Screening For Natural Tyrosinase Inhibitors From Paeonia Lactiflora Pall And Cortex Mori And Investigation Of Their Inhibitory Kinetics

Tyrosinase is a copper-containing enzyme presents in plant, animal tissues and microorganisms that catalyze the production of melanin and other pigments from tyrosine by oxidation, and it is the rate-limiting enzyme related to melanin synthesis and catecholamine metabolism of human being. Tyrosinase active substances have been widely used in the treatment of hyper pigmentation diseases, food preservatives and whitening agents in cosmetics. Due to the side effects of synthetic tyrosinase active substances, more and more people have a favor seeking for efficient non-toxic natural tyrosinase active substances for alternative. However, the conventional protocol for the discovering of novel natural tyrosinase inhibitors is time consuming,laborious and often suffers from loss of activity in the process of bioassay-guided fractionation.Online screening method for tyrosinase inhibitors based on HPLC post-column bioassay reaction exhibits the unique advantage of sensitivity and accurate detection, as well as speed and versatility.The online screening method can quickly detect tyrosinase active substances in complex natural products without pre separation or fractionation. This dissertation use HPLC based online screening for tyrosinase inhibitors from Paeonia lactiflora Pall and Cortex mori, and targeted separation for bioactive compounds from the screening experiments. The main results and conclusions are as following:1. Inhibitive effects over mushroom tyrosinase of petroleum ether extracts, chloroform extracts, ethyl acetate extracts, n-butanol extracts, and water extracts of Paeonia lactiflora Pall and Cortex mori are investigated. The results show that all extracts demonstrate different inhibitory effect on tyrosinase for both plants. The ethyl acetate extracts and n-butanol extracts of Paeonia lactiflora Pall exhibit relatively strong effective inhibitory on tyrosinase compared to commercially available Arbutin, their IC50 are 0.97 mg/mL and 0.62 mg/mL respectively. Theethyl acetate extracts of Cortex mori has moderate inhibitory activity on mushroom tyrosinase, the IC50 is 1.71 mg/mL.2. Online HPLC screening method is used to screen tyrosinase active compounds from ethyl acetate extract from the n-butanol fraction of Paeonia lactiflora Pall and ethyl acetate fraction of Cortex mori. The screening results show there are six major tyrosinase inhibitive compounds present in the ethyl acetate fraction and n-butanol fraction of Paeonia lactiflora Pall, and one active compound is found from the ethyl acetate fraction of Cortex mori extract.3. Four compounds are isolated from ethyl acetate fraction and n-butanol fraction of Paeonia lactiflora and one compound is isolated from ethyl acetate fraction of Cortex mori. Extract with various chromatography techniques including low pressure preparative liquid chromatography and preparative high performance liquid chromatography, and their structures are identified based on their1 H NMR,13 C NMR and UV. The major active compounds are gallic acid(BS-1),pentagalloylglucose(BS-2),galloyl-O-β-glucopyranosyl(BS-3),1,1’,6,6’-galloyl-O-β-D-glucopyrano(BS-4), 5, 7, 2′-trihydroxy flavanone-4′-O-β-D-glucoside(S-5).4. 96-micro well plate method is adopted for the determining of inhibitive activity against mushroom tyrosinase of five isolated active compounds. The result shows that all five isolated compounds are tyrosinase inhibitors. The pentagalloyl glucose(BS-2) has the strongest inhibitive activity, the IC50 is 0.0365mmol/L. Enzyme catalytic kinetic experiments show that the inhibitory effect of BS-2 is reversible and the inhibitory type is liner mixed inhibition.