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DETERMINATION OF ANTIBACTERIAL ACTIVITY OF WHOLE PLANT EXTRACT
OF Phyllantltus amarus Schum and thonn (kabamba maliba) AGAINST Shigella
dysenteriae.
BY
NAME: MAGIMBI ARTHUR
REG.NO: BPH/0009/141/DU
A RESEARCH REPORT SUBMITED TO THE SCHOOL OF
PHARMACY OF KAMPALA INTRERNATIONAL UNIVERSITY
IN PARTIAL FULFILMENT OF THE REQUIREMENTS
FOR THE A WARD OF BACHELOR
DEGREE OF PHARMACY.
SUPERVISOR: DR. ODDA JOHN
MAY2018
DECLARATION
I, MAGIMBI ARTHUR declare that this research is my original work and has not been
presented for the award of any degree elsewhere.
Signature ....... . .. ~ .... . .. . .... . ...... . Date .... . ..... ~"1/P.S/ ?:-PJ.{ ........ .
BPH/0009/141/DU
DEDICATION
I dedicate this to God , all my family members and dear friends for the spiritual, financial and
moral support they have given me through this period. I do this with out forgetting all lecturers
and finally my supervisor Dr John Odda for the effort they have put in to help me finish my
course.
ii
APPROVAL
I hi'> r~ ... carch prnposal is suhmilled to the school of pharmacy with the approvnl or the
... up~.:rv i:,or.
Sig11allll\' ... ~ .. • ............... .. Dale . . M.. 0.1 .... l.f.; ... ~. J. &' lk < >DDJ\ JOliN
1\.aulpala lnlcrnntiunal University western campus
TABLE OF CONTENTS
DECLARATION ............................................................................................................................ i
DEDICATION ............................................................................................................................... ii
APPROVAL ................................................................................................................................. iii
ACKNOWLEDGMENT ......................................... , ................................................................... iv
LIST OFT ABLES ...................................................................................................................... vii
ABBREVIATIONS .................................................................................................................... viii
CHAPTER ONE ........................................................................................................................... 1
1.0 INTRODUCTION ................................................................................................................. 1
1.1 BACKGROUND INFORMATION ....................................................................................... I
I .2 Statement of the pro b !em ...................................................................................................... 2
1.3 Justification for the study ...................................................................................................... 2
I .4 Research questions ............................................................................................................ 3
I .5 .2 Specific objectives .......................................................................................................... 3
CHAPTER TWO .......................................................................................................................... 4
2.0 LITERATURE REVIEW ...................................................................................................... 4
2.5 Methods for antibacterial susceptibility ................................................................................ 8
2.6 Minimum Inhibitory Concentration .................................................................................... l 0
2. 7 Minimum Bactericidal Concentration ................................................................................. I 0
CHAPTER THREE ...................................................................................................................... 11
3.0 MATERIALS AND METHODS ......................................................................................... 11
3 .I Study Design .. . . . .. .. . . .. .. .. . . . .. .. . . .. . .. . . .. .. . .. .. .. . .. .. .. .. .. .. . .. .. .. .. .. .. . . . .. .. .. .. .. .. .. .. . . .. .. .. . . .. .. .. .. . .. .. .. . .. .. ll
3.2 Study area ............................................................................................................................ ll
3.3 Plant collection and identification ....................................................................................... 11
3.4 Storage, drying and pulverization ....................................................................................... 11
3.5 Plant extraction .................................................................................................................... 12
3.6 Determination of extract yield(% yield) ............................................................................. 12
3.8 Determination of Minimum Inhibitory Concentration (MIC) using Broth dilution method
··················································································································································· 15 3.9 Determination of Minimum Bactericidal Concentration (MBC) ........................................ 16
3.10 Quality control ................................................................................................................... 17
v
3.14 Data analysis ..................................................................................................................... 18
3.15 Ethical considerations ....................................................................................................... 18
CHAPTER FOUR ....................................................................................................................... 19
4.0 RESULTS ............................................................................................................................ 19
4.1 The results for susceptibility test of Shigella dysenteriae to ethanol whole herb extract of Phyllanthus amarus ................................................ : .................................................................. 19
CHAPTER FIVE ........................................................................................................................ 23
5.0 DISCUSSION, CONCLUSION AND RECOMMENDATIONS ...................................... 23
5.1 DISCUSSION ..................................................................................................................... 23
5.2 CONCLUSION ................................................................................................................... 25
5.3 RECOMENDATIONS ........................................................................................................ 26
REFERENCES ............................................................................................................................ 27
APPENDICES ............................................................................................................................. 30
APPENDIX 1: TIME FRAMEWORK AND WORK PLAN ................................................. 30
APPENDIX 2 ............................................................................................................................... 31
Figure I: susceptablity testing agar well diffusion.( zones of inhibition) ................................. 3 I
vi
LIST OFT ABLES
Table I: Scientific classification of Shigella dysenteriae.
Table2: The results of susceptibility sample to Shigella dysenteriae on Phyllanthus amarus
extracts and controls.
Table3: The results showing minimum inhibitory concentration (MIC) of Shigella dysenteriea
against Shigella dysenteriae bacteria.
Table4: Minimum bactericidal concentration (MBC) of Phyllanthus amarus against Shigella
dysenteriae.
vii
MBC
MIC
NCCLS
!CLS
BSAC
K!U
ABBREVIATIONS
Minimum bactericidal concentration
Minimum inhibitory concentration
National Committee for Clinical Laboratory Science
Institute of Clinical Laboratory Standards
British Society for Antibacterial Susceptibility Testing
Kampala International University
viii
CHAPTER ONE
1.0 INTRODUCTION
l.lBACKGROUND INFORMATION
Medicinal plants over the century have been used as herbs for the treatment of several human
diseases and have been very important in the health care delivery at one stage or the other of
every nation (Oiuma et al., 2004). Research of recent has focused on natural plant products as
altematives to for existing drugs for disease therapy in developing countries (Aiyegoro et al.,
2007). Medicines derived from Plant have been part of traditional health care in most areas of
the world for centuries and there is increasing interest in them as sources of agents to fight
microbial diseases (Ajayi and Akintola, 2010). The emergence of multiple antibiotic resistant
organisms has constituted a global problem as far as treatment of some infectious diseases is
concerned.
