | Chocolate Brown | Characteristic | | Bitter | Opaque | 3 | AU/HP/03 | Chocolate Brown | Characteristic | | Bitter | Opaque | 4 | AU/HP/04 | ChocolateBrown | Characteristic | | Bitter | Opaque | 5 | AU/HP/05 | Chocolate Brown | Characteristic | | Bitter | Opaque |
Table 2. Results of the organoleptic properties for the Herbal Preparations

Herbal Preparation | Escherichia coli | Staphylococcus aureus | AU/HP/01 | 17.65±0.55mm | 11.85±0.11mm | AU/HP/02 | 18.25±0.12mm | 13.25±0.22mm | AU/HP/03 | 16.95±0.01mm | 13.03±0.5mm | AU/HP/04 | 19.10±0.31mm | 12.30±0.15mm | AU/HP/05 | 20.05±0.26mm | 14.25±0.21mm |
Inhibition zone diameter: In this project, the activity and effectiveness of Ark Uroplus herbal preparation was confirmed by filter paper disc diffusion assay and growth inhibition zone diameters were measured. The results after incubation of the agar plates at 37oC for 48 hours are tabulated below.
Table 3. Inhibition of test organisms by Ark Uroplus using disk diffusion test Antibiotics 500mg/ml | | Escherichia coli | Staphylococcus aureus | Tetracycline | | 25.23±0.23mm | 19.73±0.405mm | Ceftriaxone | | 30.05±0.05mm | 15.23±0.024mm | Ciprofloxacin | | 23.0-3±0.08mm | 24.2±0.12mm |

Table 4. Inhibition of test organisms by standard antibiotics using disk diffusion test

PICTURES SHOWING THE ZONES OF INHIBITION

Figure 8. Showing antibacterial activity against tested organisms. Values are expressed in mm.
Figure 8. Showing antibacterial activity against tested organisms. Values are expressed in mm.

4.1 DISCUSSION

Ark Uroplus is an antibacterial herbal preparation which is indicated for the treatment of urinary tract infections; cystitis, urethritis, pyelonephritis. Its herbal constituents are extracts from the dried leaves of Psidium guajava and the dried stem bark of Magnifera indica.
The increase of antibiotic resistance of microorganisms to conventional drugs has necessitated the search for new, efficient and cost effective ways for the control of infectious diseases. This project also sought to study the antimicrobial susceptibility testing so as to provide a reliable predictor of how an organism is likely to respond to antimicrobial therapy in the infected host. This type of information aids in selecting and developing the appropriate antimicrobial agent as well as provides data for epidemiological surveillance. The need for antimicrobial agents from natural sources contributes to the development of effective screening systems. The results of different studies provide evidence that some medicinal plants might indeed be potential sources of new antimicrobial agents (Kone et al., 2004). Plants produce a huge variety of secondary compounds as natural protection against microbial and insect attack. Indeed, many of these compounds have been used in the form of whole plants or plant extracts for food or medical applications in human because plants are the natural reservoir of many antimicrobial, antifungal as well as various therapeutic activities (Lucy and Da Silva, 1999). Acceptance of medicines from such plant origin as an alternative form of healthcare is increasing because they are serving as promising sources of novel antibiotic Prototypes. Some of the phytochemical compounds e.g. Glycoside, Saponins, Tannins, Flavonoids, Terpenoid, Alkaloids, have variously been reported to have antimicrobial activity (Okeke et al., 2001). Due to this reasons, a general question was brought forward; “does Ark Uroplus Herbal preparation have any antibacterial activity against selected microorganisms which cause urinary tract infections?”
The project was aimed at determining the activity of the herbal preparation against the selected organisms. This aim was achieved by applying the Kirby Bauer disk diffusion method to derive and measure the zones of inhibition against the growth of the microorganisms.
From the microbiological experiment carried out, the results obtained showed that Ark Uroplus herbal preparation showed a significant growth inhibition of the test organisms by showing zones of inhibition between 11mm and 20mm. Table 3 shows the values of inhibition zones of each sample of Ark Uroplus herbal preparation. The values for the zones of inhibition in the growth of Escherichia coli were large as compared to values of the inhibition of staphylococcus aureus. Escherichia coli exhibited the highest level of susceptibility to the antibacterial effect of the herbal preparation. This was also the pattern observed with the standard antibiotics. Table 4 shows the values for the zones of inhibition for the standard antibiotics. In comparison with the broad spectrum antibiotics, Ark Uroplus herbal preparation showed substantial prove that it possess antibacterial activity. Figure 8 is a graphical representation of the values obtained after the experiment, showing the levels of microorganism susceptibility in mm.

4.2 CONCLUSION

In conclusion, Ark Uroplus Herbal preparation possess antibacterial activity against Escherichia coli and staphylococcus aureus. It showed antibacterial activity against all the test organisms with a minimum zone of inhibition measuring 11mm and the maximum zone of inhibition measuring 20mm. Its use for the treatment of UTIs by Ark Clinics and Laboratory is therefore justified.

4.3 RECOMMENDATION

Due to the unstable nature of liquid herbal products it would be necessary for further research to be done to convert this product into a conventional dosage form or an appropriate pharmaceutical dosage form such as a capsule or a tablet.
More importantly there is need for detailed scientific study of traditional medical practices to ensure that valuable therapeutic knowledge of these plants are preserved and also to provide scientific evidence for their efficacies.