Studies On Screening Of Anti-HBV Active Constituents In Flavonoids And Chemical Components Of Flavonoids From Scutellaria Barbata D.don

Scutellaria barbata D.Don,which is a kind of traditional Chinese medicine and Labiatae Scutellaria plants.It contributes to antibacterial,anti-inflammatory,antiviral and anticancer significantly,with the main chemical compositions including Flavonoids,Diterpenoids and Alkaloids,etc.The whole plant of Scutellaria barbata D.Don has been widely used for treating liver diseases,such as hepatitis,liver cirrhosis,liver cancer and so on.Researches on Scutellaria barbata D.Don mostly focused on the anti-cancer and liver protection effect at home and abroad.Little research has been reported on the anti-HBV effect using pharmacological model for anti-HBV in biological active research.There are three parts of experimental reserach:Part 1:The flavonoids from Scutellaria barbata D.Don were extracted,refined and purified by AB-8 macroporous resin.According to the experimental results,the anti-HBV effect of the ethanol-extract of flavonoids from Scutellaria barbata D.Don(ESB)is better than that of the water-extract of flavonoids,removal of organic acids and other impurities.The content of total flavonoids was determined as(54.24±3.74)%,and(85.23±2.90)%in ESB before and after the purification.The.eluents with different volume fraction(0%,20%,40%,60%,80%)of ethanol-water were obtained by through AB-8 macroporous resin,according to different polarities of flavonoid monomers.Part 2:To establish a HPLC method for simultaneous determination of 9 flavonoids monomer of Scutellaria barbata D.Don.This HPLC method was developed on a Wondasil C18(250 × 4.6 mm,5 ?m).The mobile phase was composed of 1%HAc and CH3OH:CH3CN(80:20)with a linear gradient elution.The flow rate was 0.8 ml/min,and the UV detection wave length was set at 340 nm,the column temperature was maintained at 25 ?.The chromatographic separation conditions of gradient(min?B%):0?20 min 38%;20?30 min,38%-44%;30?40 min,44%-52%;40?50 min,52%-62%;50?55 min,62%-80%;55?60 min,80%-38%?This method is e accurate and reliable,used for the determination of flavonoids content of Scutellaria barbata D.Don.The content of nine flavonoids monomers are different in the different elution parts by different volume fraction of ethanol-water by macroporous resin elution,scutellarin was mainly consist in 20%elution,baicalin and scutellarin was mainly consist in 40%elution,apigenin was mainly consist in 60%elution,naringin was mainly consist in 80%elution,The content of flavonoids mainly concentrated in 20%,40%and 60%ethanol elution part.Part 3:The screening of anti-HBV activity in vitro of flavonoids from Scutellariae barbatae D.Don.A model of HBV infected HepG 2.2.15 cells in vitro was established to evaluate the anti-HBV activities of these fractions.ESB,different parts of elution by macroporous resin with different volume fractions of ethanol-water and nine kinds of Scutellaria barbata flavonoid monomers were used as the test drugs.Cytotoxicity of diffrent test drugs were evaluated by MTT colorimetric assay in vitro with IC50 as index,and the inhibition activity of HBeAg and HBsAg were evaluated by ELISA method with TC50 as index,TI index as the activity-evaluating marker.The TI index was 2.53 and 4.39 inhibited to HBsAg.HBeAg of ESB,TI index was 3.43 and 5.87 of 40%elution,TI index was 2.46 and 4.67 of 40%elution,TI index was 6.65 and 3.36 of baicalein.The results show that the ethanol extracts and total flavonoids of Scutellariae barbatae D.Don are of anti-HBV activity.The anti-HBV activity of cutellarin,baicalin,quercetin dihydrate,baicalein and wogonin is significant.