Inhibitory Effect Of Catechins On The Formation Of Melanin

Catechins are 2-phenyl benzopyran derivatives, the compound of flavanol. The large numbers of catechins are in the new shoot of tea tree, which content is 12% to 24% of the dry weight, about 70%-80% of the total tea polyphenols. According to different structures, catechin include epicatechin (EC), epigallocatechin(EGC), epicatechin gallate (ECG), Epigallocatechin gallate(EGCG). As the structure has phenolic base, catechins have excellent antioxidant activity, the same amount of substance concentration catechins of antioxidant activity were L-EGCG> L-EGC> L-ECG> L-EC. Melanin is too fast growth or uneven distributions that lead to black skin and pigmentation, resulting in freckles, age spots, melasmaand other diseases. Tyrosinase (EC,1.14.18.1) is polyphenol oxidase and a copper protein, which the binuclear copper ions play an important role in enzyme catalysis. It is a key enzyme in melanin synthesis. With the development of cosmetics and medicine industry, natural products as cosmetics and medicine conform to the development trend of returning to nature and people's consumption demand, which will become the subject of cosmetic and medicine development.This article preliminarily study effect of catechins on the formation of melanin, including inhibitory effect of catechins on potato tyrosinase; inhibitory effect of catechins on mushroom tyrosinase; effect of EGCG on the B16 melanoma cell morphology, proliferation rate, tyrosinase activity and melanogenesis. We get the following results:一,The effect of the quantity of enzyme solution, substrate concentation, temperature and pH on the activity of potato tyrosinase was determined by spectrophotometer method. The inhibitory kinetics of catechins on the activity of potato tyrosinase was studied by enzymological kinetic method in a Na2HPO4-NaH2PO4 buffer solution (pH 6.8) and at 30℃. The results showed that the optimal reaction conditions is quantity of enzyme solution 0.5 mL, substrate concentation 2 mmol/L, temperature 30℃, pH 6.8. Catechins can inhibit the activity of potato tyrosinase, and catechins concentration to 50% inhibitory rate (IC50) is 0.27 mg/mL. It was found that the inhibitory effect of catechins on potato tyrosinase is competitive inhibition mode and inhibition constant (KI) of catechins on potato tyrosinase is 0.16 mg/mL by Lineweaver-Burk plots.二,The inhibitory kinetics of catechins on the activity of mushroom tyrosinase monophenolase and diphenolase was studied by enzymological kinetic method in a Na2HPO4-NaH2PO4 buffer solution (pH 6.8) and at 30℃. The results showed that catechins can inhibit both the activity of monophenolase and diphenolase, and catechins concentration to 50% inhibitory rate (IC50) are 0.89 mg/mL and 1.13 mg/mL. It was found that the inhibitory effect of the catechins on diphenolase is in a competitive inhibition mode, and inhibition constant (KI) is 0.53 mg/mL by Lineweaver-Burk plots.三,Inverted microscope was used to observe B16 cell morphology; methylene bluemethod, L-DOPA oxidation method, NaOH decomposition method respectively was used to study the effect of EGCG on B16 cell proliferation rate, tyrosinase activity and melanogenesis. The results showed that EGCG act on the B16 at 24 h, the high concentration (>60μM) appeared the phenomenon of cell membrane fusion; after 72 h, most of cell were death at this group. EGCG have significantly inhibited B16 on cell proliferation rate, tyrosinase activity, melanogenesis, which is obvious dose-response relationship. And EGCG have inhibited B16 on cell proliferation, catechins concentration to 50% inhibitory rate (IC50) is 33.99μM.These studies have shown that catechins can inhibit tyrosinase in vitro, showed competitive inhibition; EGCG could significantly inhibit the B16 murine melanoma cell morphology, cell proliferation rate, tyrosinase activity and melanogenesis. Catechins have a certain role on the inhibition of melanin synthesis, which is expected to develop as cosmetics and medicines.