Reducing power assay

This assay was determined according to the method reported by Oyaizu (1986) with slight
modifications, using (+)-catechin as the standard. Briefly, 1 ml of reaction mixture,
containing 500 ll of the test samples in 500 ll of phosphate buffer (0.2 M, pH 6.6), was
incubated with 500 ll of potassium ferricyanide (1%, w/v) at 50 _C for 20 min. The reaction
was terminated by adding trichloroacetic acid (10%, w/v), and then the mixture was
centrifuged at 12,000g for 10 min. The supernatant solution (500 ll) was mixed with distilled
water (500 ll) and 100 ll of ferric chloride (0.1%, w/v) solution, then the optical density (OD)
was measured at 700 nm. The reducing power ability was expressed as (+)-catechin
equivalents (CE) in milligrams per gram sample.

Measurement of reducing power

Reducing power was determined according to the method of Oyaizu (1986). Various
concentrations of methanolic extracts (2.5 ml) were mixed with 2.5 ml of 200 mM sodium
phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide.The mixture was
incubated at 50 _C for 20 min. After 2.5 ml of 10% trichloroacetic acid (w/v) were added,
the mixture was centrifuged at 1000 rpm for 8 min. The upper layer (5 ml) was mixed with
1 ml of 0.1% of ferric chloride, and the absorbance was measured spectrophotometrically at
700 nm. A higher absorbance of this mixture indicates a higher reducing activity. BHT, GA
and AA were used as standards.

Determination of total phenolics

Total phenolic content was determined according to the Folin– Ciocalteu method (Quettier-
Deleu et al., 2000) using gallic acid as the standard. The extract (5 mg) was dissolved in 5 ml
of methanol/ water (50:50 v/v). The extract solution (500 ll) was mixed with 500 ll of 50%
Folin–Ciocalteu reagent. The mixture was kept for 5 min, which was followed by the addition
of 1.0 ml of 20% Na2CO3. After 10 min of incubation at room temperature, the mixture was
centrifuged for 8 min (150g), and the absorbance of the supernatant was measured at 730 nm.
The total phenolic content was expressed as gallic acid equivalents (GAE) in milligrams per
gram of sample.

DPPH radical scavenging activity (DPPH assay)

The DPPH free radical scavenging activity of the extracts from male and female flowers of B.
laxiflora was determined according to the method reported by Gyamfi, Yonamine, and Aniya
(1999). 10 ll of the test samples in methanol, yielding a series of extract concentrations of 1,
5, 10, 50, and 100 lg/ml, respectively, in each reaction, were mixed with 200 ll of 0.1 mM
DPPH–ethanol solution and 90 ll of 50 mM Tris–HCl buffer (pH 7.4). Methanol (10 ll) alone
was used as the control of this experiment. After 30 min of incubation at room temperature,
the reduction in DPPH free radicals was measured by reading the absorbance at 517 nm. (+)-
Catechin, a well-known antioxidant, was used as the positive control. Three replicates were
made for each test sample. The inhibition ratio was calculated according to the following
equation:
% inhibition = [(absorbance of control - absorbance of sample)/ absorbance of control] x 100.

Determination of DPPH free-radical scavenging activity

The method used by Takao et al. (1994) was adopted with suitable modifications from
Kumarasamy et al. (2007). DPPH (8 mg) was dissolved in MeOH (100 ml) to obtain a
concentration of 80 lg/ml. Serial dilutions were carried out with the stock solutions (1 mg/ml)
of the extracts. Solutions (2 ml each) were then mixed with DPPH (2 ml) and allowed to
stand for 30 min for any reaction to occur, and the absorbance was measured at 517 nm.
Ascorbic acid (AA), gallic acid (GA) and butylated hydroxytoluene (BHT) were used as
reference standards and dissolved in methanol to make the stock solution with the same
concentration (1 mg/ml). Control sample was prepared containing the same volume without
test compounds or reference antioxidants. 95% methanol was used as blank. The DPPH
free-radical scavenging activity (%) was calculated using the following equation:
% inhibition = (Ac –As/Ac) x 100
The IC50 value, which is the concentration of the test material that reduces 50% of the free-
radical concentration, was calculated as lg/ml through sigmoidal doseresponse curve.

