IOSR Journal of Dental and Medical Sciences (IOSR-JDMS)

e-ISSN: 2279-0853, p-ISSN: 2279-0861.Volume 13, Issue 6 Ver. I (Jun. 2014), PP 80-85
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The Antibacterial Activity of Leaf Extracts of Ocimum
Gratissimum and Sida Acuta
*Mbajiuka Chinedu Stanley
1
, Obeagu Emmanuel Ifeanyi
2,
Ochei Kingsley
Chinedum
3
and Nnadi Doris Chinenye
4

1.Lecturer,Department of Microbiology,Michael Opkara University of Agriculture,Umudike,Abia State,Nigeria.
2.Medical Laboratory Scientist,Diagnostic Laboratory Unit,University Health Services Department ,Michael
Opkara University of Agriculture,Umudike,Abia State,Nigeria.
3.Department of H.S.S. and Laboratory,FHI 360 Country Office,Plot 1073-AI Garki,Abuja,FCT,Nigeria.
4. Department of Microbiology,Michael Opkara University of Agriculture,Umudike,Abia State,Nigeria.

Abstract: The antibacterial activities of ethanolic and aqueous extracts of Sida acuta and Ocimum gratissimum
on Escherichia coli, Staphylococcus faecalis and Peudomonas aeuginosa were determined using disc diffusion
assays. All the extracts exhibited moderate to high level of inhibtion against the four microorganisms.The
antibacterial activities were measured by the diammeter by dimeter zone of inhibiton, in which ethanolic
extracts of Sida acuta exhibit the highest yield which is 18mm and the lowest yield in aqueous(cold) extract
7mm. The minimum inhibitory concentration (MIC) is 0.125mg/ml on both plant extracts using the ethanollic
extracts of Sida acuta and Ocimum gratissimum were positive on both the four microorganism and the minimum
inhibitory concentration(MIC) were also positive and gram negative organisms. This research is discussed in
relation to the accepatable herbal medicines as a means of disease control.
Keywords:Ocimum gratissimum,Sida acuta,Minimum Inhibitory Concentration, Escherichia
coli,Staphylococcus faecalis and Peudomonas aeuginosa.

