1.4.3. Dertermination of terpenoid
The extraction method of Ekwueme et al. (2015) was used with some
modifications. P. glandulosus leaves (50 g) were macerated with
methanol and water (4:1) for 24 h at 37˚C and filtered with Whatman
filter paper. Filtrate was concentrated at 40 °C and concentrate was
then acidified with 2M sulphuric acid and the mixture was then extracted
with chloroform. Non-aqueous layer was separated and evaporated to
dryness. Terpenoids content was calculated as percentage.
- In vitro antioxidant tests
- Hydrogen peroxide (H2O2)
scavenging activity
The H2O2 scavenging activity was
determined according to the method of Ruch et al . (1989). with
some modifications. The mixture containing sample (1 ml; 25-400 μg/ml),
phosphate buffer solution (PBS) (2.4 ml; 0.1 M, pH 7.4) and
H2O2 solution (0.6 ml; 40 mM) was shaken
vigorously and incubated at room temperature for 10 min. Absorbance of
the reaction mixture was determined at 230 nm. Ascorbic acid was used as
positive control. The H2O2 scavenging
activity was calculated as follows :
Where, A0 is the absorbance of the control (water
instead of sample), A1 is the absorbance of the sample,
and A2 is the absorbance of the sample only (phosphate
buffer instead of H2O2 solution). The
IC50 value represented the concentration of the
compounds that caused 50% inhibition of
H2O2.
Nitric oxide radical-scavenging activity
Nitric oxide assay was carried out following a slightly modified method
of Lee et al. (2010). Nitric oxide was generated from sodium
nitroprusside and was measured by the Griess reagent. Sodium
nitroprusside in aqueous solution at physiological pH spontaneously
generates nitric oxide, which interacts with oxygen to produce nitric
ions that can be estimated by using Griess reagent. Scavengers of nitric
oxide compete with oxygen leading to reduced production of nitric oxide
(Marcocci et al .,1994). 5mM of Sodium nitroprusside in phosphate
buffer saline (PBS) pH= 7.4 was mixed with 3.0 ml of different
concentrations (25-400 μg/ml) of P. glandulosus leaves extracts
and fractions. This reaction mixture was incubated for 150 min (25˚C).
Further mixture of sulphanilamide (1%),
H3PO4 (2%) and napthylethylenediamine
dihydrochloride (0.1%) (Greiss reagent) was added to the above reaction
mixture. Diazotization of nitrite with sulphanilamide and subsequent
coupling with napthyl ethylenediamine generates a chromophore. The
absorbance of the chromophore formed was noted at 546 nm. Ascorbic acid
was used as the reference compound. Percentage inhibition was calculated
as follows:
Where Ao was the absorbance of the control (blank,
without extract) and At was the absorbance in the
presence of the test (extract). IC50 value represented
the concentration of the compounds that caused 50% inhibition of
H2O2.