Isolation And Purification Of Tea Flavonoids By Hsccc And Their Antioxidant Activity And Inhibition Of Cancer Cells By Tea Flavonoids 3,3 ’-Bisgallic Acid Esters

Polyphenols and their derivatives undergo oxidative condensation to form theaflavins,which are soluble with ethyl acetate and produce orange yellow substances.One of the main components in black tea,theaflavins(TFs),plays a decisive role in the color,aroma,and quality of black tea.At present,the main components studied in theaflavins include theaflavin monogallate(theaflavin-3 ’-gallate and theaflavin-3’-gallate),theaflavin,and theaflavin-3,3 ’-gallate.According to a large amount of research both domestically and internationally,theaflavins have various functional effects such as anti-inflammatory,antioxidant,antibacterial,hypoglycemic,anticancer et al.We extracted,separationed,and purified theaflavins from black tea powder to study the antioxidant effect of theaflavin and the inhibitory effect of theaflavin-3,3 ’-gallate on cancer cell proliferated.The main research content is as follows:(1)The optimal process for separating and preparing theaflavins was determined through the use of three factors and three levels orthogonal experimental analysis.Firstly,the hot water extraction method was used to remove excess impurities from theaflavins.Based on the analysis of orthogonal data,the material liquid ratio was 1:30,the extraction temperature was 70 ℃,and the water bath time was 80 minutes.After optimization,the purity of theaflavins proposed by the optimized process conditions reached 30.56%.(2)This article discusses the separation coefficient K and the degree of separation α Explore two aspects and choose different separation systems.By using high-performance liquid chromatography(HPLC)to detect the upper and lower phases of each separation system,calculate and compare the separation coefficient K and separation degree α,The K and V values of the methanol ethyl acetate n-hexane water acetic acid(1:5:1:5:0.25,V/V)system can be determined α The numerical value is relatively good.And continuous chromatography separation technology is used to further separate theaflavins and catechins,aiming to improve the yield and purity of theaflavins.The purity of TF increased from 23.11%to 32.14%,while the purity of TFDG also increased from 89.68% to95.38%.(3)Studying the scavenging effects of ABTS+,DPPH ·,· OH on free radicals,experimental studies have shown that theaflavins have strong antioxidant effects,and their antioxidant effects are dosedependent and enhanced.And tyrosinase is a key rate limiting enzyme in the process of melanin accumulation.Analyzing the binding effect between theaflavin and tyrosinase,exploring the binding mode between TF,TF-3-G,TF-3-G,TFDG and tyrosinase,it was found that each monomer of TF can inhibit the corresponding activity by occupying the action site of tyrosinase activity center.It can be concluded that theaflavin can be used as a tyrosinase inhibitor to research and develop corresponding antioxidant substances.(4)Through preliminary experiments,it was found that theaflavin-3,3’-gallate ester has the strongest inhibitory effect on cancer cell proliferation.Therefore,theaflavin-3,3’-gallate ester was chosen to study its inhibitory effect on cancer cells.The CCK-8 method was used to detect its effect on liver cancer cells(Hep G-2)and colon adenocarcinoma cells(Caco-2).It was found that theaflavin-3,3’-gallate ester significantly slowed down the proliferation rate of Hep G-2 and Caco-2 cells with increasing concentration and prolonging time,And it is time-dependent and dose-dependent.There has been research confirming a certain relationship between cancer prevention and quinone reductase(QR).Combining experimental data and theory,molecular docking technology was used to verify that theaflavin-3,3’-gallate ester and QR can form a stable complex,induce QR activity,and enhance the anticancer effect,providing a basis for further research on the effect of theaflavin on cancer cells.