3.

Materials and Methods

The greatest invention of the nineteenth century was the invention of the method of
invention.
- A. N. Whitehead
________________________________________________________________________

3.1. Seed collection and Storage

3.2. Extraction
3.3. Antioxidant Activity
3.3.1. Total antioxidant activity
3.3.2. DPPH scavenging assay
3.3.3. Phenol estimation
3.3.4. Flavonoid estimation
3.4. Antibacterial Activity
3.4.1. Test organisms
3.4.2. Antibacterial susceptibility tests
3.5. Chracterization/Fractionation
3.5.1. Phytochemical screening
3.5.2. TLC
3.5.3. HPLC

33

3. Materials and Methods

3.1. Seed collection and Storage
All the seeds were obtained from fruits purchased from local market of Ahmedabad city.
During collection care was taken that seeds should be fresh, healthy and must be free
from any contamination. They were authenticated for their unambiguous identity by Prof.
Y T Jasrai, Botany Dept., Gujarat University. Seeds were washed, dried and stored in
clean airtight containers in dark. The seeds were checked at regular time intervals for any
physical, chemical or biological damage.

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.

3.3. Antioxidant Activity

3.3.1. Total antioxidant activity
Total antioxidant activity was estimated by Molybdate assay (Pilar et al., 1999). The
assay is based on the reduction of Mo (VI) to Mo (V) by the sample and subsequent
formation of a green phosphate/Mo (V) complex at acid pH. The tubes containing extract
and reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM
ammonium molybdate) were incubated at 95oC for 90 min. After the mixture had cooled

34

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.

3.3.2. DPPH scavenging assay

The capacity of the extracts to scavenge the stable 2,2-diphenyl-2-picrylhydrazyl
(DPPH) free radical was measured (Duan et al., 2007). 0.1 mL of extracts was mixed
with 2.9 mL of 0.1mM DPPH solution. The blank contained 0.1 mL of extract and 2.9
mL of DPPH solution. Negative control was prepared by mixing 0.1 mL of methanol
with 2.9 mL of DPPH solution. The radical scavenging activity was calculated in terms of
Ascorbic Acid Equivalent Antioxidant Capacity (AEAC) by using the following formula
(Lim et al., 2007).
AEAC = (A control A sample) / (A control A ascorbic acid)
concentration of ascorbic acid (mg/mL) vol extract (mL)
100/g sample
Where, A is Absorbance.
3.3.3. Phenol estimation
Folin-Ciocalteu method was used to determine total phenolic content of the sample as
described by Singleton and Rossi (1965). 0.2 mL 10 % v/v Folin-Ciocalteu reagent was
added to the 0.1 mL of the sample, and was vortexed for 5 min, followed by addition of
0.8 mL of sodium carbonate. This reaction mixture was incubated for 2 h at room
temperature. The absorbance was measured at 765 nm. The calibration curve was
prepared by employing gallic acid at concentrations of 0.4 to 1.6 mM.

3.3.4. Flavonoid estimation

Aluminum chloride colorimetric method was used for flavonoids determination (Chang
et al., 2002). 0.5 mL of each plant extract was separately mixed with 1.5 mL of methanol,
0.1 mL of 10% aluminum chloride, 0.1 mL of 1 M potassium acetate and 2.8 mL of
distilled water. The reaction mixture was allowed to stand at room temperature for 30

35

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.

3.4. Antibacterial Activity

3.4.1. Test organisms
Details of bacterial strains used as test organisms are provided in Table-1. Two strains of
S. paratyphi A (GU; GU-R3) were obtained from Microbiology Dept., Gujarat
University, Ahmedabad. Remaining all strains were procured from Microbial Type
Culture Collection (MTCC, IMTECH), Chandigarh.
All bacterial strains were maintained on nutrient agar, except A. hydrophila,
which was maintained on ampicillin starch agar (HiMedia, Mumbai) and stored at 4C.
All these strains were subcultured every month on the same media. Paraffin stocks were
prepared by overlaying agar slants with it. Glycerol stocks were also prepared by adding
glycerol (10% v/v) to the broth inoculated with organisms. Paraffin stocks were
preserved in refrigerator at 4C and glycerol stocks were stored at -20C in deep freezer
(Reimer and Carrol, 2003).

