Using Novel Micro Complex To Enhance The Stability And Anti-inflammatory Property Of Tea Bioactive EGCG

EGCG is the most abundant and bioactive catechin compound in tea leaf,which has been associated with many pharmacological functions,such as antioxidant and anti-inflammatory effects.However,EGCG is unstable due to occurrence of oxidation,polymerization,isomerization and hydrolysis reactions,and is severely degraded under the neutral or alkalescent condition of intestinal tract resulting in its low bioavailability.Our previous work indicates rice bran protein isolate is a premium carrier for EGCG,increasing the stability of EGCG during gastrointestinal digestion,and albumin is the important protein component in rice bran which interacts with EGCG.Albumin has been used in the delivery systems for bioactive polyphenols or drugs.Based on the previous study,the present study prepared EGCG-rice bran albumin isolate(RBAI)complex through self-assembly,using laboratory-made RBAI.The formula of EGCG-RBAI complex was optimized for cell culture study based on Oxygen-radical absorbance capacity(ORAC)values of samples.The inflammatory model of HT-29 cell was established by the induction of IL-1?.The gene expressions of inflammatory biomarker genes in the IL-1?induced cells with and without pretreatment of RBAI,EGCG and EGCG-RBAl complex were quantified,and the concentrations of IL-8,PEG2 and MDA as well as the activity of SOD were also measured.The major conclusions were as follows:(1)The protein content of RBAI was 300 mg/g.SDS-PAGE result indicated that albumin was the main soluble protein component of RBAI.EGCG-RBAI complex was prepared through self-assembly,and the impacts of RBAI incorporation on the stabilities of EGCG in pH 7.4 PBS and digestive fluids were investigated.After incubation in pH 7.4 PBS for 60 min,the EGCG recovery rate of EGCG-RBAI complex system was 15.9%,which was significantly higher than 7.7%of control,with the same initial EGCG amout of 18.00 jpg.After gastrointestinal digestion(2 h gastric digestion and 2 h interstitinal digestion),the recovery rate of EGCG in EGCG-RBAI complex system was 18.9%,which was much higher than 7.6%of control,with the same initial EGCG amout of 45.00 pg.These suggest that RBAI incorporation can elevate the stability of EGCG at alkalescent condition as well as bioaccessibility.(2)MTT assay was used to study the cytotoxicities of EGCG,RBAI and EGCG-RBAI complex to HT-29 cell.The results showed that the IC50 of EGCG and RBAI were 204?mol/L and 511 mg/L respectively,while the tested EGCG-RBAI complexes(20-100 p.mol/L EGCG,50 mg/L RBAI)had no obvious apoptosis effect on HT-29 cells.The inflammatory model of HT-29 cells was established:cell density 2×105/mL,induction with 35 ng/mL IL-1? in full cell culture medium for 24 h.The fold changes in mRNA expressions of important inflammation-related biomarkers NF-?B,CXCL8 and COX-2 were 3.8,5.1 and 1.1 for IL-1? group,compared with CK group,respectively.Pretreatments with EGCG(20-100?mol/L)and RBAI(50,100 mg/L)for 2 h exerted differential inhibition effects on the IL-1? induced expressions of NF-?B,CXCL8 and COX-2.(3)The optimum formula of EGCG-RBAI complex for cell study was:40 umol/L EGCG +50 mg/L RBAI,based on ORAC values.The anti-inflammatory effect of EGCG-RBAI complex was studied by using qPCR and enzyme-linked immunosorbent assays.The results showed that the expressions of MAPK14,NF-?B and STAT3 in IL-1? group were significantly up-regulated with the fold changes of 1.2,3.7,and 3.0 compared with control group,and the downstream genes CXCL8,TNFA and iNOS for NF-?B signal pathway and VEGF for STAT3 signal pathway were up-regulated accordingly.The pretreatment groups of EGCG,RBAI and EGCG-RBAI complex greatly down-regulated the expressions of inflammation-related biomarkers.Specifically,RBAI and EGCG-RBAI complex groups had higher inhibitory effect on the expressions of STAT3 than that of EGCG group.IL-1? group had the highest level of IL-8(191.8 ng/L)while CK group had the lowest level of IL-8(148.3 ng/L).There was no significant difference in IL-8 level between EGCG group(82.3 ng/L)and EGCG-RBAI group(109.3 ng/L),which were lower than IL-1? group.(4)The response to oxidative stress was evaluated by determination of Nrf2 gene expression,SOD activity and MDA level.The results showed that the fold changes in mRNA expressions of Nrf2 for the pretreatment groups of EGCG,RBAI and EGCG-RBAI complex were 2.0,1.6 and 1.4 compared with control group,which were significantly lower:than that of IL-1? group(3.3).IL-1? group had the highest activity of SOD at 15.25 U/mg protein,but no significant difference in SOD activity was observed among three pretreatment groups(13.86?14.04 U/mg protein).Thus,in our study the up-regulation of Nrf2 transcritption and SOD activity was a feedback to the altered oxidative stress of cells.