Optimizing extraction conditions for isolation of antioxidant compounds from black rice bran using ultrasonic-assisted extraction and response surface methodology

Black rice bran (BRB), a valuable byproduct from the rice milling process, possesses numerous pharmacological activities, including antioxidant potential, but information regarding highly efficient extraction methods is scarce. To enhance the extraction efficiency, ultrasonic-assisted extraction coupled with Box-Behnken design (BBD) was used in this study to maximize the total phenolic content (TPC), total flavonoid content (TFC), total anthocyanin content (TAC), and antioxidant capacity of BRB extract. The BBD results showed that 57% ethanol at 50°C and pH 3.1 were best for TPC (136.2 mg GAE/g dry weight), TAC (2.3 mg cyanidin-3-O-glucoside equivalents/g dry weight), and TFC (38.6 mg quercetin equivalents/g dry weight). In vitro antioxidant capacities of BRB extract determined by 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS⁺) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays revealed free radical scavenging potentials with EC₅₀ values of 67.62 and 1149 µg/mL, respectively. The cytotoxic effect of BRB extract on human skin HaCaT keratinocytes was determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which revealed no significant cytotoxicity and a significant increase in cell viability at a concentration of 500 µg/mL. Ultra-high performance liquid chromatography (UHPLC) analysis identified syringic acid, gallic acid, protocatechuic acid, vanillic acid, p-coumaric acid, and ferulic acid in BRB extract. This study provides the optimal conditions to extract antioxidant bioactive compounds from BRB and supports the use of BRB as a source of antioxidants. Due to BRB showing no significant cytotoxicity to human skin cells, it could potentially be further developed for topical applications.