Studies On Inhibition Effects Of Extracts From Bryopsis Plumose And Enteromorpha Linza On The Tyrosinase

Tyrosinase(EC.1.14.18.1) is widely distributed in microorganisms, plants and animals,involving in pigment biosynthesis. Tyrosinase in organisms have single phenolic enzyme activity and poly phenols enzyme activity. Single phenolic enzyme activity can make a single phenol become dipheno; polyphenols enzyme activity can make the polyphenols become quinones. After multimerizing, these quinones finally formed melanin with interacting with intracellular protein. Tyrosinase played an important role in biological melanin formation and make the body acquired immune ability when foreign substance invaded. Efficient tyrosinase inhibitor is acquired by screening natural seaweeds. The tyrosinase inhibitor can apply to developping skin care products, cosmetics, biological pesticides and food additives.In this research 11 species of marine algae were screened .The extracts of Bryopsis plumose and Enteromorpha linza have significant inhibition on tyrosinase. According to orthogonal tests, the optimum extraction conditions were determined; Using organic solvent extracting method , different components of two algaes are obtained. With kinetic analysis method ,the inhibiton mechanism and inhibition type are determined. Results are as follows:1. The screening system is 5mL volume, using 1.0mmol/L DOPA as substrate, the pH of the buffer being 6.8, the enzyme concentration 1.28ug/mL, the inhibitor final concentration 4mg/mL, and the reaction temperature and time being 37℃and 1min. The inhibition results: with the concentration of the extracts being 4mg/mL, Gymnogongrus flabelliformis, Gracilaria asiatica, Grateloupia sp., Cracilaria textorii, Symphyocladia latiuscula, and Ahnfeltiopsis flabelliformis have strong inhibition effects on tyrosinase, the inhibition ratio reaching 20%, the inhibition ratio of Gymnogongrus flabelliformis and Gracilaria asiatica reaching 40%. Bryopsis plumose , Enteromorpha prolifra and Ulva pertusa all have different inhibition effects on tyrosinase, the inhibition ratio of Bryopsis plumose reaching 78%, the inhibition ratio of Enteromorpha prolifra reaching 40.24%, Chondria tenuissima and Gymnogongrus flabelliformis show opposite effects on tyrosinase,—activating the enzyme, the activation ratio benging 18.18% and 20.45%.2. Using constant temperature water extraction method to extract the chemical composition of Bryopsis plumose and Enteromorpha. The extraction solvent is enthanol. The ratio of solid to liquid is 1:12. The extracting temperature is 40℃.First using single factor experiment to confirm the optimum extraction condition of single factor. And then through the orthogonal experiment the best extraction conditions are determined. Bryopsis plumose optimum extraction cinditions are 60℃constant temperature water extraction, 60% concentration of enthanol, 5h exracting time and 1:14 ratio of solid to liquid. Enteromorpha optimum extraction cinditions are 60℃constant temperature water extraction, 60% concentration of enthanol, 5h exracting time and 1:10 ratio of solid to liquid.3. After solvent extracting, the ethanol extract of the seaweeds were separated into three components: Ethyl acetate extract, Petroleum ether extract and water soluble components, and then separately testing the inhibition activity, inhibition type and polyphenol content. The result are as follows: The concentration corresponding to 50% inhibitory rate of Bryopsis plumose Petroleum ether extract was 2.1mg/mL, being noncompetitive reversible inhibitor and K_I being 0.33. Ethyl acetate extract was noncompetitive reversible inhibitor too, and its IC₅₀ was 0.27mg/mL, K_I being 0.13. Water soluble components was competitive reversible inhibitor, and its IC₅₀ and K_I were 1.0mg/mL, 0.91. Enteromorpha Petroleum ether extract was competitive reversible inhibitor, and its IC₅₀ and K_I were 0.29mg/mL and 0.28; Ethyl acetate extract was noncompetitive reversible inhibitor , its IC₅₀ was 0.19mg/mL and K_I was 0.08; Water soluble components was competitive reversible inhibitor, and its IC₅₀ and K_I were 7.02mg/mL, 2.95. Both the Ethyl acetate components of the seaweeds have significant inhibition activity.