Extraction, Purification And In Vitro Bioactivities Of Resveratrol From Wine Dregs

Grape residue, a common byproduct in industrial production of wine, was used as the raw material to reclaim resveratrol, which featured beneficial biological properties and physiological functions. The extraction and purification process of resveratrol, as well as the inhibitory kinetics study on tyrosinase activity and its effects on platelet aggregation in vitro, were investigated systematically, aiming to provide theoretical support for the recycling of wine dregs. The main results are listed as follows:(1) Cellulase assisted method was introduced to extract resveratrol from grape residue. Based on single factor experiments, a 4-factor,3-level Box-Behnken experimental design was conducted to evaluate the effects of the factors, including enzyme concentration, temperature, time, and the pH value on the extraction process. Furthermore, a regression model was established with response surface analysis by the Design-Expert 7.0 software. The optimal condition of enzymatic extraction was determined as follows:enzyme concentration 5 U/g residue, enzymatic hydrolysis temperature 52.34℃, enzymatic hydrolysis time 102.96 min, and the pH value 4.79, of which the extraction yield reached up to 516.53μg/g under such condition. The results indicated that the enzymatic assisted extraction of resveratrol from grape residue in wine dregs was remarkably superior to the regular ethanol extraction method.(2) NKA-9 resin was chosen to isolate and purify resveratrol based on the macroporous resin method. The optimal process for purification of resveratrol was determined as adsorbed with a sample flow velocity of 1 mL/min, and a pH value of 4, followed by a desorption by 75% ethanol with a flow velocity of 1 mL/min, and a desorption volume of 200 mL. The purity of resveratrol was 26.03% through this process.(3) The inhibitory effect of resveratrol on the activity of monophenolase and diphenolase existing in tyrosinase in vitro was investigated by the enzymatic kinetic method. The results indicated that resveratrol could inhibit both the monophenolase and diphenolase activities of tyrosinase, and the IC50 values (resveratrol concentration corresponding to 50% inhibitory rate) were 5.1 mg/mL and 5.6 mg/mL, respectively. Resveratrol was found to be a mixed inhibitor for the oxidation of L-DOPA according to the Lineweaver-Burk plot, and the equilibrium constants for binding with free enzyme Kr and with enzyme-substrate complex KIS were 3.4 mg/mL and 35.98 mg/mL respectively.(4) The suppressive effect of resveratrol on platelet aggregation which could result in thrombosis was also investigated. The results indicated that resveratrol significantly inhibited platelet aggregation induced by ADP in vitro and presented a dose-dependent relationship, of which the efficiency of inhibition increased corresponding to the concentration of resveratrol (0.0125 mg/mL～0.10 mg/mL), and the IC50 value was 0.046 mg/mL.