Comparative Study Of The Effects Of Sweated And Non-sweated Salvia Miltiorrhiza Bge. On The Cytochrome P450 Enzymes In Rats

Objective:Optimizating the extraction process and condition of Salvia Miltiorrhiza Bge.extracts by orthogonal test design combining fingerprint.Establishing a method for the simultaneous determination of seven Cyp450 enzyme substrates in rats by UPLC-MS/MS.Using Cocktail probe drug method and real-time fluorescence quantitative PCR to study the effects of sweated and non-sweated Salvia Miltiorrhiza Bge.on Cyp450 enzyme activity and mRNA expression in rats.In order to provide a certain reference value for the rational selection and rational use of sweated and non-sweated Salvia Miltiorrhiza Bge.in the clinical when it used alone or combimed with other drugs.So that Salvia Miltiorrhiza Bge.in the clinical will be a greater degree of its efficacy,better for the benefit of mankind.Methods:Orthogonal test design with multi-index of the content of monomer component,the yield of powdered extract,the total area of characteristic peaks of HPLC as indexes to optimize the best extraction process of Salvia Miltiorrhiza Bge.extracts（salvia total phenolic acids was water-soluble extracts and tanshinones was ethanol-soluble extracts）.A biological sample analysis method was established to determine the probe substrates（bupropion,omeprazole,phenacetin,dextromethorphan,midazolam,amodiaquine and diclofenac sodium）of seven main metabolic enzymes（Cyp2b1,Cyp2c11,Cyp1a2,Cyp2d2,Cyp3a1,Cyp2c7 and Cyp2c6）in rats by UPLC-MS/MS.The thirty-six SD male rats were randomly divided into six groups:normal saline（NS）,non-sweated salvia total phenolic acids（NSPA）,sweated salvia total phenolic acids（SPA）,non-sweated tanshinones（NSTN）,CMC-Na（CMC）,sweated tanshinones（STN）.The rats were administrated continuously for fourteen days.On the last day,the rats were injected with the mixed solution of the probe drugs at the tail vein.The blood samples were collected and tested by UPLC-MS/MS.The pharmacokinetic parameters were calculated by the software of DAS 2.0.Investigating the effects of sweated and non-sweated Salvia Miltiorrhiza Bge.on the activity of Cyp450 in rats.Eighteen SD male rats randomly divided into six groups.The rats were orally administered for fourteen days and sacrificed on the last day.And rats liver tissue was removed.PCR technology was used to determine the mRNA expression of rat Cyp450 enzymes in rat liver to analyze the effects of sweated and non-sweated Salvia Miltiorrhiza Bge.on the gene expression levels of seven Cyp450 enzymes in rats.Results:The best technology of salvia total phenolic acids as following:Solid-liquid ratio was 1:12 g/mL,the extraction time was 1.5 h,the alcohol content was 70%.The best technology of tanshinones as following:Solid-liquid ratio was 1:8 g/mL,the ethanol concentration was 95%,the extraction time was 0.5 h,three times.The UPLC-MS/MS method for the simultaneous determination of seven probe substrates in rats was stable and feasible.The effect of sweated and non-sweated Salvia Miltiorrhiza Bge.on Cyp450 enzyme activity and mRNA expression in rats was consistent.The Cyp450 enzyme activity and mRNA expression level in rats were not affected by salvia total phenolic acids（P>0.05）.After sweating,it still had no effect（P>0.05）.Tanshinones had no effect on the Cyp2c11 and Cyp2d2 enzyme activity and mRNA（P>0.05）,but tanshinones could significantly decreased Cyp2b1,Cyp1a2,Cyp3a1,Cyp2c7,Cyp2c6 enzyme activity(AUC_（0-t）:6.74±1.77 mg/L·min VS 9.72±2.45mg/L·min;2.44±0.19 mg/L·min VS 3.81±1.07 mg/L·min;78.59±10.40 mg/L·min VS 110.79±16.16 mg/L·min;12.99±1.81 mg/L·min VS 17±1.55 mg/L·min;13.19±9.48 mg/L·min VS 25.01±7.11 mg/L·min.)and mRNA expression level（1.003±0.080 VS 0.606±0.104;1.001±0.053 VS 0.522±0.137;1.005±0.104 VS 0.558±0.128;1.002±0.069 VS 0.580±0.118;1.004±0.101 VS 0.643±0.148）.After sweating,the inhibitory trend of tanshinones on Cyp2b1,Cyp1a2,Cyp2c6 enzyme activity(AUC_（0-t）:6.74±1.77 mg/L·min VS 10.34±1.62 mg/L·min;2.44±0.19mg/L·min VS 5.09±0.19 mg/L·min;13.19±9.48 mg/L·min VS 27.36±11.82mg/L·min)and mRNA expression（1.003±0.080 VS 0.577±0.141;1.001±0.053 VS0.294±0.083;1.004±0.101 VS 0.585±0.114）was unchanged.The inhibition effect of Cyp1a2 enzyme activity（19.61±2.88 mg/L·min VS 5.09±0.19 mg/L·min）and mRNA expression（0.522±0.137 VS 0.294±0.083）was significantly enhanced.Moreover,tanshinones could significantly reduce the Cyp2d2 enzyme activity（20.61±5.31 mg/L·min VS 27.757±3.22 mg/L·min）and the expression level of mRNA（1.001±0.066 VS 0.607±0.109）,but they had no effect on the Cyp2c11,Cyp3a1,Cyp2c7enzyme activity and mRNA expression（P>0.05）.Conclusion:Sweated and non-sweated salvia total phenolic acids had no effects on the Cyp450 enzyme activity and mRNA expression level in rats.The Cyp450 enzyme activity and mRNA expression level in rats were affected by sweated and non-sweated tanshinones,and there are similarities and differences.It was suggested that when the preparation of Salvia Miltiorrhiza Bge.or tanshinones were combined with drugs that were metabolized by this enzyme,it was important to consider the possible interaction to avoid adverse reactions.And in the clinical application of Salvia Miltiorrhiza Bge.,it should draw attention to the selection of sweated and non-sweated Salvia Miltiorrhiza Bge.,not only to avoid the occurrence of adverse reactions,but also to maximize the efficacy of Salvia Miltiorrhiza Bge..