Antibacterial activity of herbal preparations

DETERMINING THE
ANTIBACTERIAL ACTIVITY OF
HERBAL PREPARATIONS ON TWO
CAUSATIVE MICROORGANISMS
OF URINARY TRACT INFECTIONS;
ESCHERICHIA COLI AND
STAPHYLOCOCCUS AUREUS

TITLE PAGE
TABLE OF CONTENTS
ACKNOWLEDGEMENT……………………………………………………………………………………………………………… ii
CERTIFICATION …………………………………………………………………………………………………………………….iii
ABSTRACT……………………………………………………………………………………………………………………………………….iv
TABLE OF CONTENTS…………………………………………………………………………………………………………………. vi
CHAPTER 1……1.0 INTRODUCTION……………………………………………………………………………………. 1
1.1 DEFINITION OF TRADITIONAL MEDICINE, HERBAL PREPARATION AND FINISHED
HERBAL PRODUCT…………………………………………………………………………………………….. 1
1.2 DIFFERENCE BETWEEN HERBAL PREPARATIONS AND ORTHODOX DRUGS………… 2
1.3 USES OF HERBAL PRODUCTS/PREPARATIONS…………………………………………………… 3
1.4 STATEMENT OF THE PROBLEM…………………………………………………………………………. 3
1.5 AIMS AND OBJECTIVES……………………………………………………………………………………… 5
1.6 JUSTIFICATION………………………………………………………………………………………………….. 5
CHAPTER 2……2.0 LITERATURE REVIEW………………………………………………………………………………………… 7
2.1 EFFICACY REQUIREMENTS………………………………………………………………………………… 7
2.1.1 Assessment of activity and efficacy…………………………………………………………………… 7
2.2 URINARY TRACT INFECTIONS…………………………………………………………………………… 8
2.2.1 Causes of urinary tract infections…………………………………………………………………….. 9
2.2.2 Types of Urinary tract infections……………………………………………………………………… 11
2.2.3 Risk Factors……………………………………………………………………………………………………… 11
2.2.4 Symptoms of infection…………………………………………………………………………………… 13
2.2.5 Investigation and Diagnosis of infection………………………………………………………… 14
2.2.6 Treatment………………………………………………………………………………………………………. 15
2.2.7 Prevention……………………………………………………………………………………………………… 18
2.2.8 Causative microorganisms of urinary tract infections…………………………………….. 19
2.3 HERBAL PREPARATION – ARK UROPLUS………………………………………………………… 21
2.4 PHYTOCHEMISTRY OF PLANTS USED IN ARK UROPLUS PREPARATION…………… 22
2.4.1 Flavonoids……………………………………………………………………………………………………… 22
2.4.2 Alkaloids………………………………………………………………………………………………………… 23
2.5 SYNERGISM IN HERBAL PREPARATIONS………………………………………………………… 25

2.5.1 Mechanism of synergy…………………………………………………………………………………… 26
CHAPTER 3……3.0 MATERIALS AND METHODS………………………………………………………………………………………….. 27
3.1 GENERAL CLEANSING AND STERILIZATION OF ALL APPARATUS AND MATERIALS USED… 27
3.1.1 Materials and Apparatus………………………………………………………………………………………………… 27
3.2 SAMPLE COLLECTION…………………………………………………………………………………………………….. 28
3.3 METHODOLOGY…………………………………………………………………………………………………………….. 29
3.3.1 Microbiological analysis………………………………………………………………………………………………….. 29
3.3.2 Preparation of culture media……………………………………………………………………………………………29
3.3.3 Preparation of Agar plates………………………………………………………………………………………………. 29
3.3.4 Inoculation of Culture Plates…………………………………………………………………………………………… 30
3.4 ANTIBACTERIAL SCREENING…………………………………………………………………………………………… 30
3.4.1 Kirby-Bauer Disk Diffusion Test……………………………………………………………………………………….. 30
CHAPTER 4……4.0 RESULTS………………………………………………………………………………………………………………………… 32
4.1 DISCUSSION………………………………………………………………………………………………………………….. 36
4.2 CONCLUSION………………………………………………………………………………………………………………… 38
4.3 RECOMMENDATION…………………………………………………………………………………………………….. 38
REFERENCES…………………………………………………………………………………………………………………………………………….. 39

