| Insomnia is a common physiological and psychological illness,it will bring serious adverse effects on people's normal life after a long illness.At present,the clinical treatment of insomnia is still rely on BZD sedative hypnotic drugs,but the long-term use may have a certain drug dependence.Zaoren Anshen prescription(ZAP)is well known for the treatment of insomnia which consists of three herbs:Semen Ziziphi Spinosae,Radix Salviae Miltiorrhizae and Fructus Schisandrae Chinensis.In order to explore the dynamic changes of absorption,distribution,metabolism and excretion in the insomnia model rats,this study established a clinical model of insomnia by the injection of PCPA combined with chronic clamping stimulation,at the same time,a liquid chromatography-tandem mass spectrometry(LC-MS/MS)method for the simultaneous determination of the 7 major active compounds in a pharmacokinetic study after administration of ZAP in insomnia rat plasma was developed and validated:spinosin,salvianic acid A,protocatechualdehyde,6'"-feruloylspinosin,salvianolic acid B,schisandrin and deoxyschisandrin.These results will be beneficial for the clinical therapy of ZAP.The main contents and results include:1.The HPLC method for the simultaneous determination of four monoamine neurotransmitters in norepinephrine,dopamine,5-hydroxytryptamine and 5-hydroxyindole acetic acid was established.According to the content of each index component and the behavioral changes of the rats,we determined whether the clinical model of insomnia by injection of PCPA combined with chronic chimple stimulation was established successfully.The results showed that compared with the normal group,the rats in the model group had decreased in food intake,body weight,activity,falling asleep difficulty and were awakened by outside interference,by the way,the content of norepinephrine and dopamine in plasma was significantly increased,and 5-hydroxytryptamine and 5-hydroxyindole acetic acid content was significantly reduced,which indicating that the success of insomnia model.2.The HPLC method for the simultaneous determination of 7 constituents of ZAP was developed and validated,the results of method validation showed that the linearity of the seven indexes was good,the recoveries of all analytes were higher than 93.47%.The RSD of precision,stability and reproducibility were all less than 3%,which was in accordance with the requirement of chemical composition of traditional Chinese medicine.The method was applied to the determination of seven indicators in ZAP.3.The LC-MS/MS method for the determination of seven indexes in the plasma of insomnia rats was established.The results showed that the seven components could be separated from the plasma of the rats,and there was no interference with the endogenous substances.The RSD of the three groups were lower than 13.7%,the recoveries ranged from 78.3 to 94.0%,and the matrix effect was between 92.2 and 106.1%.The results met the requirements for a bioanalytical method.A high sensitivity LC-MS/MS method was provided to the pharmacokinetic study of seven indexes in the insomnia rats plasma.4.To detect the contents of seven components in insomnia rats plasma by established LC-MS/MS method.Study the pharmacokinetic effects of 7 components after oral different doses ZAP extract in insomnia rat plasma.The pharmacokinetic behavior results showed that there was a linear relationship between high dose and medium dose.All the indicator components were absorbed into the blood within 1 h.spinosin have a large area under the concentration-time curve and clearance rate in the rat.Salvianolic acid B had the highest content in the prescription,but it was rapidly decline after the peak plasma concentration,This phenomenon might reflect the generation of salvianic acid A via bond cleavage of the furan ring and two ester bonds in salvianolic acid B.schisandrin and deoxyschisandrin exhibited bimodal behaviours in their plasma concentration-time profiles,This double-peak behaviour may be caused by hepatoenteric circulation. |
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