Dpph assay

The free radical scavenging ability of the pure PEG and PEG–Au 43.5 ppm nanocomposites were measured using DPPH assay (2,2-diphenyl-1-picrylhydrazyl) (Sigma-Aldrich, Burlington, MA, USA), which was described in a previous study [32 (link)]. The nanocomposites were first dissolved in deionized water. Next, 1 mL of the nanocomposites was mixed with DPPH at ratio of 1:3 and then incubated in the dark at room temperature for 90 min. The absorbance of each sample was measured at 539 nm using an ultraviolet-visible spectrophotometer (Helios Zeta, Thermo, Waltham, MA, USA). Free radical scavenging ability was calculated based on the formula: % Inhibition ratio = [1 − (absorbance of test sample/absorbance of control)] × 100%. The absorbance value of the test sample and the control were represented as nanomaterials and deionized water, respectively.

The antioxidant capacity of 100 μg/mL A. fruticosus methanolic extract was determined using DPPH assay based on the redox potential of DDPH (Sigma–Aldrich, St. Louis, MO, USA). We prepared 1 mM DPPH solution (0.394 mg/mL) in methanol and then diluted it 1:10 to obtain a 100 μM solution (Abs at 515 nm = 0.5–0.6). Then, 500 μL each of the test sample and 100 μM DPPH solution were mixed in a cuvette. The negative control contained 500 μL each of methanol and DPPH solution. Both solutions were incubated in the dark at room temperature for 15 min, and absorbance was read at 515 nm using methanol as blank. The results were expressed as the percentage reduction in the radical absorbance [18 (link)].

The antioxidant potential of the extract was compared to trolox as a reference antioxidant agent, and the results were expressed as TEAC. We constructed the calibration curve between inhibition percentage and trolox concentrations (12.5–0.3 μg/mL). We obtained a high correlation coefficient value of 0.9915, which indicated good linearity. We calculated the TEAC of the extract by substituting the inhibition percentage of the tested extract in the regression equation of the calibration curve.

VC, NAC, and the esterified form of ATX were purchased from Sigma-Aldrich (St. Louis, MO, USA). VC, NAC, and ATX were dissolved in dimethyl sulfoxide solution (DMSO, Sigma-Aldrich) to a concentration of 10 mM. Thereafter, 10 mM antioxidant DMSO solution was dissolved in serum-free DMEM media to a final concentration of 1 mM. To determine an appropriate concentration of VC, NAC, and ATX for further experiments, antioxidants were prepared at three concentrations, 1 mM, 100 μM, and 10 μM, and investigated for their free radical scavenging activity and cell survival using diphenyl-1-picrylhydrazyl (DPPH) assay (Sigma-Aldrich) and Cell Counting kit-8 assay kit (CCK-8, Dojindo, Japan). The final concentration of the antioxidants in our study was 10 μM for all three antioxidants based on the criteria for high free radical scavenging activity and low toxicity for cells (Supplementary Data).