Antioxidant activities in a cell-free system were evaluated by free radical and NO scavenging activities. The free radical scavenging activity of AR extracts on DPPH radical was determined using the method described by Huang et al. [13 (link)], with slight modification. Briefly, DPPH ethanol solution was added to various concentrations of AR extract (0.4-50 mg/mL) in 96-well plates. After 30 min incubation at room temperature in the dark, the absorbance at 515 nm was measured by a plate reader (BioTek Inc., Winooski, VT). The free radical scavenging activity of the sample was calculated by the following formula:
Where As is the absorbance of the sample and Ab is the absorbance of the blank.
NO production was assessed by measuring the nitrite content. Briefly, Griess reagent (0.1% N-1-naphthylenediamine dihydrochloride and 5% H3PO4 solution) was added to AR extracts in a 1:1 (v/v) manner. After gentle mixing and 15 min incubation in the dark, NO levels were subsequently measured and compared with a nitrate standard curve. Absorbance values at 560 nm were measured using a microplate reader. The NO scavenging activity of the sample was calculated using the same formula as used for the DPPH scavenging activity. The IC50 values were obtained using GraphPad Prizm (Ver. 6, La Jolla, CA, USA).
Where As is the absorbance of the sample and Ab is the absorbance of the blank.
NO production was assessed by measuring the nitrite content. Briefly, Griess reagent (0.1% N-1-naphthylenediamine dihydrochloride and 5% H3PO4 solution) was added to AR extracts in a 1:1 (v/v) manner. After gentle mixing and 15 min incubation in the dark, NO levels were subsequently measured and compared with a nitrate standard curve. Absorbance values at 560 nm were measured using a microplate reader. The NO scavenging activity of the sample was calculated using the same formula as used for the DPPH scavenging activity. The IC50 values were obtained using GraphPad Prizm (Ver. 6, La Jolla, CA, USA).