Purification Of P-coumaric Acid Ethyl Ester And Caffeine By Camellia Pollen And Inhibitory Mechanism Of Tyrosinase And Aldose Reductase

Ethyl acetate phase of ethanol extract from Camellia pollen was gradiently eluted by macroporous adsorption resin,and 30%ethanol elution was obtained.After hydrolysates,it was separated by HSCCC and prep-HPLC,and identification by NMR,HPLC-MS and HPLC showed that these components were p-coumaric acid ethyl ester(p-CAEE)and caffeine,respectively.These two compounds were isolated from Camellia pollen for the first time.The molecular mechanisms of p-CAEE and caffeine on tyrosinase were elucidated by means of enzyme kinetics,UV-vis spectroscopy,circular dichroism spectroscopy,fluorescent spectroscopy and molecular dynamics simulation.The inhibitory kinetics suggested that p-CAEE and caffeine possessed inhibitory capacities on tyrosinase.The IC50 values were 4.89)μg/mL and 18.48 μg/mL,respectively,and the activities of p-CAEE and caffeine were significantly stronger than that of arbutin(IC50=51.54μg/mL).p-CAEE and caffeine were the non-competitive inhibitors of tyrosinase and the values of inhibition constants were 1.83 μg/mL and 14.57 μg/mL,respectively.UV-vis spectroscopy suggested that p-CAEE and caffeine no interaction bind with copper ions in the active center of the tyrosinase.But p-CAEE and caffeine could both change the secondary conformation of tyrosinase and quench fluorescence intensity.Molecular dynamics simulation showed that p-CAEE and caffeine could both change the binding sites of L-tyrosine and the loop conformation adjacent to the active center.The molecular mechanisms ofp.-CAEE and on aldose reductase were elucidated by means of enzyme kinetics,circular dichroism spectroscopy,fluorescent spectroscopy and molecular dynamics simulation.The inhibitory kinetics suggested that p-CAEE and caffeine possessed inhibitory capacities on aldose reductase.The IC50 values were 0.37 μg/mL and 16.20 μg/mL,respectively,and the activity of p-CAEE was far stronger than that of quercetin(IC50=0.64μg/mL).p-CAEE and caffeine were the competitive and non-competitive inhibitors of aldose reductase and the values of inhibition constants were 0.18 μg/mL and 7.19 μg/mL,respectively.pCAEE and caffeine could both change the secondary conformation of aldose reductase and quench fluorescence intensity.Molecular docking result showed that the inhibition mechanism of p-CAEE and ALR2 may be that p-CAEE changes the stereo-specific blockade effects of p-CAEE on substrates or products and flexible conformation alterations in the ALR2 active center caused by weak interactions between ALR2 and p-CAEE.Caffeine played a competitive inhibitory role by occupying the enzyme activity center,resulting in the substrate DL-glyceraldehyde can not enter the active pocket and the binding enzyme ability decreases.In this paper,two components,p-coumaric acid ethyl ester and caffeine,were obtained from Camellia pollen.It was found that these two compounds had inhibitory effects on tyrosinase and aldose reductase,which provided a basis for further study on the biological activity of Camellia pollen.