Determination,Extraction,Purifiation And Inhibition Of Tyrosinase For Glabridin

Glabridin is an unique isoflavan-flavonoid in Glycyrrhiza glabra L.,It has an efficient role in the whitening, it's a natural whitening agent.The feasibility of using Acid-catalyzed vanillin assay to determinate the content of glabridin were studied and a three-level Box-Behnken design,combined with the canonical and ridge analyses,was employed to optimise the process parameters for glabridin extraction from the Glycyrrhiza glabra L. . The optimum conditions of the purification of glabridin from Glycyrrhiza glabra L.were determined by the method of macroporous adsorbtion resins and the inhibition of glabridin on the monophenolase and the diphenolase of tyrosinase were assayed.The results were as follows:1. The feasibility of using Acid-catalyzed vanillin assay to determinate the content of glabridin were studied.The results are as follows:Acid-catalyzed vanillin assay has high sensitivity and good reproducibility,it is suitable for quantitative analysis of glabridin.Proper reaction conditions were obtained as following:0.5mL methanol sample solution was mixed with 2.5mL 2% vanillin in methanol and 2.5mL 30% H2SO4 in methanol,the reaction was carried out at 20℃for 20 minutes and the absorbance at 534nm was measured.2. A three-level Box-Behnken design,combined with the canonical and ridge analyses,was employed to optimise the process parameters for glabridin extraction from the Glycyrrhiza glabra L. one of the most valued traditional Chinese medicines.The critical factors selected for the investigation were methanol/solid ratio,duration of time and extraction temperature.The experimental results were fitted with a second-order polynomial equation by a multiple regression analysis and more than 98.08% and 91.21% of the variation could be predicted by the yield and the purity of glabridin models.The canonical analysis of surface responses revealed that the three eigenvalues had different signs,indicating a saddle stationary surface.The optimal conditions for extraction of glabridin from the Glycyrrhiza glabra L. were determined,using the ridge analysis,as extraction time,64 min;extraction temperature , 50.4°C ; at methanol/solid ratio of 1:38.5 g/ml . Under these conditions,the highest yield of glabridin reached with estimated value and verified value being of 0.52% and 0.51% .3. The static adsorption characters of a variety of macroporous adsorption resins were studied,a resin fitted to purify glabridin was selected and the process parameters of Fixed-bed adsorption and desorption were determined.The results indicated that the adsorptive capacity and the rate of desorption of 3 resins HPD-400,YPR-II and X-5 were higher than others.The adsorption kinetics behavior was accord with the secondary adsorption rate equation and the adsorption isotherm curves could be described by Freundlich equation of the three macroporous resins.HPD-400 was chosen to purify glabridin because at the same conditions the adsorption on HPD-400 was more easier to achieve than other two resins.The breakthrough curve experiment data of HPD-400 on glabridin was fitted by the Bohart-Adams equation, and the fitted formula can almost calculate breakthrough time of glabridin in given conditions.The optimum conditions of dynamic adsorption by HPD-400 were as follows:the sample concentration,0.845mg·mL^-1;flow velocity,3BV·h^-1;eluent solvent,70% ethanol;eluent velocity,2BV·h^-1.The purity of glabridin was increased from 4.5% to 21.8% at the conditions mentioned above.4. In the present paper,glabridin and several common skin-lightening agents in cosmetic (Vc, kojic acid and the licorice extract) were evaluated for their inhibition on the monophenolase and the diphenolase of tyrosinase , using absorption method.And the IC50 values were determined for comparing their abilities of inhibiting the activity of tyrosinase.The study showed that all of the three reference inhibitors,Vc,kojic acid and the licorice extract can inhibit the activity of both mono- and diphenolase.The three compounds inhibited the oxidation of L-tyrosine by the enzyme to an IC50 value of 14 mmol/L (2466μg/mL),1.14 mmol/L (162μg/mL) and 5.8μg/mL,respectively.They also inhibited the oxidation of L-DOPA by the enzyme to an IC50 value of 16.2 mmol/L (2853μg/mL),5.7 mmol/L (810μg/mL) and 70.1μg/mL,respectively.The glabridin (purity quotient of 78.8%) exhibited highly potent mono- and diphenolase tyrosinase activity inhibition with an IC50 value of 0.182 mmol/L (58.9μg/mL) and 0.07 mmol/L (22.7μg/mL) ,respectively.The inhibitor type is noncompetitive with L-DOPA as a substrate.