| Objective:Wuzhou Liupao tea is a kind of black tea,which is a famous historical tea in China.It belongs to the family Theaceae [Camellia sinensis(L.)].O.Kuntze].It has been found that Liupao tea has the effects of lowering blood fat and improving insulin resistance,but the mechanism is not clear.Tea is "the source of ten thousand medicines".It will be of great industrial significance to scientifically clarify the efficacy mechanism of Liupao tea.Taking Liupao tea as the research object,the chemical basis of Liupao tea was clarified from the aspect of chemical fingerprint,and the common components of Liupao tea extract and its fingerprint were clarified in vitro:epicatechin gallate(ECG),rutin(Rutin),epigallocatechin gallate(EGCG),ellagic acid(EA),caffeine(CAF)and gallic acid(GA).The effect of these components on the proliferation and differentiation of 3T3-L1 preadipocytes and the mechanism of improving insulin resistance of 3T3-L1 adipocytes provide a theoretical basis for the functional study of Liupao tea.Methods:(1)Establishment of HPLC fingerprint of Wuzhou Liupao tea.The characteristic fingerprints of 11 batches of Wuzhou Liupao tea samples were established by HPLC-DAD.The chromatographic column was C18-S,methanol was the mobile phase A,0.2%(v / w)phosphoric acid water was the mobile phase B.The quality of Liupao tea from different manufacturers was evaluated with GA,EGCG,CAF,ECG,Rutin and EA as quality control indexes,so as to provide a basis for further elucidating the chemical basis of Liupao tea and the quality evaluation of Liupao tea.(2)Effects of Wuzhou Liupao tea on Proliferation and differentiation of3T3-L1 preadipocytes.3T3-L1 preadipocytes were cultured routinely in vitro in control group(CG),solvent control group(1 ‰ Dimethyl sulfoxide,DMSO),rosiglitazone positive control group(ROS,10 μmol/L),Liupao tea water extract intervention group and GA,EGCG,CAF,ECG,Rutin and EA intervention groups.3T3-L1 preadipocytes were treated with each intervention group for 24 h and 48 h.CCK-8 method was used to detect its effect on the proliferation of 3T3-L1 preadipocytes.Dexamethasone(Dex),insulin(Ins)and 3-isobutyl-1-methylxanthine(IBMX)were used to induce cell differentiation.After 11 days of induction,the effects of each intervention group on cell differentiation were observed by oil red O staining.The m RNA expressions of interleukin 6(IL-6),peroxisome activated receptor γ(PPARγ),fatty acid synthase(FAS),CCAAT enhancer binding protein α(C/EBPα)and tumor necrosis factor-α(TNF-α)were detected by RT-PCR,and the protein expression of PPARγ was detected by Western-blot immunoblotting.(3)Study on the Mechanism of Wuzhou Liupao tea on Insulin Resistance of 3T3-L1 adipocytes.3T3-L1 adipocytes were induced and matured by in control group(CG)and Model Group(MG).Adipocytes were treated with Dex(1μmol/L)for 24 h,48 h,72 h and 96 h,respectively.IR model was established.With 2-(N-methylidene-7-nitro-2-methylethyl)-3-benzoxadia zole-4-amino)-2-deoxy-D-glucose(2-NBDG)to detect glucose control sumption and determine whether the model was successful or not.After successful modeling,CG,MG,ROS,Liupao tea water extract intervention group and GA,EGCG,CAF,ECG,Rutin,EA intervention group were set up.48 hours after administration,glucose consumption in each intervention group was detected by 2-NBDG,and total m RNA,was extracted from the cells of each intervention group.The m RNA expression of intracellular related cytokines Glucose Transporter 4(GLUT4),Protein Kinase B(Akt),Phosphatidylinositol 3 color kinase(PI3K)and Insulin receptor substrate-1(IRS-1),were detected by RT-PCR to observe the effect and mechanism of each intervention group on insulin resistance.Results:(1)Establishment of HPLC fingerprint of Liupao tea in Wuzhou.The fingerprints of 11 batches of Liupao tea water extract and alcohol extract were established,which were matched to 35 and 38 common peaks respectively,and 6 index components were identified as GA,EGCG,CAF,ECG,Rutin and EA.The results of similarity analysis showed that the similarity between water extract and alcohol extract of 11 batches of Liupao tea samples was more than 0.95,which met the requirements of fingerprint establishment,indicating that the quality of 11 batches of Liupao tea samples was relatively stable.Liupao tea is mainly composed of flavonoids,polyphenols and caffeine.