3.4.2. Antibacterial susceptibility tests

Disc diffusion assay (DDA) was performed by Kirby-Bauer method as per NCCLS
guidelines (Jorgensen and Turnidge, 2003). 500 L of inoculum (adjusted to 0.5
McFarland standard) was spread on surface of Muller-Hinton agar medium (HiMedia,
Mumbai, India). Sterile discs (6 mm diameter) made of Whatman paper # 1 were dipped
into the test extract of known concentration and were put onto the agar surface after
complete drying. Discs dipped into pure solvents were applied as negative control.
Commercially available discs of either streptomycin or ofloxacin (HiMedia) served as
positive control. Plates were then incubated at 35C for 24 h. After incubation plates were
observed for zones of inhibition, and their diameter were measured. Studies were
performed in triplicates.

36

Table 1. Test organisms used for antibacterial screening of plant extracts

No.

Organism

Identity

Remarks

code

(as per information provided from MTCC along with

cultures)

Aeromonas hydrophila

MTCC 1739

Isolated from tin of milk with a fishy odour

Recommended for assay of penicillin, streptomycin,

Bacillus subtilis

MTCC 619

vancomycin, and is associated with production of weak

penicillinase

Escherichia coli

MTCC 723

Pathogenic,

used

as

standard

strain

producing

colonization factor antigen I(cfx/I) and enterotoxin

Recommended for assay of antimicrobial agents in
4

Pseudomonas oleovorans

MTCC 617

aqueous metal working fluids; isolated from cutting fluid,

type strain

Salmonella typhi

MTCC 734

Pathogenic, used to make widal antigen, estimation of

TH agglutining titre, Weil-Felix test

Salmonella paratyphi A

MTCC 735

Salmonella paratyphi A

GU

Used to make widal antigen

Resistant to three different antibiotics-nalidixic acid,

Salmonella paratyphi A

GU-R3

tetracycline, and co-trimexaxole

Shigella flexneri

MTCC 1457

Recommended for assay of antibiotics, and sterility

10

Staphylococcus aureus

MTCC 737

testing

11

Staphylococcus epidermidis

MTCC 435

Associated with skin lesions

12

Streptococcus pyogenes

MTCC 442

13

Vibrio cholerae

MTCC 3906

MIC (minimum inhibitory concentration) determination was carried out using

microbroth dilution method as per NCCLS guidelines (Jorgensen and Turnidge, 2003).
Assay was performed in 96-well microtitre plates. Total volume of the assay system in
each well was kept 200 L. Cation-adjusted Muller-Hinton broth (HiMedia) was used as
growth medium. Inoculum density of the test organisms was adjusted to that of 0.5
McFarland standard. Broth was dispensed into wells of microtitre plate followed by
addition of test extract and inoculum. Extracts (reconstituted in DMSO) were serially
diluted into each of the wells. A DMSO control was included in all assays (Wadhwani et
37

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.

2.5.1. Phytochemical screening

2.5.1.1.Test for alkaloids
Dragendorff reagent was used to test the presence of alkaloids. The presence of alkaloids
was indicated by the appearance of yellow precipitate when few drops of reagent were
added to the solution (Eloff, 2004).
2.5.1.2. Test of flavones
Test solution (500 l) was mixed with 100 l of absolute alcohol, 0.02 g of p-dimethyl
amine benzaldehyde and two drops of conc. Hcl. Development of red or pink colour
indicated the presence of flavones (Borgio et al., 2008).
3.5.1.3. Test for saponins
Test solution, with distilled water (2 drops) was taken in a test tube. Development of
foamy lather indicates the presence of saponins (Borgio et al., 2008).
3.5.1.4. Test for tannins
Sample was mixed with 5% iron (III) chloride solution. Blue-black precipitates indicate
presence of tannins (Harborne, 1998).

38

3.5.1.5. Test for flavonoids

An aqueous solution of the extract was treated with 10% ammonium hydroxide solution.
Yellow florescence indicates the presence of flavonoids (Wagner et al., 1983).
3.5.1.6. Test for Phenols
Ferric chloride was used to test the presence of phenols. The presence of phenols was
indicated by appearance of green colouration when the reagent was added to extract
(Raman, 2006).

3.5.2. TLC
TLC was performed under following experimental conditions (Stahl, 1969):

Adsorbent layer : Silica gel 60 F254 plates (Merck)

Layer thickness: 0.25 mm (analytical)

Layer size: 2020 cm

Chamber type: Glass chamber; 25 x 25 x 14 cm (Merck)

Separation technique: Ascending

Chamber saturation state: Minimum 2 h

Length of run: 10 cm

Solvent composition: (a) n-butanol : water (1:1) for S. cumini extracts

(b) chloroform:acetone (90:10) for T. indica extract

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.

Samples were applied as either bands or spots.

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

39

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.

40