Studies On Content Determination And Pharmacokinetics Of Sesquiterpenes From Inulae Herba

There are about100species in Inula genus in the family Asteraceae in the wholeworld. In China, there are almost20species and lots of variable species, most of themare used as folk medicine. The main bioactive components of the Inula genus aresesquiterpene lactones, and37of which have been isolated and identified by now.Inulae Herba(Jinfeicao), a Chinese folk medicine, is derived from the aerial parts ofInula japonica Thunb. belonging to the Inula genus, and has been listed in thePharmacopoeia of the People’s Republic of China. It has been used for the treatmentof dispersing phlegm, relieving coughing, treating bronchitis, trachitis and parotitis.Pharmacological studies have shown that sesquiterpene lactones have diversebiological activities, such as antifungal, antibacterial, anti-inflammatory, antidiabetic,as well as insecticidal effects and so on. There are some papers related to the researchon the fingerprint of the Inula genus. However, there is no paper related to thedetermination of the sesquiterpene lactones in the Inula genus. Therefore，weestablished a rapid，simple HPLC-UV method，which could be used to simultaneouslydetermine5sesquiterpene lactones in the Inula genus. We apply this method toevaluating the contents of the5sesquiterpene lactones in different sources and species.We also lay a foundation for the further study of the quality evaluation of the Inulagenus.1-acetoxy-6α-hydroxyeriolanolide,1β-hydroxyalantolactone and ivangustin,which are abundant in Inulae Herba, have diverse biological activities, such asantifungal, antibacterial, anti-inflammatory, antidiabetic, as well as insecticidal effectsand so on. The three compounds have similar chemical structures. To our knowledge,there have been no reports describing a method for the determination of the threecompounds in biological samples till now. Therefore, the aim of this study was todevelop and validate a rapid, simple, and sensitive LC/MS/MS method for thedetermination of the three analytes in rat plasma for the first time. The present methodis simple, sensitive and reproducible, and thus could be successfully applied topharmacokinetics study after oral administration of Inulae Herba extract.1. Simultaneous determination of the contents of five compounds in Inula plants Purpose To determine five main active sesquiterpenes in Inula plants，to lay afoundation for the further study of the quality evaluation of the Inula genus.Method The HPLC condition was as follows column：Agilent Zorbax SB-C18(4.6mm×250mm，5μm)；mobile phase：A was acetonitrile，B was water，with gradientelution；the gradient of A phase：30%-40%(0-20min)，40%-55%(20-25min)，55%-65%（25-40min），55%-95%（40-45min），95%-95%(45-55min)； flowspeed：1.2ml/min；detection wavelength：205nm；temperature of column：25℃.Result Five sesquiterpenes (1-acetoxy-6α-hydroxyeriolanolide,1β-hydroxyalantolactone， Ivangustin, Japonicone A, Inulanolide A) were separatedat baseline within55min. Their peak areas showed a good linearship within theranges of13.4-402μg/ml，6.53-196μg/ml，6.85-206μg/ml，10.4-312μg/ml,6.65-200μg/ml，with the recovery rates being99.0％,97.6％,99.3％,97.9％,100.8%and RSD being1.0％,0.68％,2.1％,0.97%,1.9%, respectively.Conclusion The present method can be used to determine the contents ofsesquiterpenes in Inula plants； the contents of sesquiterpene lactones in differentplants have some varieties． 2. Quality standard revision of InulaeHerbaPurpose To determine the content of1-acetoxy-6α-hydroxyeriolanolide, and set upthe standard of quality control and content limit.Method The HPLC condition was as follows-column：Agilent Zorbax SB-C18(4.6mm×250mm，5μm)；mobile phase：A was acetonitrile，B was water，with gradientelution；the gradient of A phase：30%-38%(0-16min); flow speed：1.2ml/min；detection wavelength：205nm；temperature of column：30℃.Result The peak area of1-acetoxy-6α-hydroxyeriolanolide showed a good linearshipwithin the range of0.0615~2.460μg/ml，with the recovery rate being96.8%and RSDbeing2.86%.Conclusion The present method can be used to determine the content of1-acetoxy-6α-hydroxyeriolanolide, and set up the standard of quality control andcontent limit. 3. Simultaneous determination of three sesquiterpene lactones from Inulae Herbaextract in rat plasma by LC/MS/MS and its application to pharmacokinetic studyPurpose To get the pharmacokinetic parameter of the three sesquiterpene lactones,（1-acetoxy-6α-hydroxyeriolanolide,1β-hydroxyalantolactone, Ivangustin） andestablish a simple and sensitive LC-MS/MS method to determine them in rats plasma.To calculate the absolute bioavailability of the three compounds and provide a methodfor the further study of the sesquiterpene lactones.Method We established a LC/MS/MS method to determine the contents of the threesesquiterpene lactones. Plasma samples were pretreated by protein precipitation withmethanol. Chromatographic separation was accomplished on a TOSOH TSKgel ODScolumn with mobile phase consisting of methonal and0.3%formic acid (80:20, v/v).The detection was carried out by multiple-reaction monitoring (MRM) mode underpositive electrospray ionization. The quantification was performed using thetransitions of m/z309.1/185.0for1-acetoxy-6α-hydroxyeriolanolide, m/z249.0/231.1for1β-hydroxy-alantolactone and ivangustin, and m/z285.0/193.0for diazepam (IS),respectively.Result Three sesquiterpenes （1-acetoxy-6α-hydroxyeriolanolide,1β-hydroxyalantolactone， Ivangustin） were separated at baseline within6.2min.Their peak areas showed a good linearship within the ranges of4-800ng/ml，8-500ng/ml，8-500ng/ml，The lower limit of quantification was4ng/ml for1-acetoxy-6α-hydroxyeriolanolide, and8ng/ml for1β-hydroxy-alantolactone andivangustin. The absolute bioavailability of the three compounds,1-acetoxy-6α-hydroxyeriolanolide,1β-hydroxyalantolactone and Ivangustin, are18.70%、20.69%and21.39%, respectively.Conclusion The method was simple, stable, and suitable for pharmacokinetic studiesof the three sesquiterpene lactones in biological matrix.