(2)Effects of Liupao tea on the proliferation of 3T3-L1 preadipocytes.Compared with CG,DMSO and ROS had no toxic effect on preadipocytes,and the aqueous extract of Liupao tea had no toxic effect on preadipocytes at the concentration of 50 μg/m L-400 μg/m L,but had obvious inhibitory effect on cells at the concentration of 800 μg/m L,and the inhibitory effect became more obvious with the increase of concentration.According to the results of the content determination,the corresponding concentration of the control substance was converted.In the range of intervention concentration,the pre-adipocyte activity of the six control substance components was more than 80 %.(3)Effect of Liupao tea on differentiation of 3T3-L1 preadipocytes.Compared with CG,ROS can promote the expression of genes related to preadipocyte differentiation,including PPARγ,C/EBP α and FAS,.ROS can not only promote the expression of inflammatory cytokines IL-6 and TNF-α m RNA,but also increase the expression of PPARγ protein,thus promoting the differentiation of 3T3-L1 preadipocytes.The water extract of Liupao tea,Rutin,ECG,EGCG and GA can inhibit the differentiationrelated genes of preadipocytes,including PPARγ,C/EBPα,FAS and the expression of PPARγ protein.At the same time,they can also inhibit the gene expression of inflammatory factors IL-6 and TNF-α,reduce the inflammatory reaction and reduce the intracellular lipid accumulation.CAF and EA can promote cell differentiation by upregulating the m RNA expression of preadipocyte differentiation-related factors: PPARγ,C/EBPα,FAS and the expression of PPARγ protein.At the same time,the m RNA expression of inflammatory cytokines IL-6 and TNF-α was also up-regulated.(4)Effects of Liupao tea on glucose consumption in insulin resistant3T3-L1 adipocytes.Compared with CG,the glucose consumption of MG,ROS and Liupao tea extract and their six common components GA,EGCG,CAF,ECG,Rutin and EA in the intervention group were significantly lower than those in CG,group,which indicated that IR model was successful.Compared with MG,ROS and Liupao tea extract and their six common components GA,EGCG,CAF,ECG,Rutin and EA could promote glucose consumption of IR adipocytes in different concentrations.(5)Effect of Liupao tea on the expression of related genes in 3T3-L1 adipocytes of Insulin Resistance.Compared with CG,MG,ROS and Liupao tea extract and their six common components GA,EGCG,CAF,ECG,Rutin and EA could downregulate the gene expression of IRS-1/PI3K/Akt/GLUT4.Compared with MG,the m RNA expression of IRS-1/PI3K/Akt/GLUT4 could be significantly up-regulated by ROS.The water extract of Liupao tea and its six common components GA,EGCG,CAF,ECG,Rutin and EA upregulated the gene expression of IRS-1/PI3K/Akt/GLUT4 in the concentration range.Conclusion:(1)The common components GA,EGCG,ECG,CAF,EA and Rutin,were found in 11 batches of Liupao tea water extract and alcohol extract,and there were some differences among them.(2)Liupao tea regulates abnormal lipid metabolism mainly by downregulating the expression of lipid metabolism-related genes: PPARγ,C/EBPα and FAS,which inhibits the differentiation of 3T3-L1 cells in the later stage of differentiation.Liupao tea can improve chronic inflammation by down-regulating the expression of IL-6 and TNF-α m RNA,and the decrease of the expression of inflammatory factors can further improve the abnormal cell differentiation.(3)Liupao tea is mainly regulated by activating IRS-1/PI3K/Akt/GLUT4 pathway in improving glucose consumption of insulin resistance model of 3T3-L1 adipocytes. |
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