Antimicrobial Activity of Terminaliabellerica Leaf and Stem Collected From Twodifferent Sites
Antimicrobial Activity of Terminaliabellerica Leaf and Stem Collected From Twodifferent Sites
Antimicrobial Activity of Terminaliabellerica Leaf and Stem Collected From Twodifferent Sites
ABSTRACT
INTRODUCTION
India is a varietal emporium of introducing and domesticating new exotic
medicinal plants and is one of the richest plant varieties. In India, the use of
sources of medicinal plants. It exhibits a medicinal plants is centuries-old tradition
wide range in topography and climate, and approximately two million traditional
which has a bearing on its vegetation and health practitioners still use medicinal plants
floristic composition. Moreover, the agro- for curing various ailments.
climatic conditions are conducive for
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obtained. All the settled mass was collected The antimicrobial activity of extracts
together and dissolved in methanol. It was and fractions was done by agar disc diffusion
concentrated further to dryness and method19, 20. The Petri dishes were prepared
designated as Fraction I (FS I i.e. JAC I and by pouring 150 ml sterilized molten Muller
RAC I). The collected supernatant was Hinton Agar for bacteria and Sabouraud
concentrated further to near dryness and then Dextrose Agar for fungal strains, which was
dissolved in methanol. Then chloroform was then seeded with 1 ml of test culture
added to it and cooled. Light yellow waxy containing 1 × 108cfu/mL for bacteria and 2.0
sediment was separated and light buff ×105spores/mL for fungal strains as
coloured supernatant was collected. This McFarland 0.5 turbidity standard. The media
supernatant was concentrated further to were allowed to solidify. Crude acetone
dryness and designated as Fraction II (FS II extract and its Fraction I and Fraction II of
i.e. JAC II and RAC II). both stem and leaf were dissolved in 100%
DMSO (Dimethyl sulfoxide) at concentration
Antimicrobial activity of 20 mg/ml. These drugs were used to
evaluate antimicrobial activity. Sterile filter
Microorganisms tested paper disks (6 mm) were impregnated with 20
Five Gram positive bacteria viz. µl of each drug separately and allowed to
Bacillus cereus ATCC 11778, Bacillus saturated for 30 min, and were then placed on
subtilis ATCC 6633, Listeria monocytogens the surface of the agar plates, which had
ATCC 19112, Corynebacterium rubrum previously been inoculated with test
ATCC 14898 and Staphylococcus microorganisms. All plates were incubated for
epidermidis ATCC 12228; five Gram 24 and 48 hours at 370C and 280C for the
negative bacteria viz. Pseudomonas bacterial and fungal strains, respectively.
aeruginosa ATCC 27853, Proteus mirabilis Results were recorded by measuring the zone
NCIM 2241, Salmonella typhimurium ATCC of inhibition appearing around the disks. All
23564, Escherichia coli NCIM 2931and the tests were performed in triplicate and the
Klebsiella pneumonia NCIM 2719; and three mean values are presented. Pure DMSO was
fungi Candida albicans ATCC 2091, used as the negative control.
Candida glabrata NCIM 3448 and
Cryptococcus neoformans NCIM 3542 were Preparation of Inoculums
obtained from National Chemical Laboratory, From the stock slants bacteria were
Pune, India. The bacteria and fungi were inoculated in Muller Hinton Broth (MHB)
maintained on nutrient agar and MGYP and fungi were inoculated in Sabouraud
medium (Hi Media, India) respectively while Dextrose Broth (SDB). All flasks are
L. monocytogenes and E. coli were incubated for 24 hrs in shaking condition.
maintained on Brain heart infusion agar and
Luria medium (Hi Media, India) respectively
at 4°C and sub-cultured before use. The
microorganisms studied are clinically Preparation of the extracts and/or antibiotics
important ones causing several infections, for MIC and MBC study
food borne diseases, spoilages, skin infection The extracts and fractions dissolved in
and it is essential to overcome them through 100% of DMSO were first diluted to highest
some active therapeutic agents. concentration (1250 μg ml-1) to be tested, and
then serial two-fold dilution was made in a
Antimicrobial assay concentration range (0.605 to 1250 μg ml-1).