The vehicles of transmission of this etiologic agent are mainly food and water. Several other
disease-causing organisms of medical importance have also developed resistance to these
conventional antibiotics. Infectious diseases still remain an impm1ant cause of morbidity and
mortality in man, especially in developing countries. In Africa today, many resort to the use of
locally made herbal medicines prepared as infusions in hot water, decoction in cold water,
concoction with food and as tinctures with alcohol as an alternative therapy for bacterial
infections (Oluduro and Omoboye, 2010). Plant parts such as the leaves, roots, bark, shoots, fruit
peels, immature and unripe fruits have been used in most herbal preparations. According to
George and Pamplona-Roger (1998) the therapeutic value of some common plants have been
used in the treatment of various ailments including enteric fever, dysentery, diarrhoea,
convulsions, common cold, malaria, jaundice, yellow fever, dental caries, intestinal parasites,
gastroenteritis, viraL bacterial and protozoan diseases. Antiseptic, diuretic, and anti
inflammatory antibacterial properties have equally been reported (Alunas et al., 2005).
Medicines of herbal sources is readily available in our diverse vegetation, cheap and above all
carries the potential for introducing new templates into modern medicine (Akinyemiet et al.,
2005). Many countries in the world, including Ghana, practitioners of herbal medicine are still
consulted as a first choice in the treatment of ailments, due to the fact that traditional medicine
1
blends readily with the socio-cultural life of the people, and the truth that rigt medicine are more
expensive to procure and some right pharmaceutical preparations are many times faked (Abase et
a!., 20 II). There is a vast array of medicinal plants used singly or in combination with other
medicinal plants that confer synergistic effect in the treatments of various ailments.
In central uganda, Phyllanthus amarus is used for treatment of bacterial dysentery and this is
done by boiling fresh leaves of Phyllanthus amarus in water for about 30 minutes and when cool
the patient is given the resultant soup orally amounting to mug cup of about 500ml twice a day
for about a week.
Despite the wide herbal use of Phyllanthus amarus in the treatment of diarrheal diseases to
include dysentery there is a lack of scientific publication as to its efficacy against dysentery
caused by Shigella dysenteriae. The present work is an eff01i to determine the activity of leaf
extract of Phyllanthus amarus against Shigella dysenteriae.
1.2 Statement of the problem
It is estimated that 80% of people worldwide rely on herbal medicine for health care (iyenga .,
2012) and Phyllanthus amarus preparation is used to control several diseases derived for from
microbial infections which include bacterial dysentery.
The acceptance now of traditional medicine as an alternative form of health care and the
development of resistance to available antibiotics have led to widespread investigation into the
antibacterial activity of medicinal plants (Bisignano et a/.,!996). Despite the herbal use of
Phyl/anthus amarus in treatment of diarrheal diseases to include dysentery, there is lack of
scientific publication as to its efficacy hence there is need to study the antibacterial activity of
extract of Phyllanthus amarus used in central Uganda for traditional treatment of bacterial
dysentery.
1.3 Justification for the study
Phyl/anlhus amarus is a medicinal herb considered efficient for the cure of various ailments.
Despite its wide herbal use in the treatment of diarrheal diseases to include dysentery, there is
lack of scientific publication as to its efficacy against Shigella dysenteriae hence this study is
necessary to determine the activity of whole plant extract of Phyllanthus amarus against
Shigella dysenteriae
2
1.4 Research questions
What is the sensitivity of Shigella dysenteriae to various concentrations of crude extract of
Phyllanthus amarus?
What is the Minimum inhibitory concentration of whole plant extract of Phyllanthus amarus
against Shigella dysenteriae?
What is the Minimum bactericidal concentration of whole plant extract of Phyllanthus amarus
against Shigella dysenteriae?
1.50bjectives
1.5.1 Main objective
The main objective of this study is to investigate the antibacterial properties of whole plant
extract of Phyllanthus amarus on Shigella dysenteriae in vitro.
1.5.2 Specific objectives
•!• To determine the sensitivity of Shigella dysenteriae to various concentrations of crude
extract of Phyllanthus amarus.
•!• To determine Minimum inhibitor concentration (MIC)of whole plant extract of
Phyllanthus amarus against Shigella dysenteriae.
•!• To determine Minimum bactericidal concentration (MBC) of whole plant extract of
Phyllanthus amarus against Shigella dysenteriae.
3
CHAPTER TWO
2.0 LITERATURE REVIEW
2.1 Phyllanthus amarus description
Phyllanthus amarus is a member of the Euphorlliaceae family (Spurge family), with over
6500species in 300 genera. Euphorbiaceae family consist of upright or prostrate herbs or shrubs,
often with milky acrid juice and is mainly a pan-tropical family with some species either more or
less temperate. The fruit is a 3-lobed capsule containing 6 seeds and extends from the cup with
a long stalk pendant about l-2mm. The leaves are simple, alternate or opposite and leathery, and
borne on petioles 0.3 to 0.5 mm long. The flowers are very small and diclinous, often in clusters
borne in greenish cup-shaped structures with glands. The plants are monoecious or monogamous.
It has smooth cylindrical stem thick and deciduous horizontal branchlets 4-12cm long and 0.5cm
thick, with 15 to 30 leaves.
