Studies On The Anti-HBV And Anti-NIDDM Active Substances And The Resource Quality Of Swertia Punicea Hemsl

Swertia punicea Hemsl.,a perpnnial dried herb of Gentianaaeae,is used as a folktraditional medicine in China for a long time.It is frequently used for the treatment ofhepatitis,cholecystitis,pneumonia,pandemic influenza,inflammation of bone,et al.Moreover,plants of Swertia,S.chirayita and S.japonica,were reported to show significantanti-diabetic activity.In this study,material basis for anti-hepatitis B virus (HBV) andanti-diabetic activities of Swertia punicea Hemsl.was investigated.Moreover,the sourceand quality of Swertia punicea Hemsl.were evaluated for its development of medicalvalues.Firstly,to clarify the material basis for anti-HBV activity of Swertia punicea Hemsl.,activities of fractions and components from Swertia punicea Hemsl.were screened in HepG2 2.2.15 cell,a kind of HBV-producing human hepatoblastoma cell line,and theconsitituents of Swertia punicea Hemsl were studied.In this study,ethanol extract and ethyl acetate fraction of Swertia punicea Hemsl.significantly inhibited the expression of HBsAg.,which confirms the anti-HBV activity ofSwertia punicea Hemsl.and the ethyl acetate fraction was the active fraction.Study on consitituents of Swertia punicea Hemsl afforded 20 compounds,identified as,Comp.18 (1-hydrate,3- deoxy-α-D-tagatofuranose) was a new compound,CompoundsComp.16 (Campestroside),17 (Uridine)were isolated from this genus for the first time andcompounds Comp.12 (7-[(β-D-xylopyranosyl)oxy]-1,8-dihydroxy-3-methoxyxanthone),13(Norswertianolin),14 (Tetrahydrosertianolin),15 (8-hydroxy-10-hydrosweroside) wereobtained from this plant for the first time.Compounds from ethyl acetate fraction were screened for anti-HBV activity in thisstudy,as well as some compounds from other fractions.Compound 2,5 and 10 inhibitedthe expression of HBsAg and their selected index (SI)was 1.16,14.2 and 6.8,respectively.Compound 7,8,9 and 11 inhibited the expression of HBsAg and their SI was 4.5,6.2,6.87and 2.36,respectively. Moreover,to study the material foundation of anti-diabetic activity of Swertia puniceaHemsl.,we studied anti-diabetic activities of fractions and constituents of Swertia puniceaHemsl.in STZ-induced type 2 diabetic mice.As a following study,the potential mechanismof active constituent (s) was investigated.The hyperglycemia BABL/c mice which were induced by low dose of streptozotocin(STZ) and high calorie food are used to screen the hypolglycaemic activity ingredients fromS.punicea Hemsl.The effects of four fractions (ether,ethyl acetate,n-butanol and thewater-soluble),obtained from S.punicea Hemsl.,on FBG,glucose tolerance and insulinresistance were studied.The result indicated S.punicea Hemsl.was beneficial to type 2diabetes and its potential mechanism could be improvement of insulin resistance.In this study,STZ-induced type 2 diabetic male BABL/c mice treated with or withoutmethylswertianin and bellidifolin at different doses (orally,200 and 100 mg/kg/d) for 4weeks were analyzed.The results proved that methylswertianin and bellidifolinsignificantly reduced fasting blood glucose (FBG).The administration of methylswertianinand bellidifolin also improved the oral glucose tolerance and lowered fasting serum insulin(FINS).Moreover,the administration of methylswertianin and bellidifolin lowered theserum total cholesterol (TC),low density lipoprotein cholesterol (LDL) and triglyceride(TG) levels and increased the relative high density lipoprotein cholesterol (HDL)concentrations (HDL/TC).methylswertianin and bellidifolin appeared to improve insulinresistance by enhancing insulin signaling.The expression levels of insulin-receptorαsubunit (InsR-α),insulin-receptor substrate-1 (IRS-1),phosphatidylinositol 3-kinase (PI3K)were increased after the administration of methylswertianin and bellidifolin.Meanwhile,methylswertianin and bellidifolin increased hepatic glycogen content,decreasedglucokinase (GK) activities and increased glucose-6-phosphatase (G6Pase) activities.Inconclusion,these result indicated that methylswertianin and bellidifolin could be useful fortreating type 2 diabetes and the mechanism probably was the improvement of insulinresistance (IR).Lastly,To develop the medical values of Swertia punicea Hemsl.,the resource andquality of Swertia punicea Hemsl.were evaluated.The study aimed to establish HPLC fingerprint of Swertia punicea Hemsl.from Hubei Province The chronmtograms of 10 batches of Swertia punicea Hemsl.samples fromdifferent producing area were determined under such conditions as gradient elution withmethanol -0.05%aquaintance-acetonitrile,current speed at 0.8 ml/min and determinedwavelength at 256 nm.The characteristic fingerprint consisted of 13 common peaks ofSwertia punicea Hemsl.from Hubei province was established through comparing withcontrast medicinal materials.Analysis on the fingerprint by similarity analysis indicatedthat the fingerprint could be used to identify of Swertia punicea Hemsl.and evaluate itsquality.The method was stable and reliable with good reproducibility and could be used toestablish fingerprint of Swertia punicea Hemsl.and supply the reference for overall controlof its quality.In this study,a new HPLC method by using ultraviolet absorbance detection withdetected wavelength at 254 nm was presented for the simultaneous determination of fouractive components,namely Swertiamarin,Mangiferin,Swertianolin and Bellidifolin in 7species of Swertia plants (Gentianaceae).The samples were separated and detected on thecolumn of Alltima C18(250 mm×4.6 mm,5μm)under the condition of gradient elution.The limit of detection (S/N=3) is less than 0.86μg/ml and the limit of quantification(S/N=10) is less than 1.73μg/ml..All calibration curves show good linear regressionwithin test ranges.The RSD for intra- and inter-day of four investigated compounds werelower than 2% and 4.6%,respectively.The mean recovery was between 97.94% and99.84%.The method was establishded to simultaneous determination of the fouranti-hepatitis constituents relating to different kinds of chemical structures in Swertia plants.So,the method was successfully applied to quantify the four active components in differentparts of the herb and identify the specific species of genus Swertia.Moreover,the quality standard of Swertia punicea Hemsl.was established.The method,used in standard,were simple,accurate,specific and reproducible and can be used as areliable method for quality evaluation of Swertia punicea Hemsl.