Antioxidant with antimelanogenic activities of sericin hydrolysates optimized by response surface methodology in human melanin-generating mnt-1 cells

Abstract

Sericin, a protein presenting in the wastewater from silk industry, causes water pollution and ecological problem. To increase economic value to this waste product, the optimum enzymatic condition that could create sericin hydrolysates with high antioxidant and antimelanogenic capacities was generated through response surface methodology (RSM). Response surface plots demonstrate the major role of temperature on scavenging capacity of sericin hydrolysates assessed via DPPH, FRAP and ORAC assays. Alcalase®-hydrolyzed sericin consisted of sericin-related peptides in differing amounts and smaller sizes compared with unhydrolyzed sericin, as respectively demonstrated by peptidomic and SDS-PAGE analysis. Pre-incubation with 20 mg/mL sericin hydrolysates digested by Alcalase® at RSM-optimized condition (enzyme/substrate ratio: 1.5, pH: 7.5, temperature: 70°C) exhibited higher antioxidant activity against 1 mM hydrogen peroxide in human HaCat keratinocytes and melanin-generating MNT-1 cells when compared with 5 mM N-acetyl cysteine. The lower half maximum inhibitory concentration was 9.05 ± 0.66 mg/mL compared with unhydrolyzed sericin (24.54 ± 0.17 mg/mL) indicated a potent effect of Alcalase®-hydrolyzed sericin on inhibiting melanin production in MNT-1 cells. Not only inhibiting enzymatic activity but also downregulated expression of tyrosinase was evident in MNT-1 cells incubated with 20 mg/mL sericin hydrolysates. Quantitative RT-PCR revealed the decreased mRNA level of MITF, a tyrosinase transcription factor, which correlated with the reduction of pCREB/CREB, an upstream cascade, as assessed by western blot analysis in MNT-1 cells cultured with 20 mg/mL sericin hydrolysates for 12 h. Interestingly, treatment with Alcalase®-hydrolyzed sericin for 6-24 h also upregulated pERK, a molecule that triggers MITF degradation in human melanin-producing cells. The acquired information would facilitate the recycling of waste products from silk industry as an effective antioxidant and antimelanogenic compound.