Study On The Whitening Active Substance Of Coreopsis Tinctoria Nutt

Using bio-assay guided method screened 100 kinds for raw materials, compared whitening active of them. Coreopsis tinctoria Nutt. abilities of antioxidant activity and inhibition the tyrosinase were the best, which was determined as the research object. Using solvent extraction method for detect crude extract had whitening active material in the Coreopsis tinctoria Nutt.. The resin column chromatography raw crude extract was separated by macroporous adsorption, tracking their effective active site. Then, by reverse phase C18 column chromatography on silica gel and Sephadex LH-20 column for further separation and purification of the active site of chromatography, eventually got whitening active monomer compounds. Compared the abilities of cleaning DPPH-radical and inhibiting tyrosinase among the three monomer compounds. Evaluated the whitening effect to provide a theoretical basis and experimental basis for the scientific from Coreopsis tinctoria Nutt. flavonoids. The results as follow.(1) Screened raw materials which had whitening activity. According to food and medicine homology theory, selecting 100 kinds for raw materials. Crude extract by solvent extraction, detected the crude extract on DPPH-radical scavenging ability. Determined the better activity of 20 kinds of raw material for further screening. Compared ability of them on inhibit tyrosinase activity, determining whitening active. According to the results, the crude extract concentration Coreopsis tinctoria Nutt. at 0.5mg/mL, clearance rate of (87.43±0.23)%. At 5mg/mL, tyrosinase inhibition rate of (91.07±3.85)%, which was the best active of crude extract. Choosen Coreopsis tinctoria Nutt. as research development of the object.(2) Isolated and purified whitening active substances from Coreopsis tinctoria Nutt.. Crude extract was separated by macroporous resin column chromatography and compared the separate parts of the activity. Then by reverse phase C18 column chromatography on silica gel active site to got the monomer compound. Finally using Sephadex LH-20 to purified monomeric compound. According to the results, the active site of 60% ethanol eluted better than other parts. Therefore,60% ethanol portion was separated and purified, obtain three monomeric compounds, HPLC purity greater than 95%.(3) Analysed the molecular structure of monomer compounds. Using HPLC-MS and NMR techniques to identify the structure of the three active compounds. According to the chemical shifts of carbon signals and proton signals identified three monomer compounds are flavonoids. Infered the signal K1 was four hydroxyl groups by the formula monomeric compound and carbon estimation. Obtain comparative binding proton signal K1 was 3’,4’,7,8-tetrahydroxy flavanone. Infered the signal K2 was three hydroxyl groups by the formula monomeric compound and carbon estimation. Obtain comparative binding proton signal K2 was 3’,5’,7-trihydroxy flavanone. Infered the signal K3 was five hydroxyl groups by the formula monomeric compound, carbon estimation and coupling constant. Obtain comparative binding proton signal K3 was Okanin.(4) Evaluated monomer compounds whitening efficacy of the active. Three compounds were compared the abilities of antioxidant activity and inhibition tyrosinase activity in vitro of acid. Antioxidant activity results showed that K3>Vc>K1>K2>Rutin. Inhibit tyrosinase activity results showed that K3>Arbutin>K2>K1. Therefore, the whitening efficacy of the active of K3 was better than others.(5) Optimizated the extraction process of flavonoids from Coreopsis tinctoria Nutt.. Extraction process by orthogonal experiment optimization Coreopsis tinctoria Nutt. in flavonoids after as follows, using 70% ethanol,20 times the volume of solvent immersion Coreopsis tinctoria Nutt., at temperature of 80 ℃ extraction one hour, extracted a total of twice, the total flavonoids extraction rate is 10.10%.