I. Introduction
Medicinal plants are distributed worldwide,but they are most abundant in the tropical
countries(Calixto,2000).It is estimated that plant materials are presently in or have provided th models for 50%
western drugs.A relative small percentage of medical plants are used as food for both humans and other animals
species,it is possible that even more are used for medicinal purposes.
Plants based on antimicrobials have therapeutic potentials.They are used for effective treatment of infection
disease yet gently.An example is Hydratics canadensis,not only does it has antimicrobial activity but also
increase blood supply to the spleen releasing mediating compounds (Murray et al.,1995). Also, Xytopia
aethiopica has an attractive aroma and has been applied in ethno medicine in the treatment of
cough,bronchitis,dysentery and female fertilization.
Iwu (1993) reported that infectious disesae accounts for one half of all deaths in the tropical countries
irespective of efforts made in controlling the incidence of epidemic.Most food-borne disesase due to poor
hygiene can be life threatening and need antibiotic therapy but most of the causative agents have already
developed resistance to common antibiotics in many countries (Iwu, 1993).This resistance has been reported in
Ethiopia in Africa.Due to absence of modern healthcare system in most rural areas,people decided to visited
traditional healer who use medicinal plants to treat their patients with the recent adavances made in using plant
extracts in inhibiting microbial growth,it was observed that phytomedicines have antimicrobial effect against
some human pathogens such as Staphylococcus aureus,Escherichia coli,etc.
Many plants extracts have shown to acquire antibactreial properties active against many microoganisms inside
the body or in vitro for example Garcina biflavonone have been found to be active against variety or organism
like Staphylococcus aureu and Escherichia coli(Iwu,1993).It is used used in the treatment of liver disorder
bronchitis as a chewing stick and throat infections (Lamidi et al., 1995). Some extracts of green pepper,garlic
and onion have noticed to inhibit the growths of Shigella dysentria,Salmonella
typhosia(Sofowora,1983).Ocimum gratissimum and Sida acuta are also medicinal herbs in Nigeria used in
treatment of some infectious disease.
Ocimum gratissimum is used as vegetable for soup prepartions which exhibits hot and spicy taste and are
consumed during cold season.It is claimed that species and herbs assist in the concentration of the uterus in post-
partum women (Okwu, 2003).
Hpwever,it is generally assumed taht the active dietary constituents contributing to those medicinal properties
exhibited by herbs and species are the phytochemical vitamins and minerals.The Ocimum oil is active against
several species of bacterial and fungi,for example shigella,salmonella,proteus,Trichophyton rubrum etc (El-said
The Antibacterial Activity of Leaf Extracts of Ocimum Gratissimum and Sida Acuta
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et al., 2000). Ocimum gratissimum is rich in alkaloids,tannins,phylates flavnoids and Oligosaccharides and it has
tolerable cyanogenic glycoside content (Ijeh et al.,2004) which is the chemical compound active against
microorganisms.
Sida acuta is a marvelous weed that frequently dominates improved pastures,waste and disturbed places
roadsides (Mann et al., 2003). The decribed pharmacological properties of the plants involve the
antiplsamodial,antimicrobial,antioxidant,and many other properties.Some studies resulted in the isolation of
single compounds while the others just demontrated the activity of the crude extracts.
Medicinal plants as Sida acuta and ocimum gratissimum have been asserted to provide various culinary and
medicinal properties.These medicinal properties exerts bacteriostatic an dbacteriocidal effects on some
bacteria.These effects have been attributed to the peptides,alkaloids essential oil,flavonoids etc which are the
major compounds in these plants (Okigbo and Igwe, 2007).
This study was done to determine the inhibitory properties of ocimum gratissimum and Sida acuta on four
strains of bacteria of which are Staphylococcus areus,Streptococcus faecalis,Peudomonas aeruginosa and
Escherichia coli using ethanol and aqueous as solvent for extraction. It is also used to measure the zone of
inhibition of these plants on the organism.

II. Materials And Methods
MATERIALS
Materials used in this study were collected in the laboratories of Nation Root Crop Research Institute
(NRCRI) Umudike and the department of Microbiology in Michael Okpara University of
Agriculture,Umudike.Except the two plants which were collected from the laboratory of the Federal Medical
Centre,Umuahia.

METHODS
COLLECTION OF PLANT MATERIALS
The medicinal plants used in this study were the leaf of Ocimum gratissimum and Sida acuta.Fresh
samplse of Ocimum gratissimum leaves were collected from an uncultivated farmland at Nsukka in Enugu
State,while the fresh leaves of Sida acuta were collected from road side in Nsukka,Enugu State.The plants were
identified and authenticated at the Botany Department,Michael Okpara University of Agriculture,Umudike by a
taxonomist Dr.I.C.Okwulehe.The leaves of each plant was washed thoroughly under running water and dried
uder room temperature.They were grinded into powder and stored in air tight bottles.

STERILIZATION OF MATERIALS
All material were washed the detergent and rinsed thoroughly.They were placed in a rack to dry and
then autoclaved at 121
0
C for 15 minutes to kill microorganisms.

TEST MICRIORGANISM
The strain used in this work was Staphylococcus aureus, streptococcus faecalis, Pseudomonas
aeruginosa and Escherichia coli. They were all coolected at alboratory in Federal Mediacl Centre Umuahia and
their viability were tested by resuscitating them in buffered peptone broth and then sub-cultured in Nutrient agar
medium at 37
0
C for 3 hours prior to antibacterial testing.

PREPARATION AND EXTRACTION OF THE PLANTS
ETHANOL EXTRACTS
The ethanol extracts wsa prepared by weighing out twenty grams(20g) of the powdered leaves of
Ocimum gratissimum and Siad acuta in different conical fllask and adding 200ml of ethanol in each and stirring
vigorously with a glass rod and left for 24 hours. They were filterd off with the sterile filter paper(Whatman No
1 filter paper) into a clean conical flask and filterates was transferred into the sample holder of the rotary
vacuum evepoartor where the solvent was evaluated at room temperature of 28
0
C.The standard extracts obtained
were then stored in refrigerator at 4
0
C until when required for use.

