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The in vitro
antibacterial activity
of different extracts of
Cissus quadrangularis
Linn (Vitaceae) against
some Gram-negative and
Gram-positive bacteria,
were investigated. The
methanol and ethyl
acetate extract showed
high
activity against the
bacteria tested.
Cissus quadrangularis
Linn (Family: Vitaceae)
is a perennial climber,
found mostly in the
hotter parts of the
world such as India, Sri
Lanka, Tropical Africa,
South Africa, Thailand,
Java, and Philippines1.
The plant is mentioned
in the ancient systems
of medicine such as
Ayurveda, and is useful
for treatment of bloody
diarrhoea, skin
disorders, earache2,
haemorrhoids, irregular
menstruation, and
accelerates healing of
bone fracture3.
Murthy et al.4 have
previously reported the
antimicrobial
effects of the plant,
along with the values of
zones of inhibition.
Here, we report the
minimum inhibitory
concentration (MIC)
values of the different
extracts of the plant
stem using the
microtitre plate
method5,6, which is
relatively simple,
reproducible, and rapid.
The stems of Cissus
quadrangularis,
collected from the
plants maintained in the
greenhouse were washed
with water, cleaned, cut
into small sections, and
dried in an oven at 40°
for 10-12 d. The dried
material was ground to a
fine powder in a
homogeniser. The powder
was stored in a
refrigerator.
Extraction was carried
out using a Soxhlet
apparatus.
Ethyl acetate, acetone,
petroleum ether
(40-60o), methanol (HPLC
grade, Sisco Research
Laboratories, Mumbai),
ethanol, and water were
the solvents used for
extraction. The extracts
were evaporated to
obtain dry residues, and
then solutions of known
concentrations were
prepared in respective
solvents.
Bacillus subtilis (ATCC
No. 6633), Escherichia
coli (ATCC No. 25922),
Pseudomonas aeruginosa (ATCC
No. 27853), and
Staphylococcus aureus (ATCC
No. 25923), were
obtained from National
Centre for Industrial
Microorganisms, National
Chemical Laboratory,
Pune. Clinical samples
of Salmonella typhi and
Streptococcus pyogenes
were obtained from the
cultures maintained in
the department.
The bioassays were
performed in 96-well
sterile microtitre
plates (Nunclon,
Denmark). All the 96
wells of the plate were
filled with 50 μl of
sterile nutrient broth (HiMedia
Laboratories). A 50 μl
aliquot of the extract
was then added to row A,
and a 50% serial
dilution was achieved
(row A to H) by
transferring 50 μl at a
time to subsequent
wells. In all, there
were four controls;
sterility control: 50 μl
nutrient broth only,
growth control: 50 μl
nutrient broth + 50 μl
culture, ampicillin
control: 50 μl nutrient
broth + 50 μl culture +
250 μg/ml ampicillin,
solvent control: 50 μl
nutrient broth + 50 μl
culture + 50 μl solvent.
An aliquot of a 3 h
culture (50 μl) of the
test organism in
nutrient broth was added
in each well, except in
the sterility control
well. The covered
microplates were
incubated for 24 h at
37°. Bacterial growth
was detected by adding
50 μl of 0.2 mg/ml
solution of
2[4-iodophenyl]3[4-nitrophenyl]-5-phenyl-2H-tetrazolium
chloride (INT, from SRL)
in each test well, and
the plate was incubated
further for at least 30
min at 37°, to ensure
adequate colour
development. The lowest
concentration in which
there was a definite
decrease in colour was
taken as the MIC of that
extract, for that
particular organism. All
the experiments were
performed in
triplicates. The results
obtained, were subjected
to ANOVA and critical
difference analysis (CD
analysis), at 5% level
of significance.
The MIC values of
different extracts
against all the
organisms tested, are
detailed in Table 1. The
ethyl acetate, acetone,
and methanol extracts,
showed antimicrobial
properties and that B.
subtilis, P. aeruginosa,
S. typhi, S. aureus, and
S.pyogenes, were
susceptible to at least
two extracts. Petroleum
ether, ethanol, and
water extracts, failed
to inhibit the bacterial
growth of the strains
tested. E.coli did not
respond to any of the
extracts used.
ANOVA was carried out to
test the significance of
difference between the
antimicrobial properties
of the extracts (ethyl
acetate, acetone and
methanol), and
difference between the
susceptibility of
microorganisms
(P. aeruginosa, S.
typhi, S. aureus). The
analysis showed that
there is a significant
difference between the
extracts (F value=8.84;
F critical=3.42) and the
microorganisms (F
value=15.63; F
critical=3.42).
Further, CD analysis was
carried out to pinpoint
the best extract, and
the most susceptible
microorganism. The
analysis revealed that
the methanol extract is
significantly better
than all the other
extracts used
(difference of
means=2.04 and CD
value=1.80), and
Staphylococcus aureus is
significantly more
susceptible as compared
to the other tested
strains (difference of
means=2.77 and CD
value=1.80). All the
tests were carried out,
and the decisions were
taken at 5% level.
Further studies are
needed to isolate and
characterize.
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