Anti-tyrosinase activity of protein hydrolysate from spotted babylon Babylonia areolata prepared by alkaline protease

Abstract

In this research was designed to investigate the effect of protein hydrolysates from spotted babylon Babylonia areolata prepared by alkaline protease (Protease G6) on melanogenesis inhibition and antioxidant properties. The results revealed that the optimal condition of protein hydrolysates preparation for tyrosinase inhibitory activity was undiluted of Protease G6 (5.8×105 DU/g) at the hydrolysis time of 60 min with an IC50 value of 10.81 µg/ml and the optimal condition for antioxidant activity was 8-fold diluted of Protease G6 (7.25×104 DU/g) at 240 min hydrolysis time with an IC50 of 10.68 µg/ml. Spotted babylon hydrolysates of these two conditions were then fractioned using three different molecular weight cut-off membranes 10, 5, and 3 kDa, respectively. All fractions were evaluated for their antimelanogenesis effect by mushroom tyrosinase assay and antioxidant property by using DPPH, ABTS and NO radical scavenging. Spotted babylon hydrolysates MW < 3 kDa exhibited the highest as tyrosinase inhibition for monophenolase and diphenolase activity with IC50 values of 1.758 and 8.995 µg/ml ,respectively. Spotted babylon hydrolysates MW < 3 kDa also showed IC50values of 9.344, 5.689 and 10.708 µg/ml for DPPH, ABTS and NO radical scavenging. Afterward, spotted babylon hydrolystes MW < 3 kDa were further assessed inhibition type on tyrosinase, cell viability assay, melanin content ,cellular tyrosinase activity assay in B16F10 melanoma cells. Kinetic studies determined that the spotted babylon hydrolysate MW < 3 kDa behaved as an uncompetitive inhibitor for monophenolase and diphenolase activity, showing kinetic inhibition constant values (Ki ) of 2.21 and 11.86 µg/ml. The results demonstrated that spotted babylon hydrolysate MW < 3 kDa suppressed melanin content and decresed cellular tyrosinase activity, with no cytotoxicity to B16F10 murine melanoma cells. Therefore, the spotted babylon hydrolysates could be developed as tyrosinase inhibitor and formulated in skin-whitening products for cosmetic or therapeutic use.