2.1.1 Origin and distributon of Phyllanthus amarus
The plant is indigenous to tropical Americas, the Philippines and India (Chavellier, 2001). Plants
in the genus Phyllanthus are found around all tropical regions of the world; from Africa to Asia,
South America and the West Indies. The genus contains about 550-750 species in 10-11
subgenera (Unundar,l998). Some related species with medicinal importance include.
epiphyllanthus, Phyllanthus niruri Phyllanthus surinaria, Phyllanthus acuminatus and
l'hyllanthus emblica (Tirimana, 1987). The plant can be found along roads, valleys, on
riverbanks and near lakes. Phyllanthus amarus is sometimes mistaken and wrongly identified
with the closely related Phyl/anthus niruriL. In appearance, phytochemical structure and history
of use. Phyl/anthus niruri also reaches a height of 60 em with larger fruits and dark brown and
warty seeds than that of Phyllanthus amarus (Morton, 1981 ).
2.2 Traditional uses of Phy/lanthus amarus
Phyllanthus species are used ethnobotanicaly and in folk remedies in many countries.
Phyl/anthus amarus, Phyl/anthus nururi and Phyl/anthus urinaria have also been used in the
treatment of kidney related problems, gallstones, appendix inflammation and prostate problems
(Haydet, 1990). According to Foo and Wong (1992), the aerial patt of Phyllanthus amarus is
4
highly valued in traditional medicine for its healing properties. Fresh leaf juice of the plant can
be applied externally for the treatment of cuts and bruises. It is also good for treating Arthritis
and Asthma in patients (Adebisi, 1999).It has a long history of use in the treatment of liver,
kidney and bladder problems, diabetes and intestinal parasites (Foo, 1993). Phyllanthus amarus
has also shown to work as an antifungal, antibacterial and antiviral agent (Houghton et al.,
1996).Adeneye et al., (2006) reported that Phyllanthus amarus was used in traditional
medicine for its hepatoprotective, anti-diabetic, anti-hypettensive, analgesic, anti
inflammatory and antimicrobial propetties. Joy and Katted (1994) also rep01ted the anti
nociceptive, anti-lipidemic, anti-diabetic, anti-inflammatory, anti-lithic and anti-bacterial
properties of the plant. It is used traditionally in the treatment of liver ailments and kidney
stones. Whole plant is employed in some genitourinary infections. The young tender shoots are
used in chronic dysentery and the juice of the stem is mixed with oil in ophthalmology for eye
treatments.
Odugbemi (2008) and Chaudhury (2007) reported that the plant is effective for treating
gonorrhoea, genito-urinary diseases, asthma, diabetes, typhoid fever, jaundice, stomach-ache,
dysentery, ringworm, and hypertension. Kokarso (1996) confirmed the use of the plant in the
treatment of stomach disorders, skin diseases and cold. Nanden (1998) also repotted that the herb
is used to combat fever, flu and asthma in combination with other herbs. The plant when boiled
with the leaves is considered to be a diuretic and can be used in treating diabetes, dysentery,
hepatitis, menstrual disorders, and skin disorders. The herb can also be used for constipation. The
extracts from the roots can be used to treat jaundice (Heyde, 1990). In recent years, the plant has
been used successfully as a liver-protectant/detoxifier for conditions such as jaundice and
hepatitis B and can rapidly restore full functioning of a damaged liver. It has widely been
reported to offer good treatment for leprosy, hiccup, and peptic ulcer. It is anti-spasmodic, good
laxative, blood tonic, treatment of itch, flu, fever, dyspepsia, blennorrhagia, tenesmus,
gonorrhoea, malaria, uterus complaints, constipation, anorexia, carminative, tumour, colic; it
has HIV inhibitory activity, good anti - inflammation of appendix, bladder disorder (Obianime
and Uche, 2009). Meixa et al. (1995) confirm the anti-viral activity of Phyllanthus amarus
against hepatitis B virus. Phyllanthus amarus which is otherwise called Eyin-olobe in South
Western Nigeria has healing effects on hypertensive patients. It was equally found efficacious for
5
treating malaria, diabetes, kidney stones and jaundice Phyllanthus amarus can be taken for
weight loose and help to increase male fertility.
2.3 Phytochemical properties of Phyllanthus amarus
Houghton et al. (1996) isolated securing type alkaloids by Column Chromatography (CC) and
preparative Thin Layer Chromatography (TLC). Hydro-alcoholic extract of Phyllanthus amarus
showed the presence of various phyla-constituent such as alkaloids, flavonoids, saponins and
tannins (Ambaba et al.,20 II). Experiments conducted into the phytochemicals present in
Phyllanthus amarus using UV, IR, Mass and NMR spectroscopy revealed that alkaloids,
flavanoids, hydrolysable tannins, major lignans and polyphenols were present in the plant Foo
and Wong(l992). Previous studies have reported some of these phyla-components to elicit a
wide range of biological activities, which include hypolipidemic, hypoglycemic, hypoazotemia
among others. Saponin is known to elicit serum cholesterol lowering activity by causing resin
like action, thereby reducing the enterohepatic circulation of bile acids (Topping et al., 1980). In
the process, the conversion of cholesterol to bile acid is enhanced in the liver resulting in
concomitant hypocholesterolemia (Kritchevsky, 1977). The lignan constituents in the plant
consist of phyllanthin and hypophyllathin, amarinic acid, nynphyllin, phyllarurin and
ncolynan. Geranin a phytochemical in the plant has hypertensive effects and account for it's used
in hypertension conditions. This chemical can inhibit several neurotransmitter processes that
relay and receive pain signals in the brain. Geranin also has anti-ulcerous properties
(Chaudhury, 2007). Alkaloids and tannins contain in the plant contribute to the plants
effects as anti-malaria, anti-diarrhoea and analgesic agents.The major lignans, Phyllanthin and
Hypophyllanthin have been reported to exhibit anti-hepatotoxic activity.
2.4 SHIGELLOSIS
Shigellosis is a current health burden which is endemic and estimated to affect 80-165mllion
individuals annually. Ninety nine percent of infections caused by shigella occur in developing
countries, and the majority of cases and cases of deaths occur among children less than 5 years of
age ( kotloff et al., WHO. 2005). Shigella are gram negative intracellular bacterial pathogens that
inhibit the gastro intestinal tract of humans and are the causative agent of shigellosis.
