Hydroxyl free radical scavenging capacity assay kit

The freeze-dried AOS powder degraded at 25 °C for 30 min was applied for the antioxidant activity determination as described above. The assay of the ferric-reducing power of the AOS was carried out in accordance with the method described before [51 (link),52 (link)]. The absorbance of the mixture was measured at 700 nm using distilled water as the blank. Hydroxyl radical scavenging activity was determined by using the hydroxyl free-radical scavenging capacity assay kit (Solarbio, Beijing, China), and the absorbance was measured at 536 nm. Total antioxidant activity was determined by using the total antioxidant capacity assay kit with a rapid ABTS method (Solarbio, Beijing, China), and the absorbance was measured at 414 nm. Distilled water and Vc were set as the blank and positive control, respectively. Antioxidant abilities were measured with reference to the procedures of the manuals [41 (link)].

The galangal rhizome was cleaned and dried to a consistent weight, crushed, and then kept in a dry atmosphere. Salicylic acid was obtained from Tianjin Damao Chemical Reagent Co., Ltd. (Tianjin Damao Chemical Reagent Co., Ltd., Tianjin, China), and the following materials were used in this study: DEAE-52 cellulose (Greenherbs, Napier, New Zealand), Sephadex G-100 (Cytova, Marlborough, MA, USA), CCK8 assay kit (APExBIO, Houston, TX, USA), EdU cell proliferation kit (Beyotime, Nantong, China), hydroxyl free radical-scavenging capacity assay kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), and DPPH free radical-scavenging ability assay kit (Beijing Boxbio Science & Technology Co., Ltd., Beijing, China). Fetal bovine serum (FBS) and 1% penicillin–streptomycin were supplied by Guangzhou Saiguo Biotechnology Co., Ltd. (Guangzhou Saiguo Biotechnology Co., Ltd., Guangzhou, China). Transwell chambers and Matrigel matrix gel were purchased from Corning Life Sciences Co., Ltd. (Corning Life Sciences Co., Ltd., Jiangsu, China). Then, 4% tissue cell fixative solution and 0.1% crystal violet staining solution were purchased from Solarbio (Solarbio, Beijing, China). Unless otherwise stated, all chemicals used were of analytical grade.

The antioxidant effect of natto peptide was determined by investigating the scavenging capacities of 2,2′‐diphenyl‐1‐picrylhydrazyl (DPPH), 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS⁺), hydroxyl and superoxide anion free radicals, as described previously (Dong et al., 2020 (link)). For the DPPH assay, the natto peptide was incubated with DPPH buffer which was dissolved in 95% methanol in a dark environment for 30 min, then the absorbance value at 517 nm was measured by using a microplate reader. In the ABTS⁺ assay, the natto peptide was incubated with an ABTS⁺ working solution in dark for 10 min, then the absorbance value at 734 nm was determined by using a microplate reader. Moreover, the ABTS⁺ working solution was prepared by using potassium persulfate solution (2.6 mM) to dilute the ABTS⁺ stock solution (7 mM) at a ratio of 1:1 and incubated in dark for 12 h before determination. The hydroxyl and superoxide anion radical scavenging abilities were determined by using a Hydroxyl Free Radical Scavenging Capacity Assay Kit (BC1325; Solarbio) and a Superoxide Anion Detection Kit (BC1290; Solarbio), according to the manufacturer's instructions. All determinations were repeated three times.