Continued Tyrosinase Inhibition With The Chemical Composition Of The Seeds Of Resistance And Oxidation Resistance

Euphorbia lathyris was one of the plants of genus Euphorbium. We learned from the ancient Chinese medical books, "Qian Jin Yi Fang", "Wai Tai Mi Yao" and "Pu Ji Fang", that the seeds of E. lathyris were applied as cosmetics for the treatment of hyperpigmentation, such as melasma and ephelides. Modern studieds indicated that excessive production of melanin is the main reason of these skin diseases and tyrosinase is the key enzyme in biosynthesis of melanin. Based on the ancient records and modern research about component analysis of the seeds, the tyrosinase inhibitory substances from the seeds were selected through tyrosinase inhibitory experiment step by step and colorimetry was used to explore the types and mechanisms of inhibition of tyrosinase by esculetin, which was purified from the ethyl acetate phase of the seeds. At last it researched the antioxidant activity of the seeds. The specific experiments are as follows:1 Tyrosinase inhibitory experiments were carried out to compare the size of tyrosinase inhibition of ethyl acetate, butanol and water phase. The results showed that the half-inhibitory concentration (IC50) of ethyl acetate, butanol, water phase, and the positive control arbutin were 0.150 mg/mL,0.813 mg/mL,7.570 mg/mL, and 1.600 mg/mL, respectively. High-speed counter-current chromatography (HSCCC) was used to isolate and purify the potential tyrosiase inhibitor from the ethyl acetate phase. The experiment was performed with a two-phase solvent system composed of chloroform-methanol-water (v/v/v,4:3:2). Tyrosinase inhibitory experiments were conducted to test the existence of tyrosinase inhibition. As aresult, the peak I-V showed a certain extent tyrosinase inhibition activity. Then, the purity of products was tested through High Performance Liquid Chromatographic (HPLC). The results indicated that the purity of peak V was 99.04%. Based on the combined results of MS and 1H-NMR, peak V was identified as esculetin combined with data reported in the literature.2 L-tyrosine and L-DOPA were used as substrate to explore the monophenolase and diphenolase inhibition of esculetin. The results indicated that the IC50 value of esculetin for monophenolase was 0.131 mg/mL. Esculetin lengthened the lag time and decreased the steady-state activity of monophenolase. The lag time changed from 91 seconds in its absence to 385 seconds in the presence of 0.3 mg/mL esculetin. The IC50 value of esculetin for diphenolase was 0.229 mg/mL. The kinetic analysis showed that the inhibition of esculetin on the diphenolase activity of the enzyme was reversible and belonged to competitive type, and the inhibition constants (Ki) were determined to be 0.398 mmol/L.3 The scavenging activity of crude extracts (ethyl acetate and butanol phase) and esculetin was studied by three different free radical systems such as 1,1-diphenly-2-picrly-hydrazyl free radical(DPPH·), superoxide anion free radical(O2-) and hydroxide free radical(OH·), and IC50 (the value of antioxidant concentration for scavenging half of free radical) was used as the index to evaluate scavenging capacity. The results demonstrated that both crude extracts and esculetin had scavenging capacity for superoxide anion free radical, which were weaker than that of the positive control VC. The IC50 were as follows:0.62 mg/mL,2.78 mg/mL, and 0.77 mg/mL,0.48 mg/mL. Ethyl acetate phase and esculetin had stronger scavenging capacity for DPPH free radical than that of VC, except the butanol phase. The IC50 were 0.025 mg/mL,0.112 mg/mL,0.0116 mg/mL, and 0.046 mg/mL, respectively. Ethyl acetate phase and esculetin did not exhibit scavenging capacity for hydroxyl free radical at the experimental concentration range. On the contrary, they showed different degrees of promoting the formation of hydroxyl free radical, and with the increase of the concentration, the pro-oxidant effects became more apparent. The butanol phase and VC revealed certain scavenging capacity for hydroxyl free radical. The IC50 were as follows:2.453 mg/mL,0.388 mg/mL, and it indicated that the capacity of butanol phase was weaker than that of VC.