6
2.4.1Shigella diseases and symptoms
Shigellosis is a disease affecting humans is usually acquired through contaminated food and
water sources (DuPont et al., 1989). The high incidence of shigella in developing countries is
considered to be attributed to the lack of clean water, poor sanitation, malnutrition and the cost of
antibiotic treatment (Jennison&verma, 2004). Shigella infect through the oral faecal route and
infection is transmissible with as few as 100 micro-organisms due to the bacterium's ability to
survive the acidity of stomach (Small et a/.,1994). Infection is known to produce a range of
symptoms and range from watery diarrhoea to severe dysentry. Severe dysentery is characterised
by fever, abdominal pain and acute permanent bloody and mucoid diarrhoea
(Phalipon&sansonetti, 2007). In the absence of effective treatment, patients with shigellosis can
develop secondary complicatons such as septicaemia and pneumonia
2.4.2Pathogenesis and Pathology of Shigella
The initial step in pathogenesis is clearing bacterial invasion or penetration of the colonic
mucosa, the resulting focus of Shigella infection is characterized by degeneration of the
epithelium and by an acute inflammatory elements, and dependent upon the ileocecal flow. As a
result, the patient will pass frequent, scanty, dysenteric stools (Hale, 1991 ).
The virulence factors include;
A. Lipopolysaccharide (LPS): LPS plays an important role in resistance to nonspecific
host defense encountered during tissue invasion. These genes help in invasion,
multiplication, and resistance to phagocytosis by tissue macrophages. LPS enhances the
cytotoxicity of Sh ET on human vascular endothelial cells.
B. Toxins Shigella dysenteriae produce an exotoxin, Shiga toxin. Also enterotoxins
designated shETJ and shET2 have been identified, and the genetic loci encoding these
toxins have been localized to the chromosome and plasmid respectively
C. Siderophores: Ability to secrete iron from chelating compounds, 'siderophores'
which chelate iron from intestinal fluids and then are taken up to release iron
inside the bacterium for its metabolic needs.
7
2.4.3 Shigella dysenteriae description
Shigella dysenteriae is a species of rod shaped bacterial of genus shigella( Ryan, et al., 2004).
Shigella species can cause shigellosis and Are gram negative, non spore forming, facultative
anaerobic non motile bacteria(Hale, et al., 1996).
Table I: Scientific classification of Shigella dysenteriae.
KINGDOM
Phylum
Class
Order
Family
Genus
Specie
BACTERIA
Proteobacteria:
Gammaproteobactera
Enter()ba'ctefiliies< ··· ··
Enterobacteriaceae
Shigella i {/. ;;t,;J,.~'?it¥i~@;,i:, Shige/ladysenteriae
Shigella dysenteriae is spread by contaminated water and food, causes the most severe dysentery
because of its most potent and deadly shiga toxin, but other species may also be dysentery agents
(Herold. If et al.,2004).
Most commonly observed signs associated with shigella dysente1y include colitis, malnutrition,
rectal prolapsed, tenesmus, reactive arthritis and central nervous system problems. Further,
S.dysenteriae is associated with the development of hemolytic uremic syndrome, which includes
anemia, thrombocytopenia and renal failure.
2.5 Methods for antibacterial susceptibility
Antibacterial susceptibility testing standard tests can be conveniently divided into diffusion and
agar dilution methods. Agar diffusion susceptibility testing is regarded as the golden standard for
all other susceptibility testing methods.
It is important to prepare agar plates in such a way that the antibacterial concentrations in the
plates are exactly or very close to the desired concentrations. When preparing the antibacterial
solutions and agar plates for agar dilution susceptibility testing, the following guidelines will be
8
recommended as given by the national committee for clinical laboratory science (NCCLS)
(Rhoad, 2004).
The dilution procedure is going to seems to be complicated, but this method will ensure that
there is minimal risk of making out of scale dilutions for the smallest concentrations. The ethno
pharmacology research the Antibacterial Susceptibility Testing (AST) (Tyler and Eric, 2014)will
be used to determine the efficacy of potential antibacterial from biological extracts against a
number of different bacterial species. In clinical research in vitro susceptibility tests is going to
be impmiant if an organism is suspected to belong to a species that has shown resistance to
frequently used antimicrobial agents.
They might also impotiant epidemiological study on susceptility and in comparison of new and
existing antimicrobial agents (EUCAST 2003). Successful discovery of novel natural
antimicrobials has necessitated the development of new bioassay techniques which are sensitive
enough to detect small amounts of biologically active chemicals (Lampinen, 2005).
Standardized in vitro tests it will be essential for screening plants extracts or compounds by
using MlC determination of natural products in order to get results that are comparable to those
of currently used antibiotics (Devienne and Raddii, 2002). When evaluating the performance of a
susceptibility tests it should include criteria like ease of use and the ability to yield the same
result when repeated testing is done, test sensitivity and specificity (Struelens, et a/1995).
Although current standard methods, approved by various bodies like the National Committee for
Clinical Laboratory Science(NCCLS) now known as Institute of Clinical Laboratory Standards
(ICLS), British Society for Antibacterial Chemotherapy (BSAC) (Atihur 1953) and the Europe
Committee for Antibacterial Susceptibility Testing (EUCAST), exist for guidelines of
antimicrobial susceptibility testing of conventional drugs, these might not be exactly applicable
to plant extracts and modification have to be made (Hammer et al, 1999).
Dilution methods includes agar dilution and broth micro/macro dilution. The broth and agar
based methods are the conventional reference methods for AST (Tenover eta!., 1995).
9
2.6 Minimum Inhibitory Concentration
Minimum inhibitory concentration (MIC) is defined as the lowest concentration antimicrobial
agent (mg/l) that will prevent the appearance of visible growth of microorganism within a period
time. Dilution method are used to determine the MIC of antibacterial susceptibility testing. The
MlC is used in resistance surveillance, the comparative testing of new agents to establish the
susceptibility of organisms that give equivocal results in discs test. Broth dilution is a technique
use for MlC in which containers holding identical volume of broth with antibacterial solution
(Mann and Markham, 1997).
