Effects Of Muleberry Leaves On Activity And MRNA Expression Of Cytochrome P450 Enzymes In Rats

Mulberry leaves have the functions of dispersing wind and heat,clearing the liver and brightening the eyes,and suppressing liver,especially after frost.At present,mulberry leaves are widely used in the market and have broad prospects.In recent years,with the reports that the interaction between traditional Chinese medicine and chemical drugs leads to the decrease of curative effect and even the increase of toxic and side effects,it has attracted widespread attention in society.This study was mainly to investigate the effects of frost mulberry leaves on the expression of metabolic enzymes and m RNA in rats,and to provide a theoretical basis for its clinical use.Objective: The aim of this study is to investigate the effects of frost mulberry leaves on the metabolic enzymes of CYP2c11,CYP2d2,CYP1a2,CYP3a1,CYP2c6,CYP2b1 and CYP2c7 in rats from the level of enzyme activity and expression of m RNA,so as to provide reference for the combined application of frost mulberry leaves with other drugs.Method: Firstly,the contents of rutin,isoquercitrin,astragalin,chlorogenic acid and alkaloid 1-deoxynojirimycin(DNJ)in the extracts of frost mulberry leaves were determined.Secondly,the UPLC-MS/MS method is established to determine seven main metabolic enzymes in rats.48 SD male rats are randomly divided into 8 groups: low,medium and high dose group of aqueous extract of mulberry leaves(1,2,4 g/kg),saline group,low,medium and high dose group of ethanol extract from mulberry leaves,0.5% CMC-Na group.The rats in the dose group(1,2,4 g/kg)are given intragastric perfusion for 2 weeks.After the last day of administration,the rats in each group are injected with the mixed solution of probes and drugs respectively through tail vein.Blood samples are collected from the ophthalmic venous plexus at the preset time point.The plasma drug concentrations of seven probes in different groups are determined by UPLC-MS/MS method.The main pharmacokinetic parameters of probes in each group calculated by DAS 2.0 software and analyzed.The effects of different extracts from frost mulberry leaves on CYP450 activity in rats are investigated by statistical analysis.At the same time,48 SD male rats were randomly divided into 8 groups on average.After 2 weeks of continuous gastric administration,the rats are killed by dislocation of cervical vertebra on the last day.The liver tissues are taken and the relative expression levels of major cytochrome P450 enzyme subtypes in each group are determined by real-time fluorescence quantitative PCR,and the extracts from frost mulberry leaves are analyzed in rats.Whether the gene expression level of the seven CYP450 enzymes in vivo is affected.Results: The content of DNJ in the aqueous extract from mulberry leaves was 1.63 mg/m L,and the four main components of flavonoids in ethanol extract from mulberry leaves were rutin,isoquercitrin,astragalin,and chlorogenic acid.The contents were 1.16 ?g/m L,2.12 ?g/ml,1.25 ?g/m L,and 3.18 ?g/m L,respectively.Established a UPLC-MS/MS analysis method for the determination of seven probing substrates in rats: phenacetin,midazolam,amphetazone,diclofenac sodium,omeprazole,amodiquine and dextromethorphan is feasible after methodological investigation.The activity of cytochrome P450 and the expression of m RNA in rats are identical.The results shows that:(1)Aqueous extracts from mulberry leaves at medium and high doses can significantly inhibit the enzyme activities of CYP2c7,CYP2b1 and CYP2d2 and down-regulated the m RNA expression level,while low doses have no significant effect.The alcohol extracts from mulberry leaves can significantly inhibit the enzyme activities of CYP2b1 and CYP2d2 and down-regulated the m RNA expression level,but have no significant effect on the enzyme activities and m RNA expression level of CYP2c7,(2)Aqueous extract and alcohol extract from mulberry leaves significantly inhibited the enzyme activity of CYP2c6,and down-regulated its m RNA expression,and significantly increased the enzyme activity of CYP3a1 and up-regulated its m RNA expression,(3)the middle and high doses of the alcohol extracts from mulberry leaves can inhibit the enzyme activities of CYP1a2 and CYP2c11 and down-regulate their m RNA expression levels,while the aqueous extracts from the leaves of mulberry leaves can significantly inhibit the enzyme activities of CYP1a2 and down-regulate the m RNA expression levels,while the low doses of alcohol extracts and the aqueous extracts from the leaves of mulberry leaves have no significant influence on the enzyme activities and m RNA expression levels of CYP2c11.Conclusion: Different extracts of frost mulberry leaves have different effects on the activity of CYP450 enzyme and the expression of m RNA in rats,and they are related to the dosage and group.This suggests that the potential interaction should be fully considered when the drugs of frost mulberry leaves related enzyme metabolism are used in combination.At the same time,enough attention should be paid to the application of frost mulberry leaves in food.