Study On Anti-HBV Active Extract Of Compound Decotion Of Yinchenhao And Huanglianjiedu(CDYH)

In this essay, Compound decotion of Yinchenhao and Huanglianjiedu(CDYH) (Herbae Artemisiae Capillariae Decoction + Coptidis Decoction for Decoction) was selected as the main research object, transfection HBV (Hepatitis B Virus, HBV) 2.2.15 cells was choosed as a model to test the vitro efficacy of CDYH and the main active components of CDYH and studide its anti-hepatitis B virus effects in vitro experiments. Based on the results of its anti-hepatitis B virus in vitro test, we tracked its pharmacodynamics regular pattern,the activity of various parts of the CDYH is analysed by modern instruments to study its fingerprints. We combined the qualitative and quantitative study result of effective components to establish the quality standards of fingerprinting of the CDYH,which was guided by the pharmacodynamics effects of the CDYH.The compound Yin Huang Decoction of the group: artemisiae capillaris, flos, serrate rabdosia herb are principal drug;taraxaci, Gardenia florida, cortex Phellodendri, radix et rhizoma rhei, baical skullcap root, etc to compose ministerial drug. Schedul fever promoting urination from the whole side, disperse the depressed liver-energy anti-luxuriate cholagogue, polishing chylostomach. We explored the effect of Compound decotion of Yinchenhao and Huanglianjiedu(CDYH) in inhibiting the surface antigen (HBsAg) and e-antigen (HBeAg) by using the 2.2.15 cells as the cell model,which was considered a better experimental model in vitro for screeing and evaluation of anti-HBV drugs. We extacatde the chemical composition from CDYH with CHC13,Et Ac and n-butanol paragraph respectively according their polanith from low to high. And then we screened the anti-HBV activity of the extraction part by the pharmacologicall experinet in vitro with 2.2.15 cells. After analysised the above results, we finally found that the ethyl acetate extracts had a stronger activity. And its inhibition rate on HBsAg and HBsAg was 89.12%, 85.81%, 53.84%, 44.31% and 82.14%, 62.43%, 56.35%, 44.20% respectinely in the concentration of 2.5mg/ml,1.25mg/ml,0.625mg/ml,0.3125mg/ml ,with were higher than those of interferon,a positive control drug. And its inhibition rate of HBsAg and HBsAg were 50.63%, 43.83%, 34.79%, 21.96% and 57.09%, 44.20%, 32.97%, 15.47% respectively in the concentration of 10×10³ IU / ml, 5.0×10³ IU / ml, 2.5×10³ IU / ml, 1.25×10³ IU / ml.All of this procided a theoretical basis for exploring drugs of anti-HBV.Using a thin-layer chromatography (TLC) on the Compound decotion of Yinchenhao and Huanglianjiedu(CDYH) in identification of effective components. We established Compound decotion of Yinchenhao and Huanglianjiedu(CDYH) TLC method initially with the choice ofλ_S=275nm,λ_R=365nm double-wavelength scanning and singleλ=435nm wavelength scanning, which are reflective of zigzag scanning and Sx=3, Swing Width=10, slit 0.4mm×0.4mm. Spots on the preparations for the commencement were full scanned, the spots and blank spots of berberine hydrochloride, caffeic acid, ferulic acid, emodic acid were partial or full scanned. Compared with the double-wavelength scanning UV atlas, we got the corresponding peak of scaning of the test inchllding berberine hydrochloride standard peak position (approximately 40 mm～50mm), caffeic acid standard peak position (about 125 mm～135mm), ferulic acid standard peak position (about 145 mm～155mm). In addition, finished products in the emodic acid location (about 155 mm～165mm) is obvious, but rhubarb standard fluorescence was not obvious due to the impact of the forefront with solvent peak (about 165 mm～180mm), but also could be see the acromion induced by Rhein. Comparison of visible-wavelength scanning pattern, we can see that the test of the scans with berberine standard peak position (approximately 40 mm～50mm), Rhein standard peak position (about 160mm～170mm) Correspondingly, the blank - The Rhein impact had been eliminated.HPLC method was used to analyze the ethyl acetate extracts of the active ingredients of caffeic acid and ferulic acid. We measured precisly Compound decotion of yinchenhao and Huanglianjiedu(CDYH) 20mL to the separating funnel, with chloroform (20 mL) extracted three times ,and abandon chloroform solution. Thenextracted with ethyl acetate (20 mL) three times. We mergered ethyl fluid and dried ethyl acetate. Then we dissolved the residue with methanol and made its capacity to 10 mL, which were filtered to the sample vials with 0.45um membrane filters.Saved as a solution for the test. We got a better peak shape with HypersiL BDS C₁₈ column and a mobile phase of methanol-0.01mol/L potassium dihydrogen phosphate solution (adjusted to pH 3.7 phosphate),and it come out fully in 18 minutes. The contents of three batches were as follows: 99.76%, 100.2%, 99.35% and ferulic acid content is: 98.64% 101.2%, 99.55%; sample recovery of caffeic acid and ferulic acid average of 99.29% and 99.38%. The results showed that this method is simple and reliable.CDYH can be used as antidotes soup caffeic acid and ferulic acid in the control of an effective method. On this basis, using a capillary electrophoresis (HPCE) of their further analysis. HPCE in today's drug analysis had the advantages of high column efficiency, rapid analysis ,small sample size, the advantages are easying to clean than HPLC. And HPCE was more suitable for directly analysising of aqueous and non-purified samples do not need consume a large amount of high-purity organic reagent, saving operating costs. Electrophoresis in the same conditions, the determination of caffeic acid and ferulic acid reference substance, and with the Compound decotion of yinchenhao and Huanglianjiedu(CDYH) HPCE atlas of the relative ratio, in eight minutes and six minutes on the time period should be relatively the same time, a positive control Law on samples of ferulic acid and caffeic acid qualitative confirms that the peak for eight minutes and six minutes caffeic acid peak for the ferulic acid.We used rapid, high-sensitivity and selectivity in the integrated liquid chromatography - mass spectrometry technique to separate and analysis. Useing MS and LC-MS / MS scan Mode analysis, we identified the nine active ingredients. They were: Caffeic acid m/z,179 for the M-H, 160.9 for the M-H-OH and 135 M-COOH-H peak; Ferulic acid m/z,233 for the M+K,193 for the M-H and 178.3 for the (M-I-CH3); Rhein have four peaks, they are m/z 283 for the M-H,257 for the M-CO,239.6 for the M-CO₂ and 238.9 M-CO-OH peak; Aloe glycosides have three peaks, They are m/z 417 (M-H),256 for the M-glu and 227 to 256-CO peak.; Acetyl shikonin M/Z 329.0 for the M-H; Mulan spent alkali M/Z 341.1 for the M-H peak; Indigenous children theophylline M/Z 136.9 for the M-H; Bergenin of M/Z 327.1 for the M-H peak; Vary Geniposide M/Z403.3 for the M-H. Determination of these active ingredients provided the laboratory datas for its clinical application and further resareling.