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Chloramphenicol and ceftazidime were used (lowest concentration) that remained clear
as a positive control (0.0156 to 32 µg ml-1). corresponded to the MIC.
Preparation of bacterial inocula for MIC and Determination of the minimum bactericidal
MBC study concentration (MBC)
The inocula of the test organisms MBC was determined from all wells
were prepared using the colony suspension showing no growth as well as from the lowest
method 21 . Colonies picked from 24 h old concentration showing growth in the MIC
cultures grown on nutrient agar were used to assay for all the samples. Bacterial cells from
make suspension of the test organisms in the MIC test plate were sub cultured on
saline solution to give an optical density of freshly prepared solid nutrient agar plates by
approximately 0.1 at 600 nm. The suspension making streaks on the surface of the agar. The
was then diluted 1:100 by transfer of 0.1 ml plates were incubated at 37°C for 24 h
of the bacterial suspension to 9.9 ml of sterile overnight. Plates that did not show growth
Muller Hinton broth before use to yield 6 × were considered to be the MBC for the extract
105 CFU ml-1. or drug used24 . The experiment was carried
out in duplicate.
Determination of the minimum inhibitory
concentrations (MIC) Statistical analysis
The MIC was determined by the Each sample was analyzed
micro well dilution method 22 with some individually in triplicate and the results are
modifications. This test was performed in expressed as the mean value (n = 3) ±
sterile flat bottom 96 well micro test plates Standard Error of Mean (S.E.M.).
(Tarsons Products Pvt. Ltd.). 150 µl volume
of Mueller Hinton broth was dispensing into RESULT AND DISCUSSION
each well and 20 µl of varying concentrations
of the extract was added in decreasing order There is continuous and urgent need
along with 30 µl of the test organism for discovery of new antimicrobial
suspension. The final volume in each well compounds with diverse chemical structures
was 200 μl (150 μl Mueller Hinton broth, 30 and novel mechanisms of action because of
µl of the test organism suspension, and 20 μl alarming increase in the incidence of new and
plant extract/antibiotic). Three control wells re-emerging infectious diseases25 . Natural
were maintained for each test batch; the products are known to play an important role
positive control (CH/CF, MHB and test in both drug discovery and chemical biology.
organism), sterility control (MHB and In fact, many of the current drugs either
DMSO) and organism control (MHB, test mimic naturally occurring molecules or have
organism and DMSO). Plates were then structures
incubated at 37°C for 24 h. Experiments were
carried out in duplicate. After incubation, 40
µl of INT solution (0.2 mg ml-1) dissolved in
sterile distilled water was added to each well Extractive Yield
23
. The plates were incubated for further 30 The extractive yield of acetone extract
min and estimated visually for change in and fraction of T. bellerica stem of Jamnagar
color to pink indicating reduction of the dye and Rajkot regions is shown in Fig. 1. In
due to bacterial growth. The highest dilution Jamnagar region, maximum extractive yield
was in JAC II. JAC and JACI showed
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extractive value which was lower than that of against S. epidermidis (Fig. 3a). RAC, RAC I,
JACII. JHE showed lowest extractive value RAC II showed similar trend of activity like
(Fig. 1a). Unlike Jamnagar region, in Rajkot that of JAC and its fractions (Fig. 3b). The
region, maximum extractive yield was in antibacterial activity of acetone extract and its
RAC I. The extractive yield of RAC II was fractions of stem collected from both sites
much less than RAC I but more than crude showed almost similar activity towards Gram
extracts (RHE and RAC). positive bacteria.