ii
ACKNOWLEDGEMENTS
I’d like to thank the Almighty God for His divine grace and endowment. He enabled me to
undertake this project and sustained and kept me going through it all.
My earnest compliments and thanks to my supervisors, Mr. Daniel Sereboo and Dr. Addotey for
the time, effort and selfless commitment they dedicated in helping me with this project. Your
counsel and guidelines are deeply appreciated. Thank you very much sirs.
A special thank you to my parents and family for their endless love and support. God richly bless
you for all you’ve done for me.
To Central University College, SAS faculty and all staff of the Pharmacy department, thank you.
Harry, I really appreciate your assistance with my microbiological analysis.
To Dr. Wettey-Boadu, thanks for providing me with the herbal preparations and academic
materials for this project. God bless you.
Last but not least, my fellow course mates thanks for your cooperation. Thank you all.

iii
CERTIFICATION
I hereby declare that this submission is my own work towards the BSc. and that to the best of my
knowledge, it contains no material previously published by another person nor material which has
been accepted for the award of any other degree of the university, except where due
acknowledgement has been made in the text.
Daniel Nii Ayitey Adjin Sackey (PHA/09/01/0615)....……………………….
………………………
Student Signature Date
Certified by:
Mr. Daniel Sereboo ……………………….. ……………………….
Supervisor Signature Date
Certified by:
Prof. J.H. Konning ………………………... ……………………
Head of Department Signature Date

iv
ABSTRACT
This project aimed at determining the antibacterial activity of Ark Uroplus herbal preparation on
two causative microorganisms of urinary tract infections; Escherichia coli and Staphylococcus
aureus. A total of five different batches of the herbal preparation was used for the microbiological
analysis. The test organisms were inoculated on agar plates and after, employing the Kirby Bauer
disk diffusion method, filter paper disks impregnated with the herbal preparation were placed on
the agar and these were incubated at 37°C for 48 hours. Thereafter, zones of inhibition (in mm)
around the disks were measured. The same organisms were tested for susceptibility against
standard antibiotics using the same disk diffusion method. The herbal preparation demonstrated
significant antibacterial activity with zones of inhibition ranging from 11mm - 20mm. It was
concluded that Ark Uroplus herbal preparation had significant antibacterial activity against the test
organisms with a minimum inhibition zone of 11mm. Its use for treatment of urinary tract
infections by Ark Herbal Clinic and Laboratory is justified.

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)
o Upper back and side (flank) pain
o High fever
o Shaking and chills
o Nausea and/or vomiting

 Bladder (cystitis)
o Pelvic pressure
o Lower abdomen discomfort
o Frequent, painful urination
o Bloody or discolored urine
 Urethra (urethritis)
o 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
ARK UROPLUS (HP/AU/01 - HP/AU/05)
Table 1. Ingredients and pharmacological action of Ark Uroplus herbal preparation.
HERBAL
PREPARATIONS
INGREDIENTS PHARMACOLOGIC ACTIVITY
Ark Uroplus Psidium guajava leaves
Magnifera Indica Stem
bark
Antibacterial

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 121o
C 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 121o
C for 15 minutes. After sterilization it was then cooled
to 45-50o
C, 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 121o
C for 15 minutes. After sterilization it was then cooled

to 45-50o
C, 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
Chocolate
Brown
Characteristic Bitter Opaque
2 AU/HP/02
Chocolate
Brown
3 AU/HP/03
Chocolate
Brown
4 AU/HP/04
Chocolate
Brown
5 AU/HP/05
Chocolate
Brown
Table 2. Results of the organoleptic properties for the Herbal Preparations

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 37o
C for 48 hours are tabulated
below.
Table 3. Inhibition of test organisms by Ark Uroplus using disk diffusion test
Table 4. Inhibition of test organisms by standard antibiotics using disk diffusion test
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
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

PICTURES SHOWING THE ZONES OF INHIBITION

0
5
10
15
20
25
30
35
AU/HP/01 AU/HP/02 AU/HP/03 AU/HP/04 AU/HP/05 Tetra Cipro Ceftraixone
A GRAPHICAL REPRESENTATION OF THE ZONES OF INHIBITION
AND SUSCEPTIBILITY OF TEST ORGANISMS
E. coli S. aureus
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.

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