Preparation Of Tea Polyphenol Phytosteryl Esters And Investigation Of Their Antioxidant And Cholesterol-lowering Activities

Antioxidation of lipids,especially for unsaturated lipids,play an important task in food science.However,due to the potential risks of synthetic antioxidants,developing a highly efficient and multifunctional antioxidant from natural products has received much attention in current food research.Tea polyphenols,regarded as a kind of characteristic resource in China,are excellent natural antioxidants.However,the poor lipid solubility of tea polyphenols limits their antioxidant performance in lipids.Meanwhile,phytosterols,by-products from the processing of vegetable oil,have attracted much attention for its cholesterol-lowering capacity.Therefore,this study applied phytosterols as a hydrophobic portion to modify tea polyphenols for improving their physicochemical propertiesand integrating their bioactive activity.In this study,the coupling conditions of phytosterol with gallic acid were first explored and optimized.The synthetic produce was termed as galloyl stigmasterol(GSt)and galloylβ-sitosterol(GSi).Then,epigallocatechinβ-sitosterol(ESi)and epigallocatechin stigmasterol(ESt)were prepared under the coupling of epigallocatechin and phytosterols which employed the bridging strategy of succinic anhydride.Structure identification and purity analysis of galloyl stigmasterol,galloylβ-sitosterol,epigallocatechinβ-sitosterol and epigallocatechin stigmasterol were performed by nuclear magnetic resonance(NMR),fourier transform infrared spectoscopy(FT-IR),mass spectrometry(MS)and HPLC-MS.Their antioxidant and cholesterol-lowering activities were further determined.The combined antioxidant activities of galloyl stigmasterol,galloylβ-sitosterol,epigallocatechinβ-sitosterol,epigallocatechin stigmasterol or(+)-catechin-β-sitosterol with three commonly used lipid-soluble antioxidants were evaluated byβ-carotene-AAPH bleaching assay.The major results were addressed as follows.(1)Galloyl stigmasterol and galloylβ-sitosterol were prepared by employing aprotecting and deprotecting strategy on the phenolic hydroxyl groups of gallic acid.It was found that the by-product in the reaction process were produced from the intermediates which generated from the catalytic process of DMAP and phytosterols.The identity of by-product was consistent with phytosterol succinate determined by MS.As a result,the by-product was acoupling product which generated form phytosterols with protection groups stripping off from phenolic hydroxyls of gallic acid.Therefore,the by-product was successfully reduced by optimization the amount of DMAP,reaction solvent and protection groups.The final protocol of the reaction was recorded as follows:isobutyric anhydride was used to protect gallic acid for generating triisobutyric gallic acid,which was coupled with phytosterols(phytosterol orβ-sitosterol),and finally deprotection was performed by hydrazine to afford the target product.(2)The oxidation stability evaluation by the Rancimat method suggested galloyl stigmasterol and galloylβ-sitosterol had good antioxidant activity in the fat-soluble system,and there was no significant difference regarding antioxidant activity.Galloyl stigmasterol and galloylβ-sitosterol exhibited significant cholesterol-lowering activity in vitro.Compared withβ-sitosterol,galloylβ-sitosterol had significantly higher cholesterol-lowering activity,while galloyl stigmasterol had no significant difference in cholesterol-lowering activity with stigmasterol.(3)Epigallocatechinβ-sitosterol and epigallocatechin stigmasterol were prepared by employing the bridging strategy of succinic anhydride and Steglich esterification,and the products exhibited excellent ABTS·~+and DPPH radical scavenging activities.The antioxidant activities were notably higher than TBHQ inβ-carotene-linoleic acid bleaching assay andβ-carotene-AAPH bleaching assay.They also had excellent cholesterol lowering activity in vitro.(4)The combined antioxidant activity of galloyl stigmasterol,galloylβ-sitosterol,epigallocatechinβ-sitosterol,epigallocatechin stigmasterol or(+)-catechin-β-sitosterol with BHA,BHT,TBHQ,three commonly used lipid-soluble antioxidants,were evaluated inβ-carotene-AAPH bleaching assay The results revealed that galloyl stigmasterol had synergistic antioxidant effects with BHA,BHT or TBHQ,and galloylβ-sitosterol,epigallocatechinβ-sitosterol,and(+)-catechin-β-sitosterol had synergistic antioxidant effects with BHA.