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Antibacterial activity and qualitative phytochemical analysis of medicinal

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Antibacterial activity and qualitative phytochemical analysis

International Journal of Integrative Biology

Regular A journal for biology beyond borders ISSN 0973-8363

Antibacterial activity and qualitative phytochemical analysis of

medicinal plant extracts obtained by sequential extraction method

Kandiah Uthayarasa 1, Kulanthaivelu Pathmanathan 1,*,

Jeyaratnam Prince Jeyadevan 2, Emmanuel Christy Jeyaseelan 1
1
Botany, University of Jaffna, Jaffna, Sri Lanka
2
Chemistry, University of Jaffna, Jaffna, Sri Lanka

Submitted: 21 Apr. 2010; Revised: 2 Jul. 2010; Accepted: 14 Jul. 2010

Abstract
The aim of this study is to examine the antibacterial activity of sequentially extracted hexane, dichloromethane
(DCM), ethyl acetate, ethanol, methanol and water extracts of fruit of Emblica officinalis, rind of Punica
granatum and rhizome of Curcuma longa at various concentrations against Bacillus subtilis, Pseudomonas
aeruginosa, Staphylococcus aureus and Escherichia coli by agar well diffusion method. The phytochemical
analysis of each solvent extract was also carried out. Results were subjected to one-way analysis of variance
(ANOVA) and followed by Least Significant Difference (LSD) test. This study revealed that the ethanol extract
of P. granatum has significant (P<0.05) effect on all test pathogens except P.aeruginosa, which was highly
inhibited by ethyl acetate extract of E. officinalis. Dose response study demonstrated that the ethanol and
methanol extracts of all test plants, ethyl acetate extracts of E. officinalis and P. granatum, and aqueous extracts
of E. officinalis and C.longa were able to inhibit the test bacteria at concentration ranging from 0.5mg/100 µl to
10.0mg/100 µl. Phytochemical analysis revealed that ethyl acetate extract of E. officinalis has tannins, alkaloids,
flavonoids, saponins, cardiac glycoside and terpenoids while none of the phytochemicals were present in hexane
extracts of all test plants and DCM extract of P. granatum. Other test samples have at least one of the
phytochemicals tested. This study revealed that the sequential extraction of plant materials, in the initial step of
drug discovery, would be more effective than single solvent extraction. In order to address the novel
antibacterial agents, the active extracts of the present study could be subjected to next step of purification and
bioassay.

Keywords: Phytochemical analysis; antibacterial activity; sequential extraction; medicinal plants.

INTRODUCTION in traditional medicine for the treatment of diarrhoea,

Medicinal plants are successful natural sources of
materials for the treatment of various infectious
diseases of human (Stockwell, 1998) and therefore
scientists are vigorously focusing their attention to
discover natural compounds from medicinal plants with
the aim of introducing new drugs which would be more
effective than those available in the market (Parekh et
al., 2006).
Emblica officinalis (Indian gooseberry) belonging to
family Euphorbiaceae is a deciduous tree. Its fruit are
diuretic and laxative (Parmer et al., 1982), and are used
*
Corresponding author:
M.K.Pathmanathan, Ph.D.
Department of Botany,
Faculty of Science,University of Jaffna,
Jaffna , Sri Lanka
Email: sumpath17@yahoo.com
dysentery, cough and anemia (Ahmad et al., 2001).
Punica granatum (Pomegranate), a member of the
family Punicaceae, is an erect deciduous spreading
shrub or small tree, woody and thorny (Parmer et al.,
1982). The aqueous form of dried fruit coat (rind) is
taken internally for the treatment of diarrhea and
stomachache (Duraipandiyan et al., 2006). It also
possesses antioxidant, anti-malarial and antimicrobial
activity (Reddy et al., 2007). Curcuma longa (Turmeric)
belongs to the family Zingiberaceae, is a perennial herb
consisting of short and thick under ground rhizome
(Chandarana et al., 2005). Rhizome of this plant is
useful for the treatment of fever, wounds, infections,
dysentery, arthritis and liver disorders (Gul et al., 2004).
The extraction methods of plant materials may
influence the inhibitory potentiality when testing
against microbes (Valgas et al., 2007). The sequential
extraction technique has become a popular method for
the extraction of active components from natural

