Antioxidant Activity in vitro

The DPPH assay was slightly modified from a previous report.23 A mixture of anhydrous ethanol (100 μL), DPPH solution (100 μL, 0.2 mM) and sample (100 μL, 0.01–1 mg/L) was shaken vigorously and kept (25 °C, 30 min) in the dark. VC was used as a positive control. The absorbance (517 nm) was measured. The DPPH radical scavenging rate was calculated as follows:

where A₀ represents the absorbance of the negative control (DPPH plus absolute ethanol); A₁ represents the test system containing the sample (DPPH plus TSAT); and A₂ is the absorbance of the blank system (TSAT plus absolute ethanol). The 50% inhibition concentration value (IC₅₀) of the DPPH free radical scavenging rate was calculated, which represents the antioxidant capacity of TSAT. All operations are performed in parallel three times.

ABTS was determined using the method of Rafique et al with slight modifications.24 First, a series of TSAT (0.01–1 mg/mL) solutions were prepared. An ABTS solution of 7 mM and a K₂S₂O₈ solution of 2.45 mM were prepared. After being mixed in the same volume, ABTS+ was obtained by reacting (12–16 h) in the dark. The prepared ABTS+ was diluted with double distilled water to an absorbance of 0.7 ± 0.05 at 734 nm. Then, the mixture of 180 μL diluted ABTS+ solution and 20 μL sample were reacted (25 °C, 10 min) in the dark. VC was used as the positive control. The absorbance (734 nm) was measured. The ABTS+ scavenging capacity was calculated by the following formula:

where A₀ represents the absorbance of the negative control (ABTS+ plus double distilled water); A₁ represents the test system containing the sample (ABTS+ plus TSAT); and A₂ is the absorbance of the blank system (TSAT plus double distilled water). The IC₅₀ value of ABTS+ scavenging rate was then calculated. All operations were performed in parallel three times.

The tyrosinase inhibitory activity was determined according to a slightly improved version of the method previously reported by Morais et al.25 A series of 50 μL TSAT (0.01–1 mg/mL, in 50% DMSO) solutions were added to the reaction system containing 100 μL of PBS buffer (0.1 M, pH 6.8) and 50 μL tyrosinase (100 U/mL), respectively, and reacted at 25 °C for 15 min. Then, 50 μL L L-DOPA (3.5 mM) solution was added to the reaction system and incubated at 37 °C for 10 min. The absorbance (475 nm) was determined. VC was used as the positive control. The inhibition activity of TSAT was calculated by the following formula:

where A₀ represents the absorbance of the negative control with 50% DMSO instead of the sample; the system contains L-DOPA and tyrosinase; A₁ represents the test system containing the sample and contains L-DOPA and tyrosinase; and A₂ is the blank system (sample plus L-DOPA) absorbance. The IC₅₀ value was calculated to indicate the tyrosinase inhibitory activity of TSAT. All operations were performed in parallel three times.

The Fenton reaction is the most common chemical reaction that produces hydroxyl radicals. This experiment was carried out according to the instructions of the hydroxyl radical assay kit. Different concentrations of TSAT (0.01–1 mg/mL) were mixed with the reagents sequentially and left at room temperature for 20 min. Absorbance (550 nm) was measured, and VC was used as a positive control. The scavenging rate (%) was calculated as follows:

where A₀ is the absorbance of the negative control system without TSAT, A₁ is the absorbance of the reaction system containing TSAT, and A₂ is the absorbance of the reaction system containing double distilled water instead of H₂O₂. The IC₅₀ value was then calculated. All operations were performed in parallel three times.

The inhibition of superoxide anion radical kits has biological significance by simulating the reaction system of xanthine and xanthine oxidase to generate superoxide anion radicals. In this experiment, the reagents were prepared according to the kit instructions, different concentrations of TSAT solution (0.01–1 mg/mL) were added, and a water bath at 37 °C for 40 min. VC was used as a positive control. The scavenging rate (%) was calculated as follows:

where A₀ represents the absorbance of the negative control with deionized water instead of the sample; A₁ represents the test system containing the sample; and A₂ is the absorbance of the blank system. The IC₅₀ value was then calculated. All operations were performed in parallel three times.