COLD WATER EXTRACTS
The cold water extracts aws prepared by weighing out twenty grams(20g) of the powdered leaves of
Ocimum gartissimum and Siad acuta in different conical flask and adding 200ml of cold water in each stirring
vigorously with aglass rod and left for 24 hours. They were filterd off with sterile filter paper (Whatman No 1
filter paper) into a clean conical flask and the filterates was transferred into a sample holder of the rotary
vacuum evaporation where the cold water solvent was evaporated at its room temperature at 28
0
C. The standard
extracts obtained were then stored in a refrigerator at 4
0
C until when required for use.

The Antibacterial Activity of Leaf Extracts of Ocimum Gratissimum and Sida Acuta
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DISC DIFFUSION ASSAY
The disc diffusion method was adopted for the determination of the antibacterial activity of extract.
Whatman No 1 filter paper was used with slight modification. The filter paers were cut into disc of 6mm in
diameter using a perforator. The discs were treated by boiling for 30 minutes,the reason was to destroy
chemicals used in preserving the filter paper and also to avoid the inhibitor of the antimicrobial action of the
extracts on test organisms. After boiling, the disc was sterilized by autaclaving for 15 minutes and were stored
in a sterile bottle for use.

PREPARATION FOR CULTURE MEDIA
Muella Hinton agar was prepared by weighing 38 grams of the powdered agar into 100ml of distilled
water in a clean conical flask.It was soaked for 20 minutes and then covered with a foil andwasautoclaved at
121
0
C,115 atmospheric pressure for 15 minutes. The medium was cooled to 50
0
C and 20ml of the medium was
poured into a sterile glass petri dish and allowed to solidify. The sterility of the medium was tested by allowing
it to stay overnight and checking for contamination.
A flaming wire loop is being used to pick an organism and stick it well on a prepared media.After that the paper
disc is picked with a sterilized wire loop and use it to collect the extarct then place it on the media in which the
organism is stick on. Then covered well and incubated for 24 hours at 37
0
C.
After an overnight incubation at 37
0
C,the zone inhibition was measured and recorded.The test was carried out
in duplicates of different organisms with different extracts.

DETERMINATION OF MINIMUM INHIBITORY CONCENTRATION
A stock solution of 100mg/ml of ethanol and cold water extracts were prepared.0.4ml of the sloution of
Sida acuta and Ocimum gratissimum is diluted to 20ml with the nutrient broth.Serial dilutions were prepared to
obtain the following concentrations:400,200,100,50,25,12.25mg/ml.0.0ml of the suspensions of overnight
cultures of the test organisms which was adjusted to McFaralnd standard.The organism is stricked on the agar
medium,using a cut borer to make wholes on the medium.Different concentrations of the extracts were added
into the wholes and were covered and incubated for 24 hours at 37
0
C (Ekundayo and Ezeogu,2006). The zones
of inhibition were measured,and the least concentration of each extracts that inhibited microbial growth after the
incubation period wsa taken on the MIC.

CONTROL EXPERIMENT USING GENTAMYCIN AND TETRACYCLINE
Gentamycin and Tetarcycline were used as a control in order to compare the diameter of zone of
inhibition from the extracts and already standardized antibiotics and it was carried out aseptically.This is to
ensure the prescription of either antibiotics or plants herbs for antibacterial activities.
Gentamycin and tetracycline (250mg) capsule was dissolved to get a stock solution of 50,000mg/ml.0.4ml of
these solutionswee taken to make up t 10ml(0.4ml+9.6ml) of distilled water. 20ml (0.2ml) of the dilution was
dropped on each disc.Thes discs were placed on the inoculated culture medium along side the aqueous and
ethanol extracts.

III. Results
PERCENTAGE YIELD OF PLANT EXTRACT
The yields of the plant extracts(ethanol and aqueous) were calculated as percentages of the initial
powdered sample of plant materials shown in table 3 below.The ethanol extracts of Ocimum gratissimum gave
the highest yield which is 8.5g representing 40%,while the ethanol extract of Sida acuta yields 7.5g representing
35%.The percentage yields of aqueous extracts of Ocimum gratissimum and Sida acuta were 30% and 25%
respectively.