2.7 Minimum Bactericidal Concentration
Minimum bacterial concentration (MBC) is method used to determine if the extract can kill or
inhibit the growth of bacteria pathogens. The minimal bactericidal concentrations is determined
after the MlCs results were obtained. This will be done by selecting the tube that shows no
growth during the M!Cs determination. A loop (sterile wire loop) from the tube containing the
media and the extract are inoculated into a sterile nutrient broth and Sabouraud's dextrose liquid
of freshly prepared media. These will be future inoculated for another 72hrs at 37°c for bacteria,
after which they will be examined for any bacterial growth. The lowest concentration at which
no growth is observed on the plate gives the MBC (Yamamoto and Loren, 2003).
10
CHAPTER THREE
3.0 MATERIALS AND METHODS
3.1 Study Design
The study to determine Phyllanthus amarus extract activity on Shigella dysenteriae was
experimental in the laboratory and it was conducted in JAN- APRIL 20 I 8 at KIU microbiology
laboratory.
3.2 Study area
This study was conducted at Kampala International University-Western campus at the hospital
microbiology and pharmacology laboratories because of availability of trained personnel and
equipment's that were needed and experiment was conducted for three (3) months. The
extraction was carried out in pharmacology laboratory while the susceptibility of Shigella
dysenteriae on Phyllanthus amarus extract was done in the hospital microbiology laboratory in
KIU teaching hospital, according to European Committee for Antibacterial Susceptibility Testing
(EUCAST) and European Society of Clinical Microbiology and Infectious Disease (ESCMID)
2003 procedure.
3.3 Plant collection and identification
The fresh whole plant of Phyllanthus amarus was collected in January 2018 where it grows
naturally in a banana garden in wakiso district central Uganda. The collected herb was deposited
at the department of botany of Mbarara and identified by a botanist and was given a voucher
number.
3.4 Storage, drying and pulverization
The collected herb sample was washed, chopped in small pieces and dried in a shade to avoid
direct sunshine that could degrade phytochemical due to ultra violet light because the active is
not known. The herbs were air dried at room temperature by displaying it on a dry cemented
table in pharmacology lab and it was turned daily to prevent fungal attack until complete dryness
that was confirmed by weighing every week for three weeks and the constant weight was
achieved then the plant was confirmed dry.
11
The dried sample was ground into powder by grinding it using a blender (Paola and collectors,
20 II). The reason for this is to increase the surface area contact between the plant particles and
the solvent during extraction, it is because the powder can easily be mixed with the solvent,
reduce particle size powder also facilitates crude extraction and more so also increase dissolution
rate.
3.5 Plant extraction
The plant extracts was prepared by using the method of (Alade&Irobi 1993). Extraction was
performed by macerating air-dried, powdered of Phyllanthus amarus in 70% ethanol.
Plant material was grinded and weighed, the initial weight of powder was 375g and it was
soaked in 3. 7 litres of 70% ethanol and put on a mechanical shaker for 72hours. After 72 hours
of agitation on mechanical shaker, the mixture was sieved using a clean sieve and after the
filtration was made using DRs Watts filtering paper of (0.5~tm) pore size in a funnel into a
measuring cylinder which was weighed mean while the residue which remained on the filter
paper was collected and air dried pending for extraction incase if the extract was not going to be
enough.
The filtrate was put in the drying oven at 50°c for concentration, after concentration the weight
of the concentrate (extract) was 17.6g then it was stored in the refrigerator at 4°C until the time it
was needed.
3.6 Determination of extract yield (% yield)
According to Paola et al (2011) the yield% w/w from all the dried extracts for example ethanol
extract was calculated as:
% yield =weight of extract X I 00% Percentage yield for ethanol=l7.6 x 100% = 4.7%
Weight of the powder 375
12
3.7 Determination of susceptibility activity of Phyllanthus amarus on Shigella dysenteriae
The isolation of the organism from KIU teaching hospital using stool from the patient
sample
Stool culture was collected and inoculated in Deoxycholate citrate agar (DCA) and salmonella
shigella agar (SSA) a selective media for the isolation of Shigella and Salmonella.
Cultural technique was done following aseptic precautions for avoidance of any contamination
fi·mn the surrounding environment. A sterile wire loop was used for inoculation of the sample on
a sterile dried culture media mentioned above. Following inoculation, culture plates were
incubated at 37°C for 24 hour.
Identification
A cultural characteristic of the isolates was observed for size, colour, texture, and appearances;
Shigella colonies appeared colourless on the media.
Morphological characteristics
Gram stain was done by making a smear of the colonies on clean dried glass slide, using a sterile
wire loop. The slide was allowed to air dried and the smear fixed over a Bunsen burner flame
three (3) times. The slide was taken to the staining rack and was stained with crystal violet for I
minute, treated with Jugal's iodine for 30 seconds, decolourize with 50% acetone alcohol under
slow running tap water until no more colour was observed flowing from the slide. The slide was
counterstained with I% Neutral red for 2 minutes; it was washed with water, drain dried and
examined with X I 00 oil immersion lense.
Gram negative rods bacteria were observed which the morphological characteristics of Shigella
species are.
13
Biochemical test
A biochemical test was done to further confirm the isolates by inoculating the colonies on triple
sugar iron (TSI) agar.
Result
Shigella species was observed by fermentation of the butt producing acid which changed the
colour of the media to yellow while the slant remained red showing non fermentation which
indicates alkalinity of the slant.
The preparation oftest extract stock solution
The stock solution of the plant extract was achieved by diluting 512mg of the extract in IOOOml
in order to achieved 512mg/L concentration of extract.
The preparation of Mueller Hinton agar
The broth was prepared by weighing 9.5g of the powdered media that was dissolved in 250mls
of distilled water and it was sterilized in the autoclave in the temperature of 121 'c at
I 00kpa(15psi) above atmospheric pressure for IS minutes.
