Studies On Effects Of Extracts From Laminaria Japonica And Ruditapes Philippinarum On The Tyrosinase Activity And Melanogenesis

Tyrosinase(EC 1.14.18.1) is a kind of redoxidase containing copper, widely distributed in nature. It is a key enzyme involved in the melanin biosynthesis.This study would provide a basement for the developing of whitening cosmetic or tyrosinase-targeted medicine by screening new naturally occurring tyrosinase inhibitors.15 seaweeds and 5 marineanimals were screened to study their effect on the activity of tyrosinase, the result showed that the extracts from Laminaria japonica and Ruditapes philippinarum exhibited obvious tyrosinase inhibition. The optimum extraction conditions of substances from the two halobios were confirmed according to orthogonal experiment, and the inhibitory effects and mechanism of substances on tyrosinase had been elucidated; meanwhile, the biological activity such as anti-ultraviolet catalysis activity were investigated. The contents and results were as follows:1. The tyrosinase inhibitor screening model was established based on the method of spectrophotography, using 1.0mmol/L DOPA as substrate, the final concentration of tyrosinase reached to 3.14mg/L, the optimum reaction temperature and pH were 32℃and 6.8, the reaction time at the steady state was 1 minute, using 20% DMSO to dissolve the substance. The tyrosinase inhibitory activity of 15 seaweeds and 5 marineanimals were investigated according to the eatablished screening model, Laminaria japonica and Ruditapes philippinarum exhibited more larger inhibiting capacity than others. 2. The antienzyme components were extracted from Laminaria japonica by traditional extraction, using one-factor-at-a-time methodology and orthogonal experiment, the optimum extraction conditions were confirmed. The optimum extration conditions were ethanol 50%, extracting temperature 40℃, extracting time 3h, 1g:12mL for ratio of solid to liquid. The ethanol extract was leached with organic solvents to obtain the ethyl acetate-soluble fraction, the petroleum ether-soluble fraction and the substance in water, the results showed that the substance in water can inhibit the activitiy of tyrosinase obviously, the concentration corresponding to 50% inhibitory rate (IC50) was 0.16 mg/mL. Lineweaver-Burk plots demonstrate that the substance in water expresses as what a competitive inhibitor does, KI was estimated to be 0.07mg/mL. The result of the qualitative identify showed that the phenols were involved in the substance in water which were not found in the ethyl acetate-soluble fraction and the petroleum ether-soluble fraction, so the element in the substance in water which inhibited tyrosinase activity is phenols in all probability. The joint inhibitory effects of the substance in water with arbutin and kojic acid on tyrosinase activity were examined using a toxic equivalency method, results showed that the effects of the substance in water and kojic acid was synergistic.3. The antienzyme components were extracted from Ruditapes philippinarum by traditional extraction, the optimum extration conditions were ethanol 95%, extracting temperature 50℃, extracting time 4h, 1g:8mL for ratio of solid to liquid. The ethanol extract was leached with organic solvents to obtain the ethyl acetate-soluble fraction, the petroleum ether-soluble fraction and the substance in water, all of the which can inhibit the activitiy of tyrosinase obviously, the concentration corresponding to 50% inhibitory rate (IC50) was 0.43mg/mL,0.63mg/mL and 0.71mg/mL, respectively. Lineweaver-Burk plots demonstrate that the ethyl acetate-soluble fraction and the petroleum ether-soluble fraction express as what a noncompetitive inhibitor does, the KI (KIS) was 0.47mg/mL and 0.71 mg/mL, respectively; the substance in water expresses as what a competitive inhibitor does, KI was estimated to be 0.31 mg/mL. The joint inhibitory experiment showed that:the effects of the ethyl acetate-soluble fraction and arbutin was synergistic on inhibiting tyrosinase activity, the substance in water and kojic acid was synergistic.4. Choosing the substance in water from Laminaria japonica, the ethyl acetate-soluble fraction,the petroleum ether-soluble fraction and the substance in water from Ruditapes philippinarum as the stayer, studing their anti-ultraviolet catalysis activity, anti-DOPA autoxidation activity and promoting-degradation of melanin activity, the results showed that:all the four extracts exhibited extremely obvious anti-ultraviolet catalysis activity, the substance in water from Laminaria japonica and the petroleum ether-soluble fraction from Ruditapes philippinarum exhibited obvious activity of promoting degradation of melanin, the substance in water from Laminaria japonica and the substance in water from Ruditapes philippinarum exhibited extremely obvious anti-DOPA autoxidation activity.