ANTIBACTERIAL ACTIVITY OF PLANT EXTRACTS
The antibacterial activities of the extraction of two plant extracts were measured by the diameter zone
of inhibition using the disc agar assay and by using serial dilution to determine the minimum inhibitory
concentration (MIC) of the extracts.The summary of the diameter zone of inhibition produced by the plants
extract against the organisms is shown below in table 4.The ethanol extract of Sida acuta had the zone of
inhibition of 18mm on Staphylococcus aureus and Streptococcus faecalis it yielded 14mm.The diameter zone of
inhibition of the ethanol extarcts on Escherichia coli is 16mm,while Pseudomonas aeruginosa yielded
14mm.The aqueous extract of Sida acuta had diameter zone of inhibition of 10mm,on both Staphylococcus
aureus and Pseudomonas aeruginosa and 12mm on both Escherichia coli and Streptococcus faecalis.
On Ocimum gratissimum in the ethanol extract yields 13mm,15mm and 12mm on Staphylococcus aureus,
Streptococcus faecalis. Escherichia coli and Pseudomonas aeruginosa respectively while its aqeous extract
The Antibacterial Activity of Leaf Extracts of Ocimum Gratissimum and Sida Acuta
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yield 7mm,6mm,8mm,and 8mm on Staphylococcus aureus, Streptococcus faecalis. Escherichia coli and
Pseudomonas aeruginosa respectively
Table 3 shows the minimum inhibition concentration in Staphylococcus aureus using the plant extarcts in which
its ethanolic extracts had minimum inhibition concentration of 0.125 on both extracts.Tables 4,5,and 6 also
shows the minimum inhibition concentration on Streptococcus faecalis, Escherichia coli and Pseudomonas
aeruginosa. Escherichia coli had the lowest minimum inhibition concentartion which is 0.0625 on ethanolic
extracts of Sida acuta.
Gentamycin(control) show a wide zone of inhibition on both Escherichia coli and Staphylococcus aureus as
32mm and 24mm or Ocimum gratissimum and 25mm and 20mm on both organisms but in Sida acuta.While
tetracycline(control) yield 22mm and 10mm on both organisms in Ocimum gratissimum and 30mm and 26mm
on both organisms by Sida acuta

TABLE 1:THE PERCENTAGE OF THE CRUDE EXTRACTS OF Ocimum gratissimum and Sida acuta
Plant species Extracts weight of the weight of the percentage yield
Powdered sample extracts recovered of extracts
Ocimum Aqueous 20.0g 6.0g 30%
Gratissimum ethanol 20.0g 8.5g 40%
Sida acuta Aqueous 20.0g 5.0g 25%
Ethanol 20.0g 7.5g 35%

TABLE 2:ANTIBACTERIAL ACTIVITY OF THE EXTRACTS WITH DIAMETER ZONE OF
INHIBITION
Plant Extract type E.coli S.faecalis P.aeruginosa S.aureus
Species Aqueous 8mm 6mm 8mm 7mm
Ocimum Ethanol 15mm 10mm 12mm 13mm
gratissimum Gentamycin 32mm - - 24mm
Tetracycline 22mm - - 10mm
Sida acuta Aqueous 12mm 12mm 10mm 10mm
Ethanol 16mm 14mm 14mm 18mm
Gentamycin 25mm - - 20mm
Tetracycline 30mm - - 26mm

TABLE 3:THE MINIMUM INHIBITION CONCENTRATIONS ON Staphylococcus aureus by the two
PLANT EXTRACTS USED.
Plant extracts Different concentration of the extracts
1.0 0.50 0.25 0.125 0.0625 0.0315 MIC
Sida acuta Aqueoeus 10.0 7.6 5.6 0 0 0 0.25
(Cold)
Ethanol 20.6 14.6 11.0 7.3 0 0 0.125
Ocimum Aqueous 7.0 5.6 3.3 0 0 0 0.25
Gratisssimum (Cold)
Ethanol 13.0 9.5 7.6 5.3 0 0 0.125
KEYS : MIC=MINIMUM INHIBITION CONCENTRATION