The sterilized agar media was mixed in the heat preparation from the bursen burner and it was
dispensed in the sterilized culture plates where it was left to solidify in the covered plates. The
test microorganism was aseptically inoculated (approx. 1.0 x 106 colony forming units/ml) on
sterile Mueller !-linton agar by surface spreading to make uniform microbial inoculums. Using
sterile glass cork borers (6 mm in diameter), wells were carefully made on the agar plate without
distorting the media; to contain test extract.
A separate agar plate was used to test the control drugs; ciprofloxacin SJ.tg/ml against the bacteria
because it is first line for shigellosis as positive control and negative control contains solvent
used for dilution (distilled water)
Two hundred micro liters (200J.tl) of ethanol extracts in each well was dispensed into the well.
Controls were also dispensed and the plates were left on the bench for I 0 minutes in order for the
extracts not to pour away. The culture plates were then incubated at 37°C for 24 hours.
14
Using a metric ruler, the diameter of the zones of inhibition were taken in millimeters (mm) (the
diameter of the area of no growth of the microorganism around the disc) was measured for the
control in antibiotics and extract (Baris et al., 2006). The zone of inhibition was >6mm the
bacteria was said to be susceptible to the extracts and that's why the MIC was done. Since the
extract passed by satisfying the set criteria hence it was subjected to the MICas showed down
3.8 Determination of Minimum Inhibitory Concentration (MIC) using Broth dilution
method
Minimum inhibitory concentrations (MIC) of Phyllanthus amarus extract were performed on
organism Shigella dysenteriae using tube dilution method (Koneman et al., 1997). The bacterium
was inoculated in the serial dilution of eight tubes of Phyllanthus amarus extracts for ethanol
with 1 mg/ml of nutrient broth.
The preparation of MIC set np
It involved the use of I 0 test tubes in total for both ethanol extract that were dried in the hot air
drier and the test tubes were labeled from test tube I to I 0. In each test tube lml of the broth was
introduced.
Test tube I , I mls of the extract from stock of 512f,Lg/ml was introduced and it was mixed
thoroughly with I ml of the broth to give the concentration of 256 f.Lg/ml and I ml of the mixture
was transferred to test tube 2 and mixed to give the concentration of 128f,Lg/ml.
The 128f,Lg/ml of the solution was again picked and transferred test tube 3 to give the
concentration of 64f.Lg/ml. The 1 ml of 64f,Lg/ml was again picked and transferred to test tube 4 to
give the concentration of 32f.Lg/ml after thorough mixing.
The l ml of 32f.Lg/ml was again picked and transferred to test tube 5 and mixed thoroughly to give
the concentration of l6f.Lg/ml.
The I ml of l6f.Lg/ml was picked again and it was transferred to test tube 6 and it was mixed
properly to give the concentration of 8f.Lg/ml. then lml of Sf.Lg/ml was picked and transferred to
test tube 7 and it was mixed thoroughly to give the concentration of 4f,Lg/ml and finally test tube
8 I ml of 4f.Lg/ml was picked and transferred and mixed thoroughly to give the concentration of
2flg/ml.
15
Concentrations used from test tube J-8 (256flg/ml, 128J.lg/ml, 64J.lg!ml, 32J.lg/ml, l6J.lg/ml,
8J.lglml, 4J.lg/ml, 2J.tg/ml)
The bacteria were prepared as follows;
The test bacteria was made by getting the colonies from agar plate and dissolving in 2m! of
normal saline while comparing with McFarland standard giving the bacteria concentration of
l.Ox!06cfu/ml (cheesbrough et al2002). Then Jml of this bacterial solution was added to the first
test tube that contained I ml of broth which was containing 256J.lg/ml of the test extract. This
process resulted into the bacteria concentration in the first test tube (original bacterial
concentration in the broth) to become 5.0xl 05cfu/ml. This procedure of serial dilution was
repeated for all the test tubes giving the test extract concentration of 128J.lg/ml, 64;tg/ml,
32flg/ml, I6J.lg/ml, 8flg/ml, 4J.lg!ml ,2J.lg!ml and I J.lg/ml from test tube 1-8.
There were two controls i.e. Control I and control 2 which were also prepared as follows:
For control one (CJ) that had broth and bacteria and had no drug in order to find out whether the
media supported the growth of organism. On the other hand, control 2 (C2) had only broth and
no bacteria and no drug to determine whether the broth was contaminated by other organisms or
not.
Thereafter, the tubes were incubated at 37"C for 24 hours (Koneman et al., 1997). After
incubation, the tube with no turbidity next to the one showing turbidity (Microbial growth) was
considered as containing the MIC of the extract in question. The tubes that did not have growth
were showed by no turbidity in them.
All extracts that exhibited MIC of I OOJ.lg/ml and below were considered wotih further
investigation and Vice versa (Oiila., et al2001).
3.9 Determination of Minimum Bactericidal Concentration (MBC)
Following the MIC determination using the Broth dilution method, after the recommended
period of incubation, the Mueller Hinton agar was prepared and using a wire loop (O.Oiml)
samples were picked from the broth dilution that didn't have any growth after the incubation and
were spread on the agar plate that containing Mueller Hinton agar aseptically. This was followed
by incubation at a temperature of 37°C for 24 hours. The MBC was determined as the lowest
16
concentration of the extract that allowed less than 0.1% of the original inoculums of 5xl05cfu/ml
to grow. The agars that were showing no growth of the microorganism was considered as the
Minimum Bactericidal Concentration (MBC). The purpose of this test was to determine the
lowest concentration at which the ethanol extracts was able to kill Shigella dysenteriae (Baris et
al., 2006).
3.10 Quality control
Voucher specimen was prepared and deposited in the Herbarium of botany department for
correct botanical identification. Fresh plant materials was collected and dried under shade to
prevent loss of phytochemical due strong ultra violet and direct and can also cause evaporation
of volatile oil. The ethanolic extract was stored in sterile bottle to prevent contamination. The
bacteria specimen was carefully cultured and the media was prepared according to the
manufacturer's standards and it was tested to find whether it supports growth of bacteria and the
bacteria was isolated from patient specimen and it was tested for susceptibility to the standard
drug, the plates were carefully covered to avoid contamination from the environment organism
and the loop was carefully sterilized before the organism was picked.
