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.
  1. In vitro antioxidant tests
  2. 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.