International Journal of Integrative Biology IJIB, 2010, Vol. 10, No. 2, 76

©IJIB, All rights reserved
Antibacterial activity and qualitative phytochemical analysis
sources as the sequential extraction procedure involves
different solvents of various polarities that can provide
optimum effect of extraction and better activity than
direct solvent extraction (KenWojcikowski et al., 2007).
The antibacterial activity of different solvent extracts of
E. officinalis, P. granatum and C. longa have been
reported with single solvent extraction method in
various studies. In this study an attempt is made to test
the antibacterial activity of different solvent extracts of
above plants, obtained by sequential extraction method,
in some gram positive and gram negative bacteria and
also to study the phytochemical constituents present in
the test extracts.
MATERIALS AND METHODS
Collection and identification of plant
material
Emblica officinalis, Punica granatum and Curcuma
longa were identified by the Department of Botany,
University of Jaffna, Sri Lanka. The fresh and disease
free fruit of E. officinalis, rind of P. granatum and
rhizome of C. longa were collected, washed thoroughly
under running tap water, dried under sun light and
ground into powder using electric blender. The
powdered materials were stored in airtight bottles at
room temperature until used.
Extraction of plant materials
Sequential extraction method (Giridharan et al., 2002)
was employed to extract the plant powders using
different organic solvents from low polar to polar
namely hexane, dichloromethane (DCM), ethyl acetate,
ethanol, methanol and finally with water. One hundred
grams of each powder was soaked in 300 ml of hexane
into the airtight bottle separately and kept on an electric
shaker at room temperature for three days. Then these
were first filtered through double layered muslin cloth
and then using Whatman No.1 filter paper. Each filtrate
was collected into conical flask separately. This step
was repeated thrice. Hexane was removed from each
filtrate under reduced pressure using rotary evaporator
at low temperature and dried extracts were stored in
refrigerator until used. The residue was dried and used
for dichloromethane extraction followed by ethyl
acetate, ethanol, methanol and water similar to the
procedure carried out for hexane. The yield percentage
of each extract was calculated as follows:
Final weight of dried extract
% yield = ------------------------------- * 100
Initial weight of powder
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Phytochemical analysis
All the test samples were subjected to phytochemical
analysis to find out the presence of chemical
constituents according to Trease and Evans (1989) for
tannins and alkaloids, Harborne (1973) for saponins,
cardiac glycosides and flavonoids and Sofowora (1993)
for terpenoids.
Test for tannins
20 mg of dried each test sample was dissolved in 1 ml
of distilled water in a test tube and 1-3 drops of 0.1%
ferric chloride were added to the water solution. Then
the mixture was observed for blue or green colour.
Test for Alkaloids
20 mg of dried test sample was dissolved in 2 ml
methanol and a few drop of 1% HCl was added to it.
Then the mixture was heated kept in steam and after
cooling, mixture was treated with few drops of
Wagner’s reagent. The sample was then observed for
the presence of turbidity or precipitation.
Test for cardiac glycosides
20 mg of test sample was dissolved in 1 ml of glacial
acetic acid and 1-2 drops of ferric chloride solution was
added to it. This was under layered with 0.5 ml of
concentrated sulphuric acid. A brown ring of the
interface indicates a deoxysugar characteristic of
cardenolides.
Test for saponins
40 mg of test sample was dissolved with 5 ml of
distilled water and shaken vigorously for a stable
persistent froth. The frothing was mixed with 3 drops
of olive oil and shaken vigorously and then observed
for the formation of emulsion.
Test for flavonoids
20 mg of test sample was dissolved in 1 ml of distilled
water and then 0.5 ml of dilute ammonia solution was
added to it followed by concentrated H2S04. A yellow
colouration observed in each extract indicated the
presence of flavonoids. The yellow colouration
disappeared on standing.
Test for terpenoids
20 mg of each solvent test sample was dissolved in 1ml
of chloroform and 1ml of concentrated H2S04 was
carefully added to it. A reddish brown colouration of
the inter face was used to show positive results for the
presence of terpenoids.
IJIB, 2010, Vol. 10, No. 2, 77
Antibacterial activity and qualitative phytochemical analysis
Test bacteria
Gram positive bacteria, Bacillus subtilis and
Staphylococcus aureus and Gram negative bacteria,
Pseudomonas aeruginosa and Escherichia coli were
kindly provided by Department of Microbiology,
Faculty of Medicine, University of Jaffna, Sri Lanka.
All the test bacteria were maintained on nutrient agar
slope at 4ºC. Then these were sub cultured for 24 hours