3.11 Reliability of the study
After test microorganism was isolated, it was tested for effectiveness before the main research
was carried out in order to avoid the use of resistant type of the organism
The experiment had two controls that control l and control 2
3.12 limitation of the study
I didn't do phytochemical analysis due to time and financial constrains
I didn't do toxicity, safety and efficacy in lab animals and humans due to time period was shOJi.
[ also failed to determine the mechanism by which plant extract inhibit bacterial cells due time
limit and financial factors.
3.13 Time limit
The research study took 3months beginning from January of201 to April of2017.
17
3.14 Data analysis
The data collected was entered into Ms Excel and then analyzed in STATA vl4 using the
ANOVA test. A p value less than 0.05 was considered statistically significant. A post hoc
analysis (Bonferroni test) was carried out to identify the sources of differences. The results were
presented as Mean±SEM and as graphs .Based on microbiology guidelines if the mean diameter
of the zone of inhibition was >6mm then the plant extract was proceeded for the MIC.
ForMIC and MBC it was analyzed according to cutoffs.
3.15 Ethical considerations
The study was carried out after presenting research proposal and approved by research
committee of KIU-WC. All protocols in the different elaborations were observed and obeyed
such as handling of laboratory microorganisms was followed.
18
CHAPTER FOUR
4.0RESULTS The starting material was 375g of dried powered material from Phyllanthus amarus for the
extraction. The dried whole herb of Phyllanthus amarus was subjected to different 70% ethanol
solvent maceration. The percentage yield obtained was 4.7%.
4.1 The results for susceptibility test of Shigella dysenteriae to ethanol whole herb extract of Pltyllantltus amarus This was determined by agar diffusion method where the diameter zones of inhibition in mm
were measured after 24hours incubation at 37'c and it was done in triplicate (3times)
The zone of inhibition for ciprofloxacin was greater than that of all extracts since the
ciprofloxacin is the standard drug that is used as a first line in the treatment of bacterial
dysenteriae according to the (UCG 2016). The negative controls shows that solvent without
extract had no inhibitory effect on Shigella dysenteriae.
These results are summarised in the table I below.
TABLE 2 The results of susceptibility sample to Shigella dysenteriae on Phyllanthus
amarus extracts and controls
-----------Test drug Zone of Inhibition (mm) Activity Index P value
Mean±SEM
0.0000 ----~-- . --··-·-- ----- .
l28ftg/ml 11.5±0.5 0.47
256ftg/ml 12.5±0.5' 0.51
512ftg/ml 13.5±0.5' 0.55
Cipro (ftg/ml) 24.5±0.5' 1.00
Distilled water 0±0
'Indicates significant differences between the test drug and the negative control (distilled water)
19
l
0:9
0.8
&j 0.7
"' ..5 0.6
~0.5
~ OA <( 03
0.2
O.t.
0
Activity indices of the different test drugs
256 pg/m1 5:12 flalm Cipro
Test drug
MINIMUM INHIBITORY CONCENTRATION
Conventionally when test microbe is susceptible to the test extract, the MIC is usually
performed. In this study the MIC was performed according to the tube dilution method for both
ethanol and aqueous extract.
The concentration used for MIC determination was128flg/ml, 64flg/ml, 32flg/ml, l6flg/ml,
8f1g/ml, 4f1glml, 2flg/ml and lflg/ml.
The results in this study were considered only valid when there was growth in control one
(media supports growth) and no growth in control two (meaning no cross contamination of the
broth). The details of this MIC results are showed in the table 3
20
TABLE 3 The results showing minimum inhibitory concentration (MIC)of Shigella
dysenteriea against Shigella dysenteriae bacteria
Type
extract
Ethanol
Control!
Control2
128 64
+
Key; + means there was growth,
Means there was no growth.
+
Control A had broth and bacteria but without drug extract.
Control B only broth without bacteria or crude drug in it.
21
+ +
MINIMUM BACTERICIDAL CONCENTRATION
It is usually recommended after determining the MIC, the MBC is determined. In this case since
the extract exhibited MIC above the. cut off being below lOOflg/ml (Olila., et a! 200 I). MBC of
the plant extract was determined. The results of the MBC are presented in table four below.
Table 4: Minimum bactericidal concentration (MBC) of Pltyllantltus amarus against
Shigella dysenteriae.
Type of
extracts
Ethanol
Control A
Control B
tllbes. ()lg/ml)
128 64
+ + +
The original bacteria concentration was I xI 06cfu/ml as determined by comparison to Mcfarland
standard solution hence test tube I contained 5xl 05cfu/ml. since the MBC is the lowest
concentration of the extract that allowed less than 0.1% of the original inoculums of 5x105cfu/ml
to grow and colony forming unit per milliliter ( cfu/ml) on the plate at 32flg/ml is 600cfu/ml, this
makes 64fjg/ml to be the MBC since it showed no growth.
Key; + Means there was growth
-Means there was no growth
Control B the agar with no bacteria and no crude drug extract.
The results in table four, the MBC for ethanol extract were 64fjg/ml.
22
CHAPTER FIVE
5.0 DISCUSSION, CONCLUSION AND RECOMMENDATIONS
5.1 DISCUSSION This chapter presents discussion of findings of the research of antibacterial activity of
Phyllanthus amarus based on objectives of the study.