Determination of flavonoid content

The total flavonoid content was determined according to Brighente et al. (2007). 0.5 ml of
2% aluminium chloride (AlCl3) in methanol was mixed with the same volume of methanol
solution of plant extracts. After 1 h of staying at room temperature, the absorbances of the
samples were measured at 415 nm on a spectrophotometer versus blank sample. Total
flavonoids were determined as rutin equivalents (mg RU/g dry extract), and the values are
presented as means of triplicate analyses.
Determination of total antioxidant capacity
The total antioxidant activity of the methanol extracts were evaluated by the
phosphomolybdenum method (Prieto et al. 1999). The assay is based on the reduction of Mo
(VI) – Mo (V) by the antioxidant compounds and subsequent formation of a green
phosphate/Mo (V) complex at acid pH. 0.3 ml of sample extracts were combined with 3 ml of
reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mMammonium
molybdate). The tubes containing the reaction solution were incubated at 95°C for 90 min.
Then the absorbance of the solution was measured at 695 nm using spectrophotometer
against blank after cooling to room temperature. Methanol (0.3 ml) in the place of extract was
used as the blank. Ascorbic acid (AA) was used as standard and the total antioxidant capacity
is expressed as milligrams of ascorbic acid per gram of the dry extract.

Determination of the inhibitory activity toward lipid peroxidation

166 The antioxidant activity of the methanolic plant extracts were determined by the
thiocyanate method (Hsu et al. 2008). Serial dilutions were carried out with the stock
solutions (1 mg/ml) of the extracts, and 0.5 ml of each solution was added to linoleic acid
emulsion (2.5 ml, 40 mM, pH 7.0). The linoleic acid emulsion was prepared by mixing
0,2804 g linoleic acid, 0.2804 g Tween-20 as emulsifier in 50 ml 40 mM phosphate buffer
and the mixture was then homogenized. The final volume was adjusted to 5 ml with 40 mM
phosphate buffer, pH 7.0. After incubation at 37 _C in the dark for 72 h, a 0.1 ml aliquot of
the reaction solution was mixedwith 4.7 ml of ethanol (75%), 0.1 ml FeCl2 (20 mM) and 0.1
ml ammonium thiocyanate (30%). The absorbance of this mixture was measured at 500 nm,
after it was stirred for 3 min. Ascorbic acid, gallic acid, a-tocopherol and BHT were used as a
reference compounds. To eliminate the solvent effect, the control sample, which contained
the same amount of solvent added to the linoleic acid emulsion in the test sample and
reference compound, was used. Inhibition percent of linoleic acid peroxidation was
calculated using following formula:
% inhibition = (Ac –As/Ac) x 100

Disc diffusion assay

The antimicrobial activity of O. stamineus extracts was determined against nine bacterial
cultures using a paper disc diffusion assay. The bacterial strains were cultured in a nutrient
broth (Merck, Germany) for 24 h and diluted with sterilised peptone water. Then, 100 μl of
each culture (106 CFU) was spread onto the surface of Mueller–Hinton agar (Oxoid,
England) to create a bacterial lawn. Sterile blank filter paper discs of 6 mm in diameter
(Oxoid, England) were wetted with 20 μl crude extract of O. Stamineus (100 mg/ml) and left
to dry before being placed on the microbial lawn. The plates were incubated at 37°C for 24 h.
The antibacterial activity was compared with 10 μl of chloramphenicol (1 mg/ml), 20 μl of
5% lactic acid and 20 μl of 10% acetic acid. Antimicrobial activity was determined based on
the diameter of the clear zone surrounding the paper discs. Three replicate discs were
prepared for each extract in this study.

Test micro-organisms
The antibacterial activities of all extracts were determined against several food-borne
bacteria. Listeria monocytogenes Scott A (serotype 4b) was obtained from the Department of
Health and Human Services, Food and Drug Administration, (Cincinnati, Ohio, USA), and
Staphylococcus aureus (ATCC 25925), Salmonella Typhimurium (ATCC 14028) and
Escherichia coli (ATCC 1858) were obtained from American Type Culture Collection
(Manassa, USA). All bacterial growth media used in this study were supplied by Oxoid
(Basingstoke, UK). All bacteria were maintained as frozen stocks in 80% glycerol at -80 °C.
Working cultures were grown and maintained on Tryptic Soy Agar (TSA). For the
determination of antibacterial activity, a single colony of each strain was grown in 10 ml
Tryptic Soy Broth (TSB) for 18 ± 2 h at 37 °C without shaking.