TABLE 4:THE MINIMUM INHIBITION CONCENTRATIONS OF THE EXTRACTS ON
Streptococcus faecalis by two PLANT EXTRACTS
Plant extracts Different concentration of the extracts
1.0 0.50 0.25 0.125 0.0625 0.0315 MIC
Sida acuta Aqueoeus 12.0 9.6 5.6 0 0 0 0.25
(Cold)
Ethanol 14.0 10.3 6.3 0 0 0 0.25
Ocimum Aqueous 6.0 5.6 0 0 0 0 0.5
Gratisssimum (Cold)
Ethanol 10.0 7.6 6.3 5.6 0 0 0.125





The Antibacterial Activity of Leaf Extracts of Ocimum Gratissimum and Sida Acuta
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TABLE 5:THE MINIMUM INHIBITION CONCENTRATIONS OF THE EXTRACTS ON Escherichia
coli by two PLANT EXTRACTS
Plant extracts Different concentration of the extracts
1.0 0.50 0.25 0.125 0.0625 0.0315 MIC
Sida acuta Aqueoeus 12.0 9.6 5.6 0 0 0 0. 125
(Cold)
Ethanol 18.0 14.6 10.3 9.5 6.6 0 0.0625
Ocimum Aqueous 12.0 9.6 8.0 5.6 0 0 0.125
Gratisssimum (Cold)
Ethanol 15.0 12.0 9.3 6.6 0 0 0.125

TABLE 6:THE MINIMUM INHIBITION CONCENTRATIONS OF THE EXTRACTS ON
Pseudomonas aeruginosa by two PLANT EXTRACTS
Plant extracts Different concentration of the extracts
1.0 0.50 0.25 0.125 0.0625 0.0315 MIC
Sida acuta Aqueoeus 10.0 9.6 6.3 0 0 0 0.25
(Cold)
Ethanol 14.0 11.3 8.6 6.3 0 0 0.125
Ocimum Aqueous 5.0 0 0 0 0 0 1.0
Gratisssimum (Cold)
Ethanol 8.0 5.6 5.3 0 0 0 0.25

IV. Discussion
The inhibitory activities of ethanolic extarcts of Ocimum gratissimum and Sida acuta were found to be
little greater than aqueous(cold) crude extracts according to Sofowora(1993) the active principles of the plant
herb may be more soluble in ethanol as employed in traditional medicine.
In this study reported here,the aqueous(cold) extarcts of Ocimum gratissimum and Sida acuta showed the zone
of inhibition to all the organisms used which are Pseudomonas aeruginosa,Escherichia coli,Streptococcus
faecalis and Staphylococcus aureus.This result almost correspond to the findings of Lewis and Elvin(1995) who
in thier work found the weak antibacterial activity on the extarcts on both gram positive and gram negative
organisms.
The ethanol extracts of Ocimum gratissimum and Sida acuta which showed a greater zone of inhibition
to all organisms used.This also correspond to the findings of(Onajiobi,1986) who in their work found that this
plants can be useful in treating some infectious disease such as diarrhoae,haedache,skin
disease,pneumonia,fever,gum disorder etc.
The effectiveness of antimicrobial agent varies with the nature of organisms being treated.Since microorganisms
differ markedly in their susceptibility.The presence of the active principles in plants is influenced by several
factors such as age of the plants,method of extraction and extracting solvent.It is possible that the leaves of the
Sida acuta and Ocimum gratissimum contains high concentration of the antimicrobial compounds or different
antimicrobial agents based on the values of minimum inhibition concentration and the zone of inhibitions.
However,there was a based a broad spectrum activity observed on the two plants extracts which are Ocimum
gratissimum and Sida acuta as it showed high activity against both gram negative and gram positive tested.

V. Conclusion
Ocimum gratissimum and Sida acuta are plant of wide usage in traditional medicine.The results of this
work,now demonstrated that these plants were active on several bacterial starins such as Pseudomonas
aeruginosa,Escherichia coli,Streptococcus faecalis and Staphylococcus aureus.

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