Bacterial susceptibility testing
The sensitivity tests of Phyllanthus amarus to Shigella dysenteriae showed that ethanolic
extract of the plant material showed antimicrobial activity on the test organism. The test
pathogen, Shigella dysenteriae was more susceptible to the ciprofloxacin as the positive control
with zone of inhibition at 24.5±0.5mm higher than the ethanol extract at all concentrations. The
highest susceptibility was recorded with the ethanolic extract at 5l2J.tg/ml with zone of
13.5±0.5mm followed by 256J.!g/ml at 12.5±0.5mm and finally 128J.tg/ml with zone at
1 1.5±0.5mm
According to the results above, it was realized that the sensitivity increased with increasing
concentration which was in agreement with what (Oiuduro and Omoboye, 201 0). According to
Oluduro and Omoboye (20 1 0) the antibacterial activities of most plant extracts are concentration
dependent as zone of growth inhibition increased with increasing concentration of the extracts.
Ekwenye and Elegalam (2005) and Azu and Onyeagha (2007) also reported that the efficacy of
most plant extracts is concentration dependent.
Umbare et al. (2009) assessing the quality of Phyllanthus amarus leaves extract for its
hypolipidemic activity found the presence of four phyto-constituent namely alkaloids,
flavonoids, saponins and tannins in the plant sample. Flavonoids, tannins, alkaloids, steroids,
terpenoids, saponins and glycosides were also obtained by Obianime and Uche (2009) in
their comparative study of the methanol extract ofP.amarus leaves.
Oluduro and Omoboye (2010) indicated that the presence of phytochemicals in plant
extracts is a function of their antimicrobial activities against the test pathogen as they play
important roles in bioactivity of medicinal plants. They further explained that phytochemicals
Z3
exeti antimicrobial activity through different mechanisms. Chonoko and Rufai (2011) also
indicated that there was a link between the antibactei·ial activity exhibited by the plant extracts to
the presence of steroids flavonoids, tannins, alkaloids and saponins. Tannins, for example, act
by iron deprivation, hydrogen binding or specific interactions with vital proteins such as
enzymes in microbial cells (Scalbert, 1991; Akinpelu et al., 2008).
Basing on the discussion above, the difference between the activity of the extract and the
standard antimicrobial drug may be due to the mixtures of bioactive compounds which probably
have antagonistic effects against the major bioactive(s) present in the extracts compared to the
pure compound contained in the standard antibiotic ciprofloxacin. The standard drug which has
the highest zone of inhibition while distilled water had no zone of inhibition it is because water
has no active ingredients.
The policy implication of these findings is that more research should be carried out probably
using bioassay guided fraction to determine the lead compound(s) in the extract.
Minimum Inhibitory Concentration of Plzyllantlzus amarus
Following the sensitivity analysis by a standard microbiology, the MIC is usually performed if
the test organism is sensitive to the test extract /drug. In this case since Shigella dysenteriae
bacteria were active against herb extract, the MIC was carried out according to the Mann and
Markham method (Mann and Markham, 1997).
The quality control, some measures were put in place for this results to be valued included two
controls i.e. control 1 and control 2 which were also prepared as follows:
For control one that had broth and bacteria and had no drug in order to find out whether the
media supported the growth of organism. On the other hand, control 2 had only broth and no
bacteria and no drug to determine whether the broth was contaminated by other organisms or not.
Since the controls were working then the results were considered valid.
The analysis of the results showed that the MIC of ethanolic extract was 64J.lg/ml. For test
extract whose MIC is <1 OOJ.!glml is considered as good candidates to be developed into new
drugs (Olila., et al 2001). Since our MIC value was below 100J.!glml, this herb is a good
candidate for drug development.
24
The policy implication of these findings is that more research should be carried out probably
using bioassay guided fraction for lead compound in the extract.
Minimum bactericidal Concentration (MBC) of Phyllanthus amarus
During drug development for a test material as an antibacterial agent against a given bacteria if
the MIC is <I OOJlg/ml then the next step it is to do the MBC.MBC is the lowest concentration of
the extract that allowed less than 0.1% of the original inoculum of 5xl 05cfu/ml.
The analysis of the results showed that the MBC for ethanol was 64jlg/ml. This was because
original bacteria concentration was lxl06cfu/ml as determined by comparison to Mcfarland
standard solution hence test tube I contained 5xl 05cfu/ml. Since the MBC is the lowest
concentration of the extract that allowed less than 0.1% of the original inoculum of 5xl 05cfu/ml
to grow which was at 500cfu/ml (being the cut off value below which MBC is considered and
vice versa).The colony forming unit per milliliter ( cfu/ml) on the plate at 32J1glml is 600cfu/ml,
hence this makes 64Jlg/ml to be the MBC since it showed no growth.
The policy implication of these findings is that more research should be carried out probably
using bioassay guided fraction for lead compound(s) in the extract.
5.2 CONCLUSION This study found that the whole plant extract ofphyllanthus amarus for 70% ethanolic extract
was active against Shigella dysenterie at 512ftg/ml with zone of 13.5±0.5mm followed by
256Jlglm! at 12.5±0.5mm and finally l28j1g/ml with zone at 11.5±0.5mm.
Furthermore the MIC of the extract was 32Jlg/ml and the MBC for was 64Jlg/ml which indicates
that Phyllanthus amarus whole plant extract has antibacterial activity against Shigella
dysenteriae.
Based on these results, this study has scientifically validated the ethnobotanical use of
Phyllanthus amarus to treat bacterial dysentery in central Uganda.
25
5.3 RECOMENDATIONS I recommended that;
I. The more research should be carried out to determine lead compound in the Phyllanthus
amarus extract responsible for the anti-microbial activity against Shigella dysenteriae.
2. The mechanism of action by which a plant extract inhibits bacterial cells should be
studied.
3. Plant extract should be tested on different microorganisms that cause dysentery to find
out its activity against them and spectrum.
26
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Oluma, H.O, Umoh, E.U, Onekutu, A. and Okolo, J. (2004). Antibacterial potentials of eight
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29
APPENDICES
APPENDIX 1: TIME FRAMEWORK AND WORK PLAN
Concept presentation
Proposal writing
Proposal presentation
Plant collection/extraction
Antibacterial evaluation
Data analysis
Report writing
Report submission
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II/III! 111!!111
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