Study On The Extraction Process Of Compound Huzhand Prescription And The Material Base Of Anti-hepatitis B Virus

Hepatitis B was a common disease with high incidence and susceptibility to liver cirrhosis and liver cancer.It posed a serious threat to patients'physical and mental health and brought great burden to families and society.However,the current safe and effective therapeutic drugs were rather lacking.Chinese medicine treatment of Hepatitis B was superior to multi-target,multi-channel,multi-level synergy and small side effects.It played an important role in immunity enhancement,anti-hepatitis B virus,liver function improvement and liver cells protection,so this could provide a way to find novel treatment drugs of anti-hepatitis B virus from Chinese medicine.The compound Huzhang prescription was composed of Polygonum cuspidatum,Schisandra,Sarcis and the other seven herbs,which have the efficacy of clearing liver and gallbladder,and was used clinically for various symptoms of liver and gallbladder dampness and heat caused by viral hepatitis.It was applied for clinical use for more than 30 years in affiliated hospital of integrated traditional Chinese and western medicine hospital in Jiangsu province,and was proved to be a safety and effective prescription.Previous studies have shown that it could effectively inhibit the replication of HBsAg,HBeAg and HBV DNA in vitro,and could have good anti-hepatitis B virus effection.As the material basis for the compound to exert its pharmacodynamic effect in body was not clear,it seriously hampered its development into a clinical drug of advanced dosage and quality control for the treatment of HBV.Therefore,this research intended to carry out the research on the pharmacodynamic substances of compound Huzhang prescription along the axis of“in vitro component-influx component-into cell component-active component”layer by layer.The main contents are as follows.1 Study on the extraction processThe compound Huzhang prescription had complex chemical components.Many components work synergistically to exert efficacy,while select of one or more components as indicator for the evaluation of the extraction process was far from enough,thus a optimized method of extraction process based on fingerprint combined with principal component analysis?PCA?was established and then applied to optimize the compound Huzhang prescription ethanol extraction process.Different fingerprint with different ethanol extraction condition was established by HPLC,the areas of the common peaks were harvested,the total factor scores were calculated by PCA analysis.U₉?9³×3¹?uniform design experiments was arranged,the total factor scores was chosen as index,the effect of extration times,alcohol consumption,alcohol concentration and the time of extracting on the extration process were then investigated,finally the technological parameters of ethanol extraction condition were optimized by multi-nonlinear mathematic models.The results indicated that multi-nonlinear mathematic models described the good linear relationship between response indexes and factor variables.In this process,the optimized condition for compound Huzhang prescription was extracted in 90%ethanol 6 times as much as it for 3 times,1 h once.It proved that the extraction method could fully extract the active ingredients,could also provide references for optimization of the compound Chinese medicine extraction process.2 The characterization of chemical constituentsThe compound Huzhang prescription had good effection on anti-hepatitis B virus,but the material basis was not clear.So the chemical constituents of ethanol extract were analyzed by UPLC-Q-TOF/MS with the peak of quasi-molecular ion.In the process,both of the positive and negative ion mode mass spectrum were collected to analyze the chemical constituents.Based on the data of accurate molecular weight in mass spectrum,the rule of mass spectrum fragmentation,the fragment information and the chromatographic retention time,the mass spectrometry information and the references of the corresponding reference substance were all analyzed.The main components include anthraquinones,stilbenes,alkaloids,lignans and so on.Among the 87compounds,25 of these were derived from Polygoni cuspidati rhizoma et radix,25 of these were derived from Phellodendri chinensis cortex,16 of these were derived from Lysimachiae herba,17 of these were derived from Artemisiae scopariae herba,9 of these were derived from Schisandrae chinensis fructus,4 of these were derived from Sedi herba,2 of these were derived from Indigo naturalis.This provided convenience for further analysis of composition into blood.3 The characterization of chemical constituents into bloodThe compound Huzhang prescription contains various chemical constituents,but constituents that only through the gastrointestinal absorption into the body,having a medical effect on target cells in the form of prototype or metabolites could be considered as effective constituents,Therefore,the analysis of blood components was an important part of studying the material basis.In this process,the ethanol extract was given to rats by gavage,then the blank serum and the drug-containing serum was collected,respectively.The UPLC-Q-TOF/MS method was used to collect the excimer ion peak in positive and negative modes.Based on the information of chemical constituents in vitro and the reported lysis pathways in literature,MetaboLynx XS^TMM software was used to infer the mass spectral information.About 23 related metabolites in the drug-containing serum were characterized,including 17 prototype components and 6 new metabolites,which might lay the foundation for further analysis of cellular components.4 The characterization of chemical constituents into cellsThe components and metabolites of the compound Huzhang prescription that entered into the body were relatively simplified compared to those before consumption,but the ultimately function compounds should be those that directly acting to target cells.So drug-containing serum was co-incubated with HepG2.2.15cells,and blank serum control group was set to subtract the background effect.The intracellular components after affinity of HepG2.2.15 cells were collected and cell lysate was analyzed by UPLC-Q-TOF/MS.The results showed that three constituents were characterized in HepG2.2.15 cell lysate after affiliated with drug-containing serum.They were Berberine,Schisandrin and Deoxyschisandrin,which were initially concluded as the main active constituents.5 The verification of anti-hepatitis B virus activityIn order to verify whether the three compounds that enter into the cells were active constituents,the activity and intensity effect of Berberine,Schisandrin and Deoxyschisandrin on human hepatocytes which expressed HBV?HepG2.2.15 cells?were well detected by MTT assay.The results revealed that these three constituents had inhibitory activity on HepG2.2.15 cells growth,and the activity intensity was better than lamivudine,which was a positive drug.From the range of 2.5?mol.L^-1 to 320?mol.L^-1,the three compounds exhibited a significant dose-dependent inhibition of cell proliferation with IC₅₀ values of 70.85?mol.L^-1,152.80?mol.L^-1 and 167.80?mol.L^-1,respectively.In summary,firstly,this study optimized the compound Huzhang prescription extraction process through the combination of fingerprint and principal component analysis.Then,the chemical constituents,the chemical constituents into the blood and the chemical constituents into the cell were characterized by UPLC-Q-TOF/MS.Finally,the HepG2.2.15 cell model transfected with HBV gene was used to evaluate the activity of each component.On the theory of the medical effective material,it could lay the foundation of developing prescription into a potential drug prevention and treatment of hepatitis B virus.