Chemistry Reference
In-Depth Information
Antimicrobial tests were then carried out by disc-diffusion method [4] using 100 l of
suspension containing 10
8
CFU/ml of bacteria spread on nutrient agar (NA). The discs (6
mm in diameter) were impregnated with 5 l of the extracts (500 g/disc) at the
concentration of 100 mg/ml and placed on the inoculated agar. Negative controls were
prepared using the same solvents employed to dissolve the plant extracts. Ampicillin (10
g/disc) was used as a positive reference standard to determine the sensitivity of one
strain/isolate in each microbial species tested. The inoculated plates were incubated at 37
o
C
for 24 h for clinical bacterial strains. Antimicrobial activity was evaluated by measuring the
zone of inhibition against the test organisms. Each assay in this experiment was repeated
twice.
4. Evaluation of antioxidant activity
4.1. Reducing power assay
The reducing power of
E. agallocha
was determined as per the reported method [5]. Different
concentrations of plant extract (100 -2000 μg/l) in 1 ml of methanol were mixed with a
phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferrocyanide (2.5 ml, 1 %). The
mixture was incubated at 50
o
C for 20 min. A portion (2.5 ml) of trichloroacetic acid (10 %)
was added to the mixture, which was then centrifuged at 3000 rpm for 10 min. The upper
layer of the solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl
3
(0.5 ml, 0.1
%), and the absorbance was measured at 700 nm and compared with standards. Increased
absorbance of the reaction mixture indicated increased reducing power.
4.2. Metal chelating effect
The chelation of ferrous ions by the extract was estimated as per the method of Dinis [6].
Different concentrations of the extract (100-2000 g/l) were added to a solution of 1 mM
FeCl
2
(50 l). The reaction was initiated by the addition of 1 mM ferrozine (0.1 ml) and the
mixture was finally quantified to 1 ml with methanol, shaken vigorously and left standing at
room temperature for 10 min. After the mixture had reached equilibrium, the absorbance of
the solution was measured spectrophotometrically at 562 nm. All analyses were done in
triplicate and average values were taken. The percentage of inhibition of ferrozine-Fe
2+
complex formation was calculated using the formula given below: % Inhibition [(
A
0
- A
1
)/A
0
x
100], where
A
0
is the absorbance of the control and
A
1
is the absorbance in the presence of
the sample of
Excocaria
extract. FeCl
2
and ferrozine complex formation molecules are present
in the control.
4.3. Nitric oxide radical inhibition activity
Nitric oxide, generated from sodium nitroprusside in an aqueous solution at physiological pH,
interacts with oxygen to produce nitrite ions which were measured by Griess reaction [7]. The
reaction mixture (3 ml) containing sodium nitroprusside (10 mM) in a phosphate buffer saline
and the extract (100 - 2000 g/l) were incubated at 25°C for 150 min. After incubation, 0.5 ml