The extractive yield of acetone extract Antibacterial activity of extracts and
and fraction of T. bellerica leaf of Jamnagar fractions of T. bellerica stem from Jamnagar
and Rajkot regions is shown in Fig. 2. In and Rajkot regions against Gram negative
Jamnagar region, maximum extractive yield bacteria is shown in (Fig.4). All the four
was in JAC II. JAC and JAC I had similar extracts of stem collected from both the
extractive value which was lower than that of regions showed antibacterial activity against
JAC II. The hexane extract (JHE) showed K. pneumonia. JHE and RHE did not show
lowest extractive value (Fig. 2a). In Rajkot activity against any other Gram negative
region, maximum extractive yield was in bacteria (Fig. 4). The remaining three extracts
RAC II similar to that of JAC II. Here also, from both the regions showed similar type of
RAC and RAC I had almost similar extractive inhibition against all the other bacteria. P.
values but were very much less than RAC II. aeruginosa was resistant to all the extracts of
RHE like JHE had minimum extractive yield both the sites. K. pneumoniae was the most
(Fig. 2b). From the above results, it can be susceptible Gram negative bacteria towards
concluded that leaf and stem collected from all the extracts and fractions (Fig. 4b). From
two different sited showed different trends, these results it can be concluded that Gram
The extractive yield of stem was different in negative bacterial were more susceptible that
stem collected from two different sites but it Gram positive bacteria but the susceptibility
did not affect the yield of leaf. The extractive was not very much different from stem
yield depends on many factors like plant part, collected from two different sites.
solvent used, temperature, soil conditions, The antibacterial activity of T.
climatic conditions, etc. There are many bellerica leaf acetone extract and its fractions
reports where different solvents showed from Jamnagar and Rajkot regions against
different extractive yield26-30. Gram positive bacteria are shown in Figs. 5-6.
Antibacterial activity of T. bellerica leaf
Antimicrobial activity acetone extract and its fractions from
The antibacterial activity of T. Jamnagar and Rajkot regions against Gram
bellerica stem acetone extract and its positive bacteria is shown in Fig. 5. JHE and
fractions from Jamnagar and Rajkot regions RHE did not show any activity against any of
against Gram positive bacteria are shown in the Gram positive bacteria studied (Fig. 5).
Figs. 3-4. The antibacterial activity of T. JAC and RAC and their fractions showed
bellerica stem acetone extract and its similar type of antibacterial activity against C.
fractions from Jamnagar and Rajkot regions rubrum and S. epidermidis i.e. leaf collected
against Gram positive bacteria is shown in from two different sites showed same type of
Fig. 3. (JAC, JAC I, JAC II showed activity inhibition. B. cereus, B. subtilis and L.
against C. rubrum and S. epidermidis while B. monocytogenes were resistant towards all the
cereus, B. subtilis and L. monocytogenes were extracts of both the sites. The antibacterial
resistant towards all the four extracts. JAC activity of acetone extract and its fractions of
and JAC II showed highest zone of inhibition leaf collected from both sites showed almost
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>32 µg ml-1 respectively (Tables 1-3). site. Gibbons38 suggested that isolated
Amongst all the tested extracts, JACI showed phytochemicals should have MIC < 1000 µg
least MIC value against CR – Gram positive ml-1. However, Madikizela.39 found in their
bacteria; while JACI, JACII and RAC and study, antibacterial MIC values equal to or
RACII showed least MIC value against PA – less than 1000 µg ml-1 for crude extracts were
Gram negative bacteria; Amongst fungal considered active. China40 found in their
strains, JAC showed least MIC value against study the petroleum ether extract of S.
