2.5. In- vitro anti-oxidant assays

The antioxidant capacity of leaves and stem of C. argentea were determined using DPPH, ABTS, ferric reducing antioxidant power (FRAP) and phosphomolybdate (Total antioxidant capacity).

DPPH free radical scavenging assay was determined as outlined by Unuofin et al. (2018). 1 mL of 0.135 mM DPPH radical was mixed with 1 mL each of the test samples (previously prepared with methanol), standard antioxidant (BHT) and control at four concentrations (62.5, 125, 250 and 500 μg/mL). Subsequently, the mixture was vortexed and kept in the dark 25 °C for 30 min. The rate of absorbance was then spectrophotometrically measured at 517 nm, using methanol as the blank and positive control. Finally, the scavenging ability of the plant extract was calculated using the equation:

Where; Abs control is the absorbance of DPPH + methanol; Abs sample is the absorbance of DPPH radical + sample/or standard.

The method described by Khan et al. (2012) was used to determine the ABTS radical scavenging activity of the plant extract with little modifications. ABTS stock solution was prepared by mixing 7 mM ABTS and 2.45 mM potassium persulfate in equal quantity. The resultant stock was incubated in the dark for 18 h at 25 °C to form a green-coloured ABTS radical (ABTS⁺). This solution was further diluted by mixing 1 mL of the ABTS⁺ solution with 50 mL of methanol to obtain a working solution with absorbance of 0.700 ± 0.006 at 734 nm. The extracts and standards (Rutin and BHT) of concentrations ranging from 12.5 to 200 μg/mL were then mixed with 1 mL of ABTS solution and kept in the dark for 7 min. Absorbance was measured at 734 nm and the percentage inhibition of ABTS⁺ by the extract was calculated from the equation:

The ferric reducing power of the plant extracts was determined by using the method described by Jayanthi and Lalitha (2011). The FRAP mixture consisted of 2.5 mL of 0.2 M phosphate buffer reagent (Mixture of 62.5% monobasic and 37.5% dibasic: pH 6.6) and 2.5 mL of 1% potassium hexa-cyanoferrate (w/v). To this was added 1mL of the standard (Gallic acid and ascorbic acid) and the C. argentea extracts at five concentrations (50, 100, 200, 500 and 1000 μg/mL). The mixture was incubated at 50 °C for 20 min in a water bath. Subsequently, 2.5 mL of 10% Trichloroacetic acid (TCA) (w/v) was added to the mixture, after which the mixture was centrifuged for 10 min at 3000 rpm. Then, 2.5 mL of distilled water and 0.5mL 0.1% freshly prepared FeCl₃ were added to the supernatant from the centrifuged solution. The resultant solution was allowed to stand for 10 min at 25 °C. The percentage inhibition of the radicals by the plant sample was measured at 700 nm against the methanol blank.

Ferrous sulphate equivalent concentration in mM was calculated from the standard graph and expressed as mg Fe (II) equivalent/mg.

The complex formation method using phosphomolybdenum as described by Olugbami et al. (2015) was used for the determination of the total antioxidant capacity. Briefly, 0.3 mL of the plant extract and standards (Gallic acid and ascorbic acid) at five concentrations (50, 100, 200, 500 and 1000 μg/mL) was added to 3 mL of reagent solution (4 mM ammonium molybdate, 28 mM sodium phosphate and 0.6 M sulfuric acid). The mixture was incubated at 95 °C for 90 min in a water bath. After cooling, absorbance of the solution was measured at 695 nm against the control (methanol) and the percentage inhibition was calculated as:

The concentration at which 50% of the radicals were scavenged by the extracts (IC₅₀) was calculated from the plot of the percentage inhibition against the concentration used. The results are expressed as half minimal inhibitory concentration (IC₅₀). This is the amount of the extract or standard necessary to decrease the initial concentrations of DPPH, ABTS, FRAP radicals by 50% and the total antioxidant capacity (TAC). The lower the IC₅₀ value the higher the antioxidant activity. The synthetic antioxidants (Rutin, BHT and ascorbic acid) were used as positive controls in all the assays for comparison IC50 activities between the activities of test extract and standard antioxidants.