2.5 Antioxidant activity assays

ABTS radical scavenging activity was assayed according to instructions from the Beyotime Institute of Biotechnology. ABTS solution was prepared by mixing the stock solution and oxidant solution in equal volumes and allowing them to react at room temperature in the dark for 16 h. A 10-μL aliquot of CCE solution was added to 200 μL of fresh ABTS solution. The solution was then diluted with 5-mM phosphate-buffered saline (pH 7.4) to obtain an absorbance of 0.7 ± 0.05 at 734 nm. The absorbance was measured at 734 nm after 6 min at room temperature. Ascorbic acid was used as a reference; the ABTS radical scavenging activity was calculated using Eq 1.

where A_t is the absorbance of the sample, B is the absorbance of the sample and distilled water, and A₀ is the absorbance of the blank control.

The free radical scavenging activity of CCE was analyzed by a 2, 2-diphenyl-1-picrylhydrazil (DPPH) assay as described by Sharma et al. [15]. First, the CCE solution (0.5, 1, 2, 3 and 4 mg/mL) was prepared by diluting CCE in distilled water and add 0.4 mL solution in the test tubes respectively. Finally, 3.6 mL of DPPH solution (0.1 mM) was transferred to the test tubes, which were gently stirred and incubated in the dark at 25°C for 30 min. The absorbance was read at 517 nm using a spectrophotometer, and the scavenging activity (%) of each concentration was calculated in triplicate (Eq 2). Distilled water was used as the blank control.

where A₀ is the absorbance of the blank control, A_s is the absorbance of the sample, and A_c is the absorbance of the sample and ethanol mixture.

The ferric reducing ability of CCE was determined by FRAP assay as per instructions from the Beyotime Institute of Biotechnology. Stock solutions were the detective buffer, 2, 4, 6-tripyridyl-s-triazine (TPTZ) solution, TPTZ dilution, 0.5 mL of 10-mM FeSO₄ solution, and 0.1 mL of 10-mM Trolox solution. A working solution was prepared by mixing TPTZ dilution, detective buffer, and TPTZ solution in a ratio of 10:1:1 (v/v), respectively. The working solution was warmed to 37°C before use. CCE samples (5 μL) with concentrations ranging from 0.3 to 1.5 mM were mixed with 180 μL of FRAP working solution and incubated at 37°C for 5 min. The absorbance of the reaction mixture was measured at 593 nm. FRAP was expressed as FeSO₄ concentrations, which were calculated using standard curves.