| In this study,we used three different fermentation stages teas(Huangshan Maofeng,DaHong Pao and Keemun black tea)as raw materials,the tea polyphenol were extracted by methanol.The qualitative and quantitative analysis of the major catechins in the three kinds of tea polyphenol extracts was performed by HPLC.The obtained tea polyphenol extracts were given to C57BL/6J mouses 7 days respectively,Real-time reverse transcription polymerase chain reaction(real-time PCR)and Western blot were used to investigate the dose-effect relationship between the mRNA and protein expression of Cyp450s(Cyp3a11,Cyp2e1,Cyp2c37,Cyp1a2)and PXR in mouse liver;C57BL/6J mouses were inoculated with the three kinds of tea polyphenol extracts at the appropriate dose respectively 7,14,28 days,and the time-effect relationship between the mRNA and protein expression of Cyp3a11,Cyp2e1,Cyp2c37,Cyp1a2 and PXR in mouse liver were investigated;Polyphenol extract affected on CYP450 through human pregnane X receptor transfected into HepG2 cells and luciferasea ctivities were measured by ransient co-transfection reporter gene assays.To provide a theoretical basis for the mechanism of tea polyphenol extracts on the regulation of drug metabolizing enzyme of liver.The main conclusions are as follows:(1)The components and contents of Huangshan Maofeng,DaHong Pao and Keemun black tea polyphenol extracts were detected by Folin-Ciocalteu and HPLC.The results showed that: The contents of total phenolics in Huangshan Maofeng,DaHong Pao and Keemun black tea were 434.11±8.76、401.94±2.90、282.19±6.47 mg GAE/g DW,respectively.In Huangshan Maofeng polyphenol extracts,they were mainly contain-ing epigallocatechin gallate(EGCG),epicatechingallate(ECG),catechin(C),epigallia(EGC),respectively 265.57,133.05,69.27 μg/mg,64.92 μg/mg;DaHong Pao polyphenol ertracts were with the higher content of EGCG,ECG,EGC and gallic acid(GA),173.36,73.14,23.74,13.92 μg/mg,respectively.Keemun black tea polyphenol ertracts has EGCG,C,gallic acid(GA),82.94,28.55,20.45 μg/mg respectively.(2)The dose-effect relationship of Huangshan Maofeng,DaHong Pao and Keemun black tea polyphenol extracts on the expression of Cyp450s(Cyp3a11,Cyp2e1,Cyp2c37,Cyp1a2)and PXR in mouse liver were investigated.The results showed that: The mRNA expression of Cyp3a11 and Cyp2c37 were significantly induced and the mRNA expression of Cyp2e1,Cyp1a2 were significantly inhibited by Huangshan Maofeng and DaHong Pao polyphenol extracts(P<0.05);the protein expression of Cyp3a11,Cyp2e1,Cyp2c37 and Cyp1a2 was significantly induced(P<0.05).Keemun black tea polyphenol extracts could significantly affected the mRNA and protein expression of Cyp3a11,Cyp2e1,Cyp2c37 and Cyp1a2(P<0.05).Overall,Huangshan Maofeng,DaHong Pao and Keemun black tea polyphenol extracts could significantly induced the mRNA and protein expression of PXR(P<0.05).(3)The time-effect relationship of Huangshan Maofeng,DaHong Pao and Keemun black tea polyphenol extracts on mouse liver Cyp450 s and PXR was discussed.The results showed that: The expression of mRNA and protein of Cyp3a11,Cyp2e1,Cyp2c37 and Cyp1a2 were significantly affected at 7,14 and 28 days after intragastric administration by Huangshan Maofeng polyphenol extracts(P<0.05).DaHong Pao polyphenol extracts significantly induced the mRNA expression of Cyp3a11,Cyp2c37 but inhibited the expression of Cyp2e1,Cyp1a2(P<0.05)after gavaging 7 days,the mRNA expression of Cyp3a11,Cyp2e1,Cyp2c37,Cyp1a2 were significantly induced after 14,28 days(P<0.05);they has a significant effect on the protein expression of Cyp3a11,Cyp2e1,Cyp2c37,Cyp1a2(P<0.05).The Keemun black tea polyphenol extracts has a significant effect on the protein expression of Cyp3a11,Cyp2e1,Cyp2c37 and Cyp1a2 mRNA after gavaging 7,14,28 days(P<0.05)and show a downward trend;they has a significant effect on the protein expression of Cyp3a11,Cyp2e1,Cyp2c37(P<0.05)but has no effect on the Cyp1a2 protein,all of which show a downward trend.Huangshan Maofeng,DaHong Pao and Keemun black tea polyphenol extracts could significantly affected the mRNA and protein expression of PXR after gavaging 7,14,28 days(P<0.05).(4)The polyphenol extract 100,200 and 300 μg/mL could significantly enhanced luciferase activity in CYP3A4 gene,indicating that tea polyphenol extract may act as a PXR ligand to activate CYP3A4 DNA response elements and thereby upregulate CYP3A4 expression.In conclusion,Huangshan Maofeng,DaHong Pao and Keemun black tea polyphenol extracts could significantly affected the mRNA and protein expression of CYP450 s,and thus may influence the metabolism of other substrates on CYP450 s,which may be related to the activation of PXR. |
