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3.2. Extraction
Seeds were extracted in different solvents (Merck, Mumbai, India) water, methanol,
ethanol (50%), acetone, hexane, chloroform, and chloroform:methanol (2:1) mixture,
using the microwave assisted extraction (MAE) method (Kothari et al., 2009). One gram
dry seed powder was soaked into 50 mL of the solvent and subjected to microwave
heating (Electrolux EM30EC90SS) at 720 W. Total heating time was kept 90, 70, 120,
180, 300, 180, and 50 second for methanol, ethanol, acetone, chloroform, hexane, water,
and chloroform:methanol mixture, respectively, with intermittent cooling. This was
followed by centrifugation (at 10,000 rpm for 15 min.), and filtration with Whatman
paper # 1 (Whatman International Ltd., Maidstone, England). Solvent was evaporated
from the filtered extract and then the dried extracts were reconstituted in: (i) their
respective solvents for disc diffusion assay, and (ii) dimethyl sulfoxide (DMSO) for broth
dilution assay. Reconstituted extracts were stored in autoclaved glass vials under
refrigeration for further use.
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to room temperature, the absorbance of each tube was measured at 695 nm. The standard
curve was prepared by using the known concentrations (0.5-13 mM) of gallic acid. The
antioxidant capacity of extracts was expressed in terms of g of gallic acid equivalent
(GAE)/g of dry extract. Ascorbic acid was used as positive control.
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min and the absorbance of the reaction mixture was measured at 415 nm. The calibration
curve was prepared by using quercetin at concentrations of 12.5 to 100 g/mL in
methanol.
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No.
Organism
Identity
Remarks
code
Aeromonas hydrophila
MTCC 1739
Bacillus subtilis
MTCC 619
Escherichia coli
MTCC 723
Pathogenic,
used
as
standard
strain
producing
Pseudomonas oleovorans
MTCC 617
Salmonella typhi
MTCC 734
Salmonella paratyphi A
MTCC 735
Salmonella paratyphi A
GU
Salmonella paratyphi A
GU-R3
Shigella flexneri
MTCC 1457
10
Staphylococcus aureus
MTCC 737
testing
11
Staphylococcus epidermidis
MTCC 435
12
Streptococcus pyogenes
MTCC 442
13
Vibrio cholerae
MTCC 3906
al., 2009). Gentamicin (HiMedia) served as positive control. Plates were incubated at
35 C for 16-20 h, before being read at 655 nm in a plate reader (BIORAD 680). MIC
was recorded as the lowest concentration at which no growth was observed.
Concentration at which growth was inhibited by 50% was recorded as IC50 value.
After reading the plates for MIC, subculturing was made on nutrient agar from the
wells showing no growth. After incubation the concentration which killed 99.9% of the
population (compared with the growth control) was reported as MBC (minimum
bactericidal concentration). Total activity was calculated as (Eloff, 2004);
Total activity (mL/g) = extraction efficiency (mg/g) / MIC (mg/mL).
2.5. Characterization/Fractionation
Active extracts were subjected to phytochemical screening, UV-vis spectroscopy, and
separation through TLC and/or HPLC.
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3.5.2. TLC
TLC was performed under following experimental conditions (Stahl, 1969):
Length of run: 10 cm
Margin between start point and plate edge: 2.0 cm above the lower edge of
the plate and 1.5 cm was left from each side.
After completion of the solvent run, plates were examined under ultraviolet light (254
and 365 nm; Uvitec-LF-206.LS, UK). The retardation factor (Rf) was calculated and
tabulated. Quercetin (SD Fine chemicals, New Delhi) was run as a marker along with S.
cumini extracts.
The separated constituents were recovered by scraping off the adsorbent at the
appropriate places on the developed plate, and the powder was reconstituted in methanol,
followed by centrifugation (Eppendorf 5417R) at 14,000 rpm for 15 min. This step was
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carried out twice to ensure complete removal of the adsorbent. The supernatant was then
stored in sterile glass vials (15mL, Merck) under refrigeration.
3.5.3. HPLC
HPLC was performed in gradient HPLC PU-2080 plus system (Jasco, Japan)
incorporated with a Jasco PU 2080 Plus pump and a Jasco MD-2015 Plus UV
detector. The column used was HiQ Sil C18, 4.6 250 mm, 5 micron. All the solvents
(CDH, New Delhi) used were of HPLC grade. Injection volume was 20 L in all cases
with run time of 20 min and flow rate either 0.7 mL/min or 0.5mL/min as required. The
results were recorded using the software Borwin : Version 1.50 (Jasco) at detection
wavelength of 270 nm. Quercetin and gallic acid (SRL, Mumbai) were run as standards.
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