| Suan-Zao-Ren(SZR)decoction,a classical traditional Chinese formula containing five Chinese herbs Ziziphi Spinosae Semen,Poria,Chuanxiong Rhizoma,Anemarrhenae Rhizoma and Glycyrrhizae Radix Et Rhizoma,was originally described in Jin Kui Yao Lue,written in ancient China Eastern Han Dynasty by Zhongjing Zhang,which has been used for treatment of insomnia,restlessness and anxiety.Effects of SZR decoctions with different extraction solvents on sleeping number,sleeping latency and time in mice induced by sodium pentobarbital were studied.As a result,water was selected as the optimal extraction reagent.Furthermore,effects of SZR decoctions extracted by various polar solvents on sleeping number,sleeping latency and time in mice induced by sodium pentobarbital was investigated.Finally,the extracts of n-buty alcohol and 75%alcohol were found to be effective fractions.A rapid and simple method for simultaneous determination of 5-HT,5-HIAA,Glu,GABA,DA,NE,Ach and Try in rat brain using ultra-fast liquid chromatography and tandem mass spectrometry(UFLC-MS/MS)without derivatization,ion-pairing reagent or pre-concentration was developed.After addition of the internal standard isoproterenol hydrochloride,brain samples were extracted by protein precipitation(PPT)with methanol,and separated on an ODS-EP column and analyzed in a single chromatographic run in less than 9.0 min,using gradient elution with the mobile phase consisting of methanol and 0.01%acetic acid in water at a flow rate of 1.2 ml/min.The detection of the analytes was performed on 4000Q UFLC-MS/MS system with turbo ion spray source in positive ion and multiple reaction-monitoring mode.The developed method provided excellent linear calibration curves for the assay of analytes(R2?0.9915).Limits of quantification were in the range of 1.0 ng/ml to 1.0 ?g/ml for the analytes in rat brain.Intra-and inter-day precision and accuracy of analytes were well within acceptance criteria(15%).Mean extraction recoveries of analytes and IS from rat brain were all more than 86.0%.A simple,fast and sensitive UFLC-MS/MS method for simultaneous determination of 8 neurotransmitters in rat plasma has been developed and completely validated.Using.MRM mode allows for a reliable analysis with high sensitivity and selectivity.Following the optimization of chromatographic condition,the compounds are assayed in less than 9.0 min with symmetric peak shapes,which is suitable for high sample loads.A simple sample PPT method with 0.1%formic acid in water is applied to sample extraction,obtaining the recoveries for all analytes and IS above 83.0%.The developed method provided excellent linear calibration curves for the assay of analytes(R2?0.9841).Limits of quantification were in the range of 1.0 ng/ml to 1.0 ?g/ml for the analytes in rat plasma.Intra-and inter-day precision and accuracy of analytes were well within acceptance criteria(15%).The effect in the treatment of SZR decoction and its effective fractions on liver yin deficiency,insomnia and their composite model in rats,which were injected with hydrocortisone once daily for 5 d and PCPA once daily for 4 d,respectively,were studied.The improvement of the rats in different models were evaluated based on weight of rat,level changes of 5-HT,5-HIAA,Glu,GABA,DA,NE,Ach and Try in rat brain and plasma,cAMP and cGMP in rat plasma,and T3 and T4 in rat serum.The results showed that effective fractions of n-buty alcohol and 75%alcohol had definitely therapeutic effect on liver yin deficiency and insomnia.A UFLC/MS/MS-QTrap method combined with liquid-liquid extraction sample preparation were employed for screening and identifying the main constituents of SZR decoction.18 compounds were identified including chrysophanol,spinosin,mangiferin,betulinic acid,glycyrrhizin,liquritin,ferulic acid,tumulosic acid,oleanolic acid,senkyunolide A,swertisin,isoswertisin and senkyunolide I.Possible MS/MS fragmentation mechanism of spinosin,mangiferin,ferulic acid and tumulosic acid were studied.To compare the pharmacokinetics of spinosin,mangiferin and ferulic acid in normal and insomnic rats orally administrated with the prescription,a sensitive and reliable UFLC-MS/MS method for simultaneous quantitation of these main active ingredients in Suan-Zao-Ren decoction was developed.The compounds and IS were separated on a SHIM-PACK XR-ODS column protected by a high pressure column pre-filter at 35 ?.The mobile phase were composed by 0.1%formic acid in water and methanol using a 6.0 min UFLC gradient program.Efficient and symmetrical peaks were obtained at a flow rate of 0.4 ml/min with a sample injection volume of 2 ?l.All the plasma samples were extracted by isopropanol-ethyl acetate(1:1,v/v)using liquid-liquid extraction technique.The mean extraction recoveries of spinosin,mangiferin,ferulic acid and IS from rats plasma were all more than 87.0%.The linear range was 1.0-500 ng/ml for spinosin,6.0-3000 ng/ml for mangiferin and 1.0-500 ng/ml for ferulic acid,with the lower limit of quantitation of 1.0,6.0 and 1.0 ng/ml,respectively.Intra-and inter-day precision and accuracy of spinosin,mangiferin and ferulic acid were well within acceptance criteria(15%),indicating satisfactory results for accuracy and precision of the analytes.The validated method was successfully applied to the pharmacokinetics of spinosin,mangiferin and ferulic acid in rat plasma after oral administration of SZR decoction.The results demonstrate that there were statistically significant differences(p<0.05)in pharmacokinetic parameters including AUC0-t,AUC0-?,Cmax and T1/2 for spinosin and mangiferin,but no significant difference for ferulic acid between two groups.After oral administration of SZR decoction,the insomnic group exhibited a remarkable decrease(P<0.05)in the value of AUC0-t,AUC0-? and Cmax,and increase(p<0.05)of T1/2 associated with spinosin and mangiferin. |
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