before use.
Preparation of test sample
The working concentrations of 0.1, 0.5, 1.0, 10.0, 20.0,
30.0, 40.0, 50.0 and 60.0 mg/100µl were prepared
using the solvent mixture of dimethyl sulfoxide
(DMSO) and acetone (1:1 v/v).
Determination of antibacterial activity
In vitro antibacterial activity of different crude extracts
obtained by sequential extraction was studied using
agar well diffusion method (Nair et al., 2005). Briefly
15 ml of autoclaved nutrient agar was cooled down to
40ºC and mixed with 1 ml of bacterial suspension (1 x
106cells/ml). This was poured into a sterile Petri dish
and allowed to set. Wells were made on it using sterile
cork borer of 8 mm diameter and each well was filled
with 100 µl of each extract dissolved in acetone and
DMSO (1:1 v/v). 100 µl of Streptomycin (0.5mg/ml)
and mixture of acetone and DMSO (1:1 v/v) were used
as standard and control, respectively. The plates were
incubated at 37 ºC for 24 hours and antibacterial
activity was recorded by measuring the zone of
inhibition around the well. Each experiment was
repeated thrice and the mean value was calculated.
Experiments were carried out under biological safety
cabinet.
Statistical analysis
The diameter of zone of inhibition (excluding well) of
triplicates was expressed as mean ± standard deviation
(SD). The data were subjected to one-way analysis of
variance (ANOVA), P value < 0.05 was considered as
significant and mean values were compared by using
Least Significant Difference (LSD) test using computer
software, SAS system for windows (version 8.0).
RESULTS
Yield Percentage
The yield percentage of sequential extraction varied
with the plant materials used. The yield of hexane
extract of all three plants was found to be lesser than
the other solvent extracts. Furthermore, the yield
increased with increasing polarity up to ethanol and
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decreased to methanol and again increased to aqueous
extract in both E. officinalis and C. longa whereas in P.
granatum it increased up to methanol and then
decreased to aqueous extract (Table 1 [Supplementary
data]). The maximum yield was obtained from ethanol
extract for E. officinalis and C. longa and methanol
extract for P. granatum.
Antibacterial activity of different extracts
of test plants
The results demonstrated that 10.0 mg/100 µl of
ethanol and methanol extracts of all three plants, ethyl
acetate extract of E. officinalis and P. granatum and
aqueous extract of E. officinalis and C. longa were able
to inhibit the growth of all the test bacteria at various
degrees (Table 2 [Supplementary data]). Among the test
samples ethanol extract of P. granatum showed
significantly (P<0.05) higher inhibition on all test
pathogens except P. aeruginosa, which was highly
inhibited by ethyl acetate extract of E. officinalis.
Ethanol extracts of E. officinalis can be considered as
equally potent (P>0.05) as P. granatum on B. subtilis.
Among the different solvent extracts of E. officinalis
ethyl acetate, ethanol and methanol extracts exhibited
higher effect on S.aureus while aqueous extract of same
plant showed higher inhibition on B. subtilis (Table 2).
Among the methanol extracts, C. longa showed lower
effect against all the test bacteria compared to other two
plants. Although aqueous extract of E. officinalis and C.
longa were able to inhibit the growth of all pathogen
tested, E. officinalis possessed higher inhibitory effect
than C. longa except E.coli but aqueous extract of P.
granatum did not show any inhibition on any pathogen
tested (Table 2). Among the ethyl acetate extract of test
plants, E. officinalis showed higher inhibition against
all the test bacteria and this inhibitory effect differed
significantly (P<0.05) from other two plants on all test
pathogens (Table 2).
Hexane and DCM extracts of E. officinalis and C.
longa failed to inhibit the test bacteria except S. aureus,
which was inhibited by both hexane extract of E.
officinalis and DCM extract of C. longa. But both
hexane and DCM extracts of P. granatum were able to
inhibit S. aureus and P. aeruginosa and also the later
extract showed inhibition on B. subtilis (Table 2).
Effect of standard and control
The experiment performed with the standard antibiotic
revealed that the order of the sensitivity to streptomycin
was found to be B. subtilis, P. aeruginosa, S. aureus
and finally E.coli. There was no inhibition exerted by
solvent mixture that was used as control. Further all the
active test samples from E. officinalis, Ethanol and
methanol extracts of P. granatum and aqueous extract
of C. longa showed better effect than standard
IJIB, 2010, Vol. 10, No. 2, 78
Antibacterial activity and qualitative phytochemical analysis
antibiotic on E. coli (Table 2). Inhibitory effect of
streptomycin on B. subtilis, and P. aeruginosa was
found to be higher than all other test samples. But 10.0
mg / 100µl of methanol extract of P. granatum,
methanol and ethyl acetate extracts of E. officinalis
