Studies On The Separation, Purification And Pharmacokinetics Of Active Ingredients Against Liver Injury From Radix Gentianae

Gentian, the dry roots and rhizomes of a family of herbs such as Gentianamanshurica Kitag., Geniana scabra Bge., Geniiarur triflora Pall. and Gentianarigescens Franch. is widely used in traditional medicine for liver protection. Recently,more effects of Gentian including diuretic, anti-inflammatory, antioxidant andantihypertensive have been scientifically proven. Although it is well established thathepatoprotection is the major clinical activity of Gentian, the toxic ingradients intraditional medicine can cause negative effects. Therefore, further exploring thepharmacological properties and in vivo pharmacokinetics of the active ingredientsfrom gentian are important for the determination of its efficacy and safety as ananti-hepatotoxic drug.1. Separation and identification of iridoid glycosides in Radix GentianaeIridoide glycosides that commonly found in medicinal plants are responsible formany pharmaceutical effects. With reversed phase ODS column chromatography andpreparative high performance liquid chromatography separation techniques, a total ofsix iridoid glycosides were separated from70%ethanol-Radix Gentianae extract.Their structures that identified through NMR and MS analysis were as follows:loganic acid (1), gentiopicroside (2),8-epiloganic acid (3), swertianlarin (4),6’-O-β-D-glucopyranosylgentiopicroside (5), and sweroside (6).8-epiloganic acid and6’-O-β-D-glucopyranosylgentiopicroside were separated from Gentianamanshurica Kitag for the first time.2. Isolation of gentiopicroside from Gentian with HSCCCSeparation and purification of gentiopicroside from Gentian extract wassuccessfully preformed by high-speed counter-current chromatography (HSCCC) withtwo-phase solvent system composed of n-butyl alcohol-water (1:1, v/v). The upper phase was used as stationary phase and lower phase was the mobile phase. At850rpmand a flow rate of2mL/min, the retention time of gentiopicroside was120.0-133.6min. The purity of gentiopicroside obtained by HSCCC was determined as98.48%byHPLC analysis. HSCCC method is simple, efficient, low toxicity, low costs andprovides an effective separation of the product, therefore it has been considered as anoptimum method for gentiopicroside purification.3. Quantitation and purification of active parts of Radix GentianaeSimultaneous determination of gentiopicroside, swertiamarin and sweroside inRadix Gentianae was achieved by reversed phase high performance liquidchromatography. This method is simple, accurate with high reproducibility andstability, which can be applied as a quality control strategy for isolating the activeingredients from Radix Gentianae. The resins with different pore size that used toseparate and purify iridoid glycoside from Radix Gentianae were screened by staticand dynamic adsorption， desorption assays. HPD300macro porous resin with thehighest adsorption and desorption efficiency was selected for purifiyingGentiopicroside, swertiamarin and sweroside from Radix Gentianae. This optimizedprocess was simple, stable and feasible, which supports further pharmacology and invivo pharmacokinetic investigations.4. Anti-hepatotoxicity effect of the active parts in Radix GentianaeIn vitro activities: A CCl4-induced injury model in hepatic cells was established.MTT assay was employed to evaluate the anti-hepatotoxicity effect of the active partsin Radix Gentianae. Cells treated with the active ingredient showed an increasedsurvival rate compared to the control cells, indicting a hepatoprotective effect onCCl4-induced injury hepatic cells. However, a toxic effect that causing cell death wasshown with treatment concentration higher than20mg/L. In addition, activities ofALT and AST in the growth medium as well as the levels of SOD and MDA in theactive ingredient treated cells were determined, suggesting the antioxidant and radicaldeoxygenization activities of the active parts in Radix Gentianae.In vivo activities: A CCl4-induced acute liver injury model in mice wasdeveloped. By monitoring the levels of serum biochemical indicators of liver function and liver histopathological changes, the preventive and protective effect of the activeparts in Radix Gentianae was shown on liver injury mice model.5. Pharmacokinetics of active ingredients from Radix Gentianae in liver injuryrat modelA rapid and sensitive method based on ultra-fast liquid chromatography-tandemmass spectrometry (UFLC-MS/MS) has been developed for the simultaneousdetermination of gentiopicroside and swertiamarin in rat plasma. The method wasfully validated and applied to a pharmacokinetic study involving oral administrationof a Radix Gentianae extract to groups of male and female rats. Theconcentration-time curves of gentiopicroside and swertiamarin were both described bytwo compartment model. The Tmaxof gentiopicroside and swertiamarin was between2h to3h and the t1/2was between0.8h to2h. Compared with male rats, the Cmax，AUC0-t， AUC0-∞and t1/2of gentiopicroside and swertiamarin in female rats weresignificantly improved（p<0.05). However, no significant change of Tmax（p﹥0.05）was obtained, indicating the pharmacokinetic properties of gentiopicroside andswertiamarin are remarkably different between male and female rats.6. Pharmacokinetics and distribution of active parts from Radix Gentianae in ratmodelSince pharmacokinetics of gentiopicroside was highly influenced by otheringredients of Radix Gentianae, a high performance liquid chromatography methodwas developed to investigate the pharmacokinetic properties of gentiopicroside fromRadix Gentianae extract to avoid interference.This well established high performance liquid chromatography method was alsoused to study the pharmacokinetic and tissue distribution of gentiopicroside after oraladministration of the active parts of Radix Gentianae in rats. The concentration-timecurve of gentiopicroside was described by two compartment model. The sequence ofgentiopicroside distribution in rats was as follows: liver>kidney>lung>heart>spleen.