Effect Of Active Components Of Chinese Herbs On Cytochrome P450 Enzymes Gene Expression

Cytochrome P450 superfamily is the most important drug metabolisming enzymes in human. They are monooxygenases major presented in the liver and intestine, and most on the the endoplasmic reticulum in cells,catalyzing by a variety of endogenous compounds. Cytochrome P450 enzymes have many subfamilies, CYP1A1, CYP2E1 and CYP3A is the most important enzymes can be induced. CYP1A1, CYP2E1 metabolically activates a large number of procarcinogens to reactive intermedites that can interact with cellular nucleophiles and can ultimately trigger carcinogenesis, CYP3A metabolism 60% clinical commonly used drugs, so they are very important in drug metabolism / toxicology research.Nowadays, with the promotion of Chinese herbal medicine around the world, with the great development of combination of Chinese and Western medicine, it has accelerated combination use of the traditional Chinese medicine and western medicine to treat disease. More and more clinical and pharmacological experiments show that many herbs have an impact on the CYPs. The metabolism of Chinese herbal medicines is like synthetic medicine mostly through CYPs metabolism. Although Chinese herbal medicine have very complex ingredients, its monomers or active ingredients in the body metabolism are the same as western medicine through CYPs metabolism,and may have an impact on those enzymes. Therefore, the research on the Chinese herbal medicines'impacts on the CYPs and herb-drug Interactions are undoubtedly very important to promote the rational drug use, avoid adverse reactions of combination using drugs as far as possible and ensure the safety and efficacy of clinical medication. Flavonoids are one of the principal groups of phytoestrogens and synthesized by all vascular plants. They are present throughout our daily diet in edible vegetables, fruits, nuts, herbs and plant-derived beverages, such as juice, tea coffee, and red wine. They have a variety of pharmacological function, including the role of anti-infective, anti-allergic, antibacterial, antiviral, anti-diabetic, anti-osteoporosis, anti-thrombosis, anti-carcinogenic substances, inhibiting proliferation, protection of the liver, cardiovascular protection. In vivo, in vitro and epidemiological investigation and clinical trials have shown that plant-derived flavonoids play an important role for tumor prevention and treatment, and the mechanism involving in the various stages of tumor development.By using HepG2 cells in vitro system and fluorescent dye SYBR GREEN,in this study we quantitate mRNA expression in the HepG2 cell by real-time quantitative PCR. For many genes, including most of the commonly used internal reference"housekeeper"genes, such as GAPDH gene, there exist genomic DNA sequences termed"processed pseudogenes". Processed pseudogenes are sequences present in the genome that are identical or nearly identical to the mRNA sequences. So we designed an mRNA-specific reverse transcription primer named universal RT, and one pair of PCR primers relation to reverse transcription primer universal RT, named SP and AP. In real-time PCR those primers rule out the genome DNA contamination interference mRNA quantitation and rule out the false positive results.In this research we study the effect of the active components of eight Chinese herbs (baicalin, baicalein, sodium aescine, ligustrazine hydrochloride, artemisinin, gastrodine, caffeine, salicyl pseudoephedrine) on cytochrome P450 enzymes (CYP) 1A1, 2E1, 3A4 and 3A5 mRNA expression. The mRNA expression levels of four CYP isoenzymes were determined by quantitative reverse-transcriptase polymerase chain reaction. baicalin, baicalein and artemisinin induce CYP1A1 expression at different concentrations. Compared with baicalin and baicalein, artemisinin was weaker inducing agent. The expression of CYP3A4 gene was significantly induced by sodium aescinate, baicalein and artemisinin. HepG2 cells should be an appropriate in vitro system for investigating potential human CYP inducing agents. CYP1A1 and CYP3A4 expression could be significantly induced by baicalin, baicalein, artemisinin and sodium aescinate, which would give the evidence for herb–drug interactions based on cytochrome P450 and toxicology.In this research we study the effects of fifteen flavonoids on CYP1A1 mRNA expressions and on DMBA-induced CYP1A1 mRNA expressions. The fifteen flavonoids are baicalin, baicalein, flavone, 6-hydroxyfalvone, 7-hydroxyfalvone, scutellarin, scutellarein, chrysin, luteolin, apigenin, morin, fisetin, quercetin, wogonin and kaempferol. Among the fifteen flavonoids, the effects of 6-hydroxyfalvone, morin, baicalin, scutellarein, scutellarin and wogonin had not been reported. The fifteen flavonoids varied largely on inducing CYP1A1 mRNA expressions. CYP1A1 mRNA expressions were significantly induced by flavone, quercetin, baicalein, scutellarein and chrysin. In the concentrations of 50uM, they induced CYP1A1 mRNA expressions more than 10 folds different from control DMSO. CYP1A1 mRNA was inhibited by 7-hydroxyfalvone, fisetin, apigenin, luteolin and kaempferol in different levels. Most of the fifteen flavonoids to some extent inhibited the DMBA-induced CYP1A1 mRNA expressions. Among them, DMBA-induced CYP1A1 mRNA expressions were significantly inhibited by flavone, quercetin, baicalein, scutellarein and chrysin.According to the 15 flavonoids studied, if the ring A with three hydroxyls or with two hydroxyls and an another group, or if the ring B has two hydroxyls especially with catechol group,or if AC-ring with at least three hydroxyls, this kind of flavonoids may have a very important role on inducing CYP1A1 mRNA expression. The more hydroxyls have on flavonoids,the greater possibility of that flavonoids inducing CYP1A1 mRNA expression. If AC-ring with less than three hydroxyls or the ring B with only one hydroxyl, it is very possible that this kind of flavonoids inhibit CYP1A1 mRNA expressions.In low concentrations, flavonols tend to inhibit DMBA-induced CYP1A1 mRNA expression, or in high concentrations the flavonoids in AC-ring with the 5 position and 7 position hydroxyls can strongly inhibit DMBA-induced CYP1A1 mRNA expressions, A ring or two of the hydroxyl groups and is replaced by a non-hydroxy group of flavonoids than all the hydroxyl group of flavonoids, more inclined to inhibit DMBA-induced CYP1A1 mRNA expression. The flavonoids on the ring A with two hydroxyls and with a non-hydroxy substituent group compared to the flavonoids with three hydroxyls,the former flavonoids are more inclined to inhibit DMBA-induced CYP1A1 mRNA expression.