Chemical Characterization And Biological Activity Of Flavonoid Extracts From Rhus Verniciflua

Lacquer tree(Rhus verniciflua) is an important economic forest species in China. Raw lacquer from this species is a material for traditional paint. Most research and applications have focused on the raw lacquer and seeds, but there were little research on the actives of lacquer tree branchs, barks and woods. In the present study, the extraction, separation, HPLC-MS,chemical components, anti-tyrosinase and antioxidant activity of flavonoids from lacquer tree were studied. It is provided theoretical basis for comprehensive utilization and the industry scale development of lacquer tree. The main research contents and conclusions were summarized as follows.(1) Screening an effective antioxidant activity parts of lacquer treeThe chemical compositions and antioxidant activity of leaves, branchs, barks, seeds and woods of lacquer tree were tested, including total polyphenol, flavonoid, tannin, urushiol and polysaccharide contents. The result showed that leaves ethanol extracts exhibited a higher polyphenol, tannin, and urushiol contents then other parts, which were 42.51%, 25.53% and9.19%, respectively. The woods powder ethanol extracts exhibited a higher flavonoid contents,which was 17.52%. The leaves water extracts exhibited a higher polysaccharide, which was30.49%. Ethanol extracts exhibited a significantly higher antioxidant activities than water extracts. The ethanol extracts of green leaves, yellow leaves and woods showed more obvious antioxidant activities than other parts. The wood was found to have the higher flavonoid content and antioxidant activities. Thus, wood was selected for further experiments.(2) Separation and structure characterization of flavonoidsThe total flavonoid extraction yield and anti-tyrosinase activity were an independent variable, solvent extracts of lacquer tree wood were made using decompressing inner ebullition,and a single factor experiment and Box-Behnken design were used to optimize extraction conditions. The RVWEE was partitioned with different polarity solvents. Seven flavonoids of EAE were identified by HPLC-MS, FC and Pre-HPLC, there were fustin, garbanzol, fisetin,sulfuretin, 3,7-dihydroxy flavanone-4′-rhamnose, quercetin and butin. Besides, ethoxy 3-hydroxy benzoic acid, gallic acid, 3, 4- dihydroxy almond acid, gallic acid cetyl ester,protocatechuic acid, ethyl gallate, 1-hexadecanoic acid-2,3- acetic acid glyceride and benzyl propionate heptyl ester were also identified. The ethoxy 3- hydroxy benzoic acid, gallic acid cetyl ester,1-hexadecanoic acid-2,3- acetic acid glyceride, benzyl propionate heptyl ester and3,7-dihydroxy flavanone-4′-rhamnose were identified from lacquer tree for the first time.(3) The antityrosinase activity of the different parts extracts and the inhibitory mechanisms of lacquer treeThe inhibitory effect of the different parts extracts, the four partitioned extracts and purified compounds on tyrosinase were evaluated to screen the higher anti-tyrosinase activity flavonoids. The results showed that the IC50 of EAE was 308 mg/L, with the lag period of the enzyme being obviously lengthened, EAE acted as a mixed type inhibitor. The anti-tyrosinase rates of RVF from strong to weak were sulfuretin, butin, quercetin, fisetin, gallic acid, fustin and garbanzol.The inhibitory mechanism of RVF(fisetin, butin and sulfuretin) on tyrosinase were investigated. The results showed that flavonoids were acted as potent inhibitors of monophenolase and diphenolase activities of the enzyme. The lag period of the enzyme was obviously lengthened; A kinetic analysis showed that fisetin, butin and sulfuretin were competitive, non competitive and competitive inhibitors, the KI were 261.76mmol/L, 3.08mmol/L and 72.40 mmol/L, respectively. The RVF were not combined with the copper ions of tyrosinase from the UV-visible spectrum.(4)The antioxidant activities of RVFThe antioxidant activities of EAE and RVF were evaluated by in vitro experiments. The EAE showed strong antioxidant activities in scavenging DPPH, ABTS and hydroxyl radicals,the IC50 were 19.90 mg/L, 29.74 mg/L and 37.95mg/L, respectively. The total reducing power and ferric ion reducing power presented a linear positive correlation with concentration of EAE.The RVF showed potent antioxidant activities, the antioxidant activities of RVF from strong to weak were quercetin, fisetin, sulfuretin, butin, fustin, garbanzol. Among them, quercetin,fisetin, and sulfuretin showed higher antioxidant activities in scavenging radicals than Vc and BHT.