关键词: 二葡糖基没食子酸, 酪氨酸酶活性抑制, 动力学, 竞争抑制, 抗氧化

Abstract:

A whitening agent, diglucosyl gallic acid, was synthesized through acetylation reaction, salt formation, acidification and other processes using methyl gallate and pentaacetyl glucose as raw materials. The structure of the final product was confirmed by IR, MS, ¹H NMR and ¹³C NMR. The effect of diglucosyl gallic acid on tyrosinase activity, the inhibitory mechanism of tyrosine diphenolase activity and the antioxidant capacity were investigated. The results show that when L-tyrosine is used as a substrate, diglucosyl gallic acid can not only prolong the lag time of the tyrosine monophenolase reaction, but also cause a further increase in the lag time with the increase of concentration, and its half-inhibitory mass concentration IC₅₀ is 1.46 mg/mL; When L-dopa is used as a substrate, the progress curve of the inhibitory effect of diglucosyl gallic acid at different mass concentrations on tyrosine diphenolase all passes through the origin of the coordinate axis, indicating that the progress has occurred and shows a retardation effect, and the IC₅₀ of diglucosyl gallic acid against tyrosine diphenolase is 2.68 mg/mL; the enzyme activity was plotted against the enzyme concentration. As the mass concentration of diglucosyl gallic acid increases, the slope of the straight line continues to decrease and all straight lines intersect at the origin, which indicates that the inhibitory effect of diglucosyl gallic acid on tyrosine diphenolase is a reversible process, namely the inhibitory effect is through the binding of diglucosyl gallic acid with the enzyme to inhibit the activity of tyrosine diphenolase, thereby reducing the efficiency of the enzyme to catalyze the oxidation of L-dopa to produce dopa pigment, rather than reducing the effective enzyme amount; Lineweaver-Burk double reciprocal mapping method was used to judge the inhibitory effect type and inhibitory constant of diglucosyl gallic acid. As the mass concentration of diglucosyl gallic acid increases, the vertical intercept of all straight lines is 2.645. Moreover, the slope of the straight line （Michaelis constant K_m） also shows an increasing trend, while V_max remains basically unchanged, which can indicate that the type of inhibitory effect of diglucosyl gallic acid on tyrosine diphenolase is competitive inhibition. Gallic acid has a better scavenging effect on DPPH free radicals than V_C does under the same mass concentration, while diglucosyl gallic acid still has a certain scavenging effect on DPPH free radicals compared to V_C under the same mass concentration.

Key words: diglucosyl gallic acid, inhibition of tyrosinase activity, kinetics, competitive inhibition, antioxidant