Disc diffusion assay

The antimicrobial activity of extracts was evaluated using a slightly modified agar disc
diffusion method (Barry, 1976). A bacterial culture grown for 18 h was serially diluted in 9
ml of 0.1% peptone to obtain 105 cfu/ml and 100 μl spread on the surface of Mueller Hinton
(MH) agar in Petri plates. An aliquot (10 μl) of herb and spice extract was pipetted on a
sterile paper disc (Whatman No. 1, 5.5 mm paper disc) on the agar surface. Except in the case
of hexane extracts the discs were air dried to evaporate the ethanol. A disc impregnated with
an aliquot (10 μl) of Streptomycin (Fluka, Switzerland) served as a positive control on the
same plate. An additional negative control disc was impregnated with 10 μl of sterile distilled
water (for water extracts), 90% ethanol which was evaporated (for ethanol extracts) or
DMSO (for hexane extracts). The plates were inverted and incubated for 18 h at 37 °C.
Microbial inhibition was determined by measuring the diameter of the clear zone of
inhibition of growth around each disc and recorded as diameter of inhibition zone (DIZ) in
millimeter. All assays were performed using a randomized complete block design with two
replicates.
Broth dilution assay
To determine the minimal inhibitory concentration (MIC), quantitative serial dilutions of
water, ethanol and hexane extracts of each spice and herb were tested against four food-borne
pathogens with some modifications of the method described by Hufford and Clark (1988).
Twofold serial dilutions of extracts were made with MH broth. After adding 20 ll of herb and
spice extracts to the first tube containing 1 ml of MH broth, serial transfers were made
through to the fourth tube. Since different spice extracts have different solubilities, one drop
of Tween 80 (14.5 mg) was added to ethanol and hexane extracts followed by vortexing to
solubilise the extract. Most of the ethanol and hexane extract required heating in a water bath
at 50°C for 5 min to solubilise the content before performing serial dilutions. A 0.5 ml aliquot
(5 x 105 cfu/ml) of test micro-organism was added to each tube. A positive control tube
contained MH broth and micro-organism and a negative control tube contained 10 μl of
Streptomycin in MH broth and microorganism was maintained. A further control tube was
prepared with broth and spice extracts to confirm broth or spice extracts were not
contaminated. Tubes were subsequently incubated at 37°C. The tubes were visually examined
for the lowest concentration of extract that showed inhibition of microbial growth (indicated
by a clear solution) after 24 h and 48 h. The concentration in the lowest serial dilution of the
spice and herb extracts at which growth did not occur on broth was recorded as the MIC.
Finally the pH value of the inoculated broth containing the MIC of each spice and herb
extract was measured. Buffering of broth containing G. quaesita extracts was done using
sodium hydroxide and subjected to broth dilution assay as above to determine the MIC
values.

Free Radical Scavenging Activity (Diphenyl-picrylhydrazyl assay)

The free-radical scavenging capacity of different extracts (I, II, III) of leaves of O. indicum
were evaluated with the DPPH stable radical, by the methodology described by Blois in 1958.
Briefly, 0.1mM alcoholic solution of DPPH in methanol was prepared and 2mL of this
solution was added to 0.3mL of different extract concentrations (1-100 µg/mL) and allowed
to react at room temperature. After 30min, the absorbance values were measured at 517nm
against the blank, which did not contain the extract. L-ascorbic acid was used as control. The
radical scavenging activity (percent inhibition) was expressed as percentage of DPPH radical
elimination calculated according to the following equation:
 Percentage Inhibition (%) = (Acontrol-Asample/Acontrol) × 100

Where,
Acontrol is the absorbance of the control (L-ascorbic acid) and Asample is the absorbance
of reaction mixture (in the presence of sample).
All tests were run in triplicates (n = 3), and the average values were calculated.

IC50 Value
Inhibition Concentration (IC50) was introduced by Brand-Williams and his colleagues [21] for
the interpretation of the results from DPPH method. The discoloration of sample was plotted
against the sample concentration in order to calculate the IC50 value. It is defined as the
amount of sample necessary to decrease the absorbance of DPPH by 50%.