CA, RHE and RACII against CG and JACI, grandiflora flower showed MICs values
JACII, RHE and RAC against CN. The stem ranged from 13-250 µg ml-1 against the tested
collected from Jamnagar site showed better pathogenic strains.
activity i.e. lower MIC values than that From the above, it can be concluded
collected from Rajkot site. that T. bellerica leaf and stem showed
For leaf extracts and its fraction MIC promising antibacterial activity especially
varied from 78 to >2500 µg ml-1 while MBC towards Gram negative bacteria. This is very
was >2500 µg ml-1 for the Gram positive promising because it is reported that plant
bacterial strains (Table 4). C. rubrum was extracts are more active against Gram positive
susceptible bacterial pathogen to JAC and its bacteria and the search is always going on for
both fractions and RAC and its both fractions plants extracts which are able to inhibit the
(MIC: 78 µg ml-1). L. monocytogenes was dangerous Gram negative bacteria. However,
susceptible to RAC and its both fractions yield and antibacterial activity of T. bellerica
(MIC: 78 µg ml-1) i.e. both C. rubrum and L. leaf and stem was affected by the site of
monocytogenes showed same MIC values but collection. More work is needed to confirm
their susceptibility was towards different its reason. Work in this direction is in
extracts. For the Gram negative bacterial progress.
strains the MIC varied from <39 to >2500 µg
ml-1 while MBC was >2500 µg ml-1. P. Conflict of Interest Statement
aeruginosa was the most susceptible bacterial
pathogen to RAC II (MIC: <39 µg ml-1) We declare that we have no conflict of
closely followed by P. mirabilis; it was the interest.
susceptible pathogen to RAC and RAC I
(MIC: 78 µg ml-1). For the fungal strains, ACKNOWLEDGEMENT
MIC varied from 39 to >2500 µg ml-1 while
MBC was >2500 µg ml-1 C. glabarata was The authors thank Prof. S.P. Singh, Head,
susceptible to RHE (MIC: 39 µg ml-1). For Department of Biosciences, Saurashtra
the standard antibiotics (CH and CF) MIC University for providing excellent research
and MBC ranged from 1 to 32 µg ml-1 and 8 facilities. The author(DD) are thankful to
to >32 µg ml-1 respectively. Amongst all the University Grants Commission, New Delhi
tested extracts, JAC and RAC and their for providing financial support as meritorious
fractions showed least MIC value against C. Junior Research Fellowship.
rubrum - Gram positive bacteria; while RAC
and RACI showed least MIC value against P.
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(A) (B)
Figure 1. Extractive yield of extracts and fraction of T. bellerica stem of Jamnagar and Rajkot regions. (J =
Jamnagar; R = Rajkot; HE = hexane extract; AC = acetone extract; ACI = Fraction I of acetone extract; ACII =
Fraction II of acetone extract)
(A) (B)
Figure 2. Extractive yield of extracts and fraction of T. bellerica leaf of Jamnagar and Rajkot regions. (J = Jamnagar;
R = Rajkot; HE = hexane extract; AC = acetone extract; ACI = Fraction I of acetone extract; ACII = Fraction II of
acetone extract)
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(A) (B)
Figure 3. Antibacterial activity of extracts and fractions of T. bellerica stem from Jamnagar and Rajkot sites against
Gram positive bacteria. (J = Jamnagar; R = Rajkot; HE = hexane extract; AC = acetone extract; ACI = Fraction I of
acetone extract; ACII = Fraction II of acetone extract)
(A) (B)
Figure 4. Antibacterial activity of extracts and fractions of T. bellerica stem from Jamnagar and Rajkot sites against
Gram negative bacteria. (J = Jamnagar; R = Rajkot; HE = hexane extract; AC = acetone extract; ACI = Fraction I of
acetone extract; ACII = Fraction II of acetone extract)
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(A) (B)
Figure 5. Antibacterial activity of extracts and fractions of T. bellerica leaf from Jamnagar and Rajkot sites against
Gram positive bacteria. (J = Jamnagar; R = Rajkot; HE = hexane extract; AC = acetone extract; ACI = Fraction I of
acetone extract; ACII = Fraction II of acetone extract)
(A) (B)
Figure 6. Antibacterial activity of extracts and fractions of T. bellerica leaf from Jamnagar and Rajkot sites against
Gram negative bacteria. (J = Jamnagar; R = Rajkot; HE = hexane extract; AC = acetone extract; ACI = Fraction I of
acetone extract; ACII = Fraction II of acetone extract)
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