The Inflammatory Inhibition Of Theaflavin Prepared By MPLC In VSMC

Theaflavins are the important compounds which not only contribute to black tea taste,but also provide the health effects after drinking.It have been shown that theaflavins are excellent natural phytochemicals with many biological activities including anti-oxidation,free radical scavenging,anti-bacteria,anti-allergy,preventing atherosclerosis,and anti-radiation.However,it's a little hard to prepare theaflavin monomer from black teas,which has impeded the development of theaflavin pharmacological researches.Therefore,it is necessary to explore a simple,efficient and low-cost method to prepare theaflavins monomer and then to find out the novel theaflavins biological activities.In this paper,tea pigments with high theaflavin content were prepared using tea polyphenols as raw materials in in vitro oxidation system based on previous studies;and then MPLC was applied to purify three major theaflavin monomers.Moreover,the effects of three theaflavin monomers on inflammation inhibiton were studied in rat vascular smooth muscle cells induced by Ang II.The main results achieved in this paper are as follows:1.Tea pigments with high theaflavin content were prepared using tea polyphenols as raw materials and crude enzymes from fresh tea leaves in in vitro two-phase liquid oxidation system.The content of theaflavins is up to 49.9%.2.A MPLC system with C18 silica columns has also been applied based on previous studies and successful to separate theaflavins from the tea pigments.After 280 min separation,three kinds of theaflavins as theaflavin(TF1),theaflavin-3-gallate(TF2a),and theaflavin digallate(TF3)were prepared.Their purity is up to 94.6%,97.1%,and 95.6%,respectively.The total yield and recovery of three theaflavins are up to 34.0% and 66.8%,respectively.3.The effects of EGCG and theaflavins on the pro-inflammatory cytokine m RNA expression was detected by real-time fluorescence quantitative PCR in Ang II-induced VSMC inflammation model.Compared with the Ang II model group,the effects of 40?M EGCG,40?M and 100?M TF1 and 100?M TF3 on IL-6 m RNA,expression was decreased by 44.3%,22.7%,51.4%,and 38.4%respectively.It's suggested that TF1 and TF3 has similar inhibitory effects as EGCG on IL-6 expression in VSMC inflammation.4.Our results showed that EGCG?TF1?TF3 significantly inhibited ROS generation by Ang II in VSMCs.5.The differential expression of IKK,p38/MAPK,TLR4 proteins by western blotting was further detected to explore the anti-inflammatory mechanism of theaflavins on VSMC inflammation model induced by Ang II.Compared with the Ang II model group,the effects of 40?M EGCG,100?M TF1 and 40?M and100?M TF3 on IKK phosphorylation was decreased by 44.4%,22.5%,51.4%,and 38.2% respectively.Moreover,40?M EGCG and 100?M TF3 significantly inhibited Ang II induced TLR4 protein expression;However,the phosphorylation of p38/MAPK have no significant difference compared with the Ang II model group.Taken together,those data suggest that theaflavins and EGCG could inhibit NF-?B activity throuth down-regulate the phosphorylation of IKK,not p38/MAPK signaling and also decrease the endocellular ROS to exert the anti-inflammatory effects induced by Ang II in VSMC.