Isolation Of Tyrosinase Inhibitors From Corn Stalk And Tyrosinase Inhibitory Activity Enhancement

With the increase of energy consumption and the proposal of biomass refining,the applications of lignocellulosic biomass have drawn extensive attention.Although the lignocellulose is rich in bioactive substances,however,the biological activities of lignocellulose has not been widely used in practical production due to the complicated physical/chemical structure.In this study,the tyrosinase inhibitors in corn stalk were isolated,and then the inhibitory activity and inhibition mechanism were investigated.The purpose of this present work is,therefore,not only to screen the efficient tyrosinase inhibitors from underused corn stalk but also to provide a theoretical basis for expanding the sources of natural inhibitors and the value-added application of corn stalk.(1)For the first time in literature,this study revealed the anti-tyrosinase performance of hydrolysates from post-washing liquor of steam exploded corn stalk.It showed strong anti-tyrosinase activity with an IC50 of 2.17 mg/mL at steam explosion severity of 4.20.Through hydrolysates distribution profiles and model substances analysis,phenolic compounds(especially H-unit monomers and phenolic acids)were confirmed to be major inhibitors in hydrolysates.An enrichment process of phenolic compounds by ethyl-acetate fractionation of hydrolysates was employed.Ethyl-acetate soluble fraction exhibited the higher anti-tyrosinase activity with an IC50 of 0.29 mg/mL,which was even better than p-coumaric acid(IC50,0.31 mg/mL).Additionally,the mixed-type inhibition of hydrolysates on tyrosinase was illustrated by kinetic studies and the interaction between hydrolysates and tyrosinase was confirmed by fluorescence quenching analysis.(2)Organosolv ethanol lignin(OEL)from corn stalk was divided into four fractions by multiple organic solvent successive extraction and the anti-tyrosinase activities of four obtained fractions were compared.The results revealed that the successive extraction realized the fractionation of the heterogeneous OEL into four relatively homogeneous parts with gradually increasing molecular weight and decreasing phenolic OH content.The four fractions exhibited obviously different anti-tyrosinase activities and the dichloromethane fraction had the highest tyrosinase inhibition rate(75.12%,0.5 mg/ml),whose value was comparable to the positive controlp-hydroxybenzaldehyde.Besides,the relationships between the anti-tyrosinase activity and the lignin properties(purity,molecular weight and phenolic OH)were discussed to elucidate the possible formation mechanism of differences among the four lignin fractions in anti-tyrosinase activity.(3)In order to improve the anti-tyrosinase activity of lignin and evaluate the effect of carboxyl group content on tyrosinase,the inhibitory activities of carboxymethylated lignin obtained from different conditions were investigated.Compared with untreated alkali lignin,the carboxylic group content of carboxymethylated lignin acquired from the optimum condition(3 g NaOH,4 g sodium chloroacetate,70?,90 min)increased by 0.89 mmol/g and the tyrosinase inhibition rate increased by 43.56%.The results determined by 31P NMR showed that the sodium chloroacetate can band not only to phenolic hydroxyl groups but also to aliphatic hydroxyl groups.The inhibition types and mechanisms of carboxymethylated lignin on tyrosinase were studied by enzyme kinetics and fluorescence spectroscopy,respectively.The fluorescence quenching analysis showed that the carboxymethylated lignin exhibited inhibitory activity by changing the hydrophobic environment of amino acid residues in tyrosinase.Therefore,all results indicated that the carboxymethylation is an effective way to obtain the efficient tyrosinase inhibitors from lignin of corn stalk.