showed almost equal effect on S. aureus compared to
standard antibiotic (50.0 µg / 100µl) used in this study.
Dose response study of solvent extracts of
test plants
Dose response study performed with the various
solvent extracts of test plants revealed that these
samples have antibacterial activity in concentration
dependent manner on all test pathogens (Table 3, 4 and
5 [Supplementary data]). Minimum Inhibitory
Concentration (MIC) value for most of the solvent
extracts ranged from 0.5mg/100 µl to 10.0mg/100 µl.
The result also revealed that the concentration of 0.1
mg / 100µl or less (data not shown) had no effect on
test pathogens.
Phytochemical screening
Phytochemical analysis revealed that ethyl acetate
extract of E. officinalis has tannins, alkaloids,
flavonoids, saponins, cardiac glycoside and terpenoids
while none of the phytochemical was present in hexane
extracts of all test plants and DCM extract of P.
granatum. Other test samples have at least one of the
phytochemical tested (Table 6 [Supplementary data]).
DISCUSSION
In this study comparatively most of the polar solvent
extracts revealed better activity than low polar solvent
extracts (Table 2). The higher inhibitory effect of polar
solvent extracts is due to the more solubility of active
components in polar solvents and the polarity of
solvents may play an important role in the inhibitory
effect of plant extracts (Parekh et al., 2006). Yield
percentage of different solvent extracts obtained from
sequential extraction clearly revealed that the
compounds dissolved in polar solvents are
quantitatively higher than compounds dissolved in low
polar solvents (Table 1). Though, yield of ethanol
extract of C. longa was greater than ethanol extract of E.
officinalis the antibacterial effect of ethanol extract of E.
officinalis was higher than C. longa (Table 2). It reveals
that the amount of yield does not always influence in
inhibiting the growth of pathogen but the active
ingredients found in the extract play major role.
In an earlier study it was reported that the alcohol
extract of E. officinalis (single solvent extract) had
inhibitory effect on B. subtilis and S. aureus but did not
inhibit the growth of E. coli at the concentration of
15.0mg / 100 μl by agar well diffusion method (Ahmad
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et al., 2001). The present study carried out with similar
assay method using 1.0 mg / 100μl of ethanol extract of
same plant showed better inhibitory effect against
above three bacteria including E. coli (Table 3). In
another investigation made by Panthi et al. (2006), it
was found that methanol extract of E. officinalis (single
solvent extract) had the ability to inhibit the growth of
S. aureus but failed to inhibit the growth of P.
aeruginosa and E. coli at the concentration of 100.0
mg/100μl by disc diffusion method. Interestingly the
present study with lesser concentration (10.0mg/100μl)
clearly demonstrated that methanol extract of E.
offcinalis inhibited the growth of all test bacteria
including P. aeruginosa and E. coli (Table 2). In
another investigation it has been reported that direct
ethanol extract (1:1) of pericarp (rind) of P. granatum
was able to inhibit the growth of P. aeruginosa and B.
subtilis but was unable to inhibit the growth of S.
aureus and E. coli by paper disc method (Nascimento
et al., 2000). However, in the present study ethanol
extract of P. granatum inhibited the growth of all above
bacteria including S. aureus and E. coli even at the low
concentration (Table 4). Chandarana et al., (2004)
reported that the disc diffusion assay of heated and
unheated aqueous extract of C. longa showed inhibitory
effect on E. coli and B. subtilis but did not inhibit the
growth of S. aureus. In the present study sequentially
extracted aqueous extract of C. longa inhibited the
growth of above bacteria including S. aureus at low test
concentration (Table 5) These vast differences of
inhibitory effect between past studies and the present
study might be due to either method of extraction or
assay or sometimes both. It is obvious that the number
of compounds present in the ethanol, methanol and
aqueous extracts in the present study are lesser than the
alcohol, methanol and aqueous extracts used in the
former cases (single solvent extraction) where
antagonistic effect of chemical compounds might have
played a role to minimize the antibacterial effect.
Because compounds present in crude mixture may
interfere with the action of the other (Adwan et al.,
2008). Therefore the efficacy of an antibacterial effect
of a plant material depends not only on the type of
solvent, type of assay and the dose used but also on the
method of extraction of plant materials.
An earlier study (Voravuthikunchai et al., 2005)
revealed that 2.5 mg/disc of ethanol and aqueous
extracts of P. granatum, which were obtained by the
successive extraction using chloroform, ethanol and
boiling water, exhibited inhibitory effect on different
strains of E. coli by disc diffusion method but the
chloroform extract failed to inhibit the growth of all the
tested strains of E. coli where as in present study 1
mg/100 μl of ethanol and methanol extract of P.
granatum showed inhibitory effect on E. coli but
aqueous extract did not show any effect (Table 4). In
the present study, after the ethanol extraction the
residue was continuously extracted with methanol and
IJIB, 2010, Vol. 10, No. 2, 79
Antibacterial activity and qualitative phytochemical analysis
then by water. Therefore, the absence of the inhibitory
effect of aqueous extract may be due to the absence of
the active constituents which would have been
extracted out in methanol.
Negi et al. (2003) reported that the ethyl acetate extract
of rind of P. granatum obtained by sequential
extraction using ethyl acetate, acetone, methanol, and
aqueous did not show any considerable effect on B.
subtilis, S. aureus, P. aeruginosa and E. coli but in the
present study the ethyl acetate extract showed
considerable effect on all these pathogens (Table 4).
Although the sequential extraction method was
employed in both cases, previous was begun with ethyl
acetate and the later was begun with hexane and then
followed by DCM before using ethyl acetate. Therefore
the failure of inhibitory effect of ethyl acetate extract in
the previous case may be due to the interference of
compounds that might have been extracted out in the
later case. Hence, it is suggested that sequential
extraction could be more efficient when it is carried out
with more number of solvents in an increasing polarity
order.
Alkaloids present in the fruit of E. officinalis were
found to be active against B. subtilis and S. aureus
(Rahman et al., 2009) and in another investigation it
was found that monomeric flavonoids was the active
constituents of fruit of E. officinalis (Ahmad and Beg,
2001). But in the present study ethyl acetate, ethanol
and methanol extracts of E. officinalis have both
alkaloids and flavonoids (Table 6) and also these
extracts exhibited good antibacterial activity against all
test bacteria (Table 2). In another instance Ahmad and
Beg (2001) reported that alcoholic extract of P.
granatum possess alkaloids, flavonoids, glycoside and
tannins but not saponins. Also, phytochemical
screening of ethanol extract of pericarp (rind) of P.
granatum revealed the presence of flavonoids and
tannins (Voravuthikunchai et al, 2005). In the present
study the tannins, alkaloids, cardiac glycoside and
saponins were present in the ethanol extract of P.
granatum but flavonoids were found in ethyl acetate
extract. The reason for the absence of flavonoids in the
ethanol extract of the present study might have been
due to the fact that it was already extracted out in the
ethyl acetate extract (Table 6). Therefore, in order to
find the active constituent(s) responsible for the
antibacterial effect, further purification and bioassays
should be done.
In previous studies (Hashemi et al., 2008; Okigbo et al.,
2009) it was demonstrated that the direct aqueous
extract of C. longa possessed flavonoids, terpenoids,
tannins and saponins but in the present study aqueous
extract possessed saponin only (Table 6). The absence
of flavonoids, terpenoids and tannins in the present
study might be due to the separation of these
phytochemicals into other solvents used before the
International Journal of Integrative Biology
©IJIB, All rights reserved
aqueous extraction. Therefore, it further reveals that
sequential extraction of plant material could be useful
as far as further purification is concerned. In an earlier
study it was pointed out that curcumin present in the C.
longa is soluble in the organic solvent but insoluble in
water however the aqueous extract showed antibacterial
effect on test bacteria (Chandarana et al., 2005). In the
present study also the aqueous extract showed
antibacterial effect on test bacteria, and phytochemical
analysis demonstrated that the aqueous extract of C.
longa possessed saponins. Therefore, further
purification and characterization of aqueous extract of
C. longa may provide other novel active compounds.
CONCLUSION
In vitro antibacterial activity of different solvent
extracts of E. officinalis, P. granatum and C. longa
revealed that the ethyl acetate, ethanol, methanol and
aqueous extracts of E. officinalis, ethanol and methanol
extract of P. granatum and aqueous extract of C. longa
exhibited higher inhibitory effect against tested bacteria.
Phytochemical analysis of above extracts revealed the
presence of tannins, alkaloids, flavonoids, saponins,
cardiac glycoside and terpenoids. Therefore, these
active extracts could be subjected to further isolation
and purification of active compounds to discover novel
lead antibacterial agents.
Acknowledgement
The authors wish to thank the National Science Foundation,
Sri Lanka for providing financial assistance (Grant No:
RG/2006/HS/05) for this work.
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