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Research Article
ISSN: 0974-6943
with 0.01% mercuric chloride solution. The leaf and stem were chopped
separately into small pieces and shade dried at room temperature for seven
days.
Extraction
The extraction of leaf and stem was carried out with different solvents in
their increasing order of polarity viz., hexane, benzene, ethyl acetate, acetone, methanol and ethanol by soaking the plant material in the respective
solvents overnight at room temperature one after the other 4 . The contents of
each flask were subjected to reflux below the boiling point of the respective
solvents viz., Hexane (68o C), Benzene (80o C), Ethyl acetate (77o C), Acetone
(55o C), Methanol (65o C) and Ethanol (78o C) for 6-8h in order to extract the
active compounds into the solvent. Each extract was vacuum filtered and the
filtrates were concentrated by vacuum distillation. The concentrated extracts
were incubated at 37o C for 3-4 days to facilitate complete evaporation of the
volatile solvent leaving behind the dried plant extract. The dried plant extract
of100mg each was dissolved in 1000l of 1:10 diluted DMSO (in sterile
distilled water) so as to obtain the final concentration of 10mg /100l 5 .
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Zone of inhibition in mm
Hexane
Benzene
Ethyl acetate
Acetone
Methanol
Ethanol
Gentamicin
20
18
16
14
12
10
8
6
4
2
0
Esch
S
E
S
erich nteroba Klebsiella Pseudom Bacillus Lactobac taphylo treptoco
c
cter
ia co
aero pneumo onas aer subtilis illus delb occus au ccus pyo
li
ruec
gene
ugino
gene
niae
reus
kii
s
s
sa
Test culture
Fig-1. Antibacterial activity of Leaf extracts in Hexane, Benzene, Ethyl acetate,Acetone,Methanol and Ethanol of
Avicennia marina on selected bacteria in comparison with Gentamicin.
Antibacterial activity by the Stem extracts of Avicennia marina
Zone of inhibition in mm
Hexane
Benzene
Ethyl acetate
Acetone
Methanol
Ethanol
Gentamicin
20
18
16
14
12
10
8
6
4
2
0
Esch
E
P
S
K
S
L
erich nteroba lebsiella seudom Bacillus actobac taphyloc treptoco
cter
i
o
ccu
l
ia co
o
l
s
p
n
u
cc
ubt
neu
as
s de
aero
li
lbrue us aure s pyoge
g e n e m o n i a e aerugin i l i s
us
nes
c
osa
kii
s
Test culture
Fig-2. Antibacterial activity of Stem extracts in Hexane, Benzene, Ethyl acetate, Acetone, Methanol and Ethanol of
Avicennia marina on selected bacteria in comparison with Gentamicin.
of culture medium from each broth tube showing no apparent growth was
picked upon and sub-cultured on fresh Mueller Hinton agar medium. After
incubation at 37o C for 24 hrs, plates showing no visible growth of bacteria
were considered for Bactericidal effect and plates with visible growth of
bacteria as Bacteriostatic 8 .
RESULTS AND DISCUSSION
The data on antibacterial activity of the Leaf and stem extracts of A. marina
L in, hexane, benzene, ethyl acetate, acetone, methanol, ethanol and gentamicin against eight bacterial species is given in Figs 1 and 2. Hexzane, benzene
and acetone extracts of A. marina L. leaf were effective only against gram
negative test cultures used, where as, ethyl acetate and methanol extracts are
active only on gram positive cultures used. While the ethanol extracts were
active on all the test cultures used except E. coli and E. aerogenes.
Leaf extract of A. marina L. in ethyl acetate and methanol were active only on
gram positive cultures used. The effect of these extracts is higher than gentamicin against gram positive test cultures except leaf extract in ethyl acetate
against B. subtilis and was found to be equivalent to gentamicin.. Hexane
and Acetone extracts of A. marina L leaves were active only against E. coli
and E. aerogenes, with same zone size. The efficacy of these extracts were
less than that of the gentamicin. Whereas, the benzene extracts were effective only against the gram negative test cultures used. The zone size in the
case of K. pneumonia and P. aeruginosa to ethanol extracts of leaf were
same and is less than that of the gentamicin. However, the effectiveness
against B. subtilis, L. delbrueckii, S. aureus and S. pyogenes is higher than
gentamicin.
The stem extracts of A. marina in hexane, ethyl acetate, acetone, methanol
and ethanol were effective on all the gram positive test organisms used. The
efficacy of the extracts on these organisms was higher than gentamicin.
Where as, benzene extract does not inhibit any one of the gram positive test
cultures used, but showed effect on the growth of gram negative test cultures
used. E. coli and E. aerogenes were senstive only to the stem extract in
benzene. Where as, K. pneumonia and P. aeruginosa were sensitive to the
stem extracts in benzene, acetone and ethanol. The size of the inhibition
zones in the case of K. pneumonia and P. aeruginosa were same to the
benzene and acetone extracts and are higher than that of gentamicin, however, same zone size is exhibited by the ethanol extract also but it is lower
than that of gentamicin.
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Hexane
Benzene
Ethyl acetate
Acetone
Methanol
Ethanol
5.0
5.0
-
5.0
5.0
5.0
5.0
-
2.5
2.5
2.5
2.5
5.0
5.0
-
1.25
1.25
1.25
1.25
5.0
5.0
1.25
1.25
1.25
1.25
Table-2. MIC (mg/100l) of the Stem extracts of Avicennia marina against the bacterial test organisms
Organism
Hexane
Benzene
Ethyl acetate
Acetone
Methanol
Ethanol
2.5
2.5
2.5
2.5
5.0
2.5
2.5
2.5
-
2.5
2.5
2.5
2.5
1.25
1.25
2.5
2.5
2.5
2.5
1.25
1.25
1.25
1.25
2.5
2.5
1.25
1.25
1.25
1.25
Table-3. Bactericidal /Bacteriostatic activity of Leaf extracts of Avicennia marina against the bacterial test organisms
Organism
Hexane
Benzene
Ethyl acetate
Acetone
Methanol
Ethanol
BS
BS
-
BS
BS
BS
BS
-
BS
BS
BS
BS
BS
BS
-
BC
BC
BC
BC
BC
BC
BC
BC
BC
BC
BS Bacteriostatic
BC - Bactericidal
Table-4 Bactericidal /Bacteriostatic activity of Leaf extracts of Avicennia marina against the bacterial test organisms
Organism
Hexane
Benzene
Ethyl acetate
Acetone
Methanol
Ethanol
BS
BS
BS
BS
BS
BS
BS
BS
-
BS
BS
BS
BS
BS
BS
BS
BS
BS
BS
BC
BC
BC
BC
BC
BC
BC
BC
BC
BC
BS Bacteriostatic
BC - Bactericidal
The positive extracts for antibacterial activity, were further tested to determine the Minimum Inhibitory Concentration (MIC) at different concentrations viz., 1.25, 2.5 and 5.0 mg /100l and the data is given in tables 1 and
2. The value of MIC was found to be in the range of 1.25 to 5.0 mg /100l
for leaf and stem extracts of A. marina L. against all the bacteria tested. The
MIC of leaf extracts of A.marina L. in hexane, benzene acetone and ethanol
was found to be 5mg/100l for all the gram negative bacteria tested. Whereas,
the ethyl acetate extract exhibited 2.5mg/100l MIC value for the gram
positive test cultures. Leaf extracts of methanol and ethanol showed a MIC
value of 1.25mg/100l on gram positive test cultures.
The hexane, benzene and acetone extracts of stem showed the MIC value of
2.5mg/100l against all the gram positive bacteria tested, where as, methanol
and ethanol extracts exhibited a MIC value of 1.25mg/100l against the same
gram positive bacteria. Regarding gram negative bacteria, the benzene extract
showed a MIC value of 5.0mg/100l against E.coli and 2.5mg/100l on the
rest of the gram negative bacteria tested. The acetone and ethanol extracts of
stem exhibited the MIC values of 1.25mg/100l and 2.5mg/100l, respectively on K. pneumonia as well as P. aeruginosa.
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