Study On Anti-HBV Effects And The Chemical Constituents Of Different Polar Parts Of Ethyl Acetate Extract Active Fractions Extracted From Folium Artemisiae Argyi

Folium Artemisia Argyi is the dry leaf of Artemisia argyi Lévl. et Vant, and it iscommonly used clinically. It is reported in some literature that Folium ArtemisiaArgyi mainly contain essential oil, flavonoid, sterols, terpenoid, chomones, traceelements and so on; Folium Artemisia Argyi can regulate Qi and the Blood, expelcold dampness, warm channels and blocking blood and relieve pain; modernscholarship has established that Folium Artemisia Argyi has the effects of antitussive,antiasthma,expectorant anti-bacterial, antiviral, anti-allergic, anti-cancer andenhancing the immune response and so on. There were no reports about the anti-HBVactivity and anti-HBV active fractions of Folium Artemisia Argyi.Our team’sresearch found that the essential oil and ethyl acetate extract of Folium ArtemisiaArgyi had anti-HBV activity in vitro and in vivo, the content of which has beenawarded two national patents. In order to develop new anti-HBV drugs, we detectedthe anti-hepatitis B virus effect of the different polar fractions of ethyl acetate extractsand analyzed their chemical constituents.The preparation of the different polar fractions of ethyl acetate extracts ofFolium Artemisia Argyi. Firstly, we prepared ethyl acetate extracts of FoliumArtemisia Argyi.Having weighed1Kg shattered Folium Artemisia Argyi, we extracted the volatile oil by steam distillation, and then prepared the essential oil and aqueous extract of Folium Artemisia Argyi. Then we successively recycled-extracted the gruff of Folium Artemisia Argyi with petroleum ether, ethyl acetate and ethanol, extracting it twice, and for two hours each time. We merged the extract together, and then concentrate, evaporated and vacuum dried it, gaining ethyl acetate extract of20.6g. Secondly, by recycled-extracting the ethyl acetate extract successively with petroleum ether, petroleum ether-ethyl acetate(1:1,V:V) and60%ethanol,we prepared four different polar fractions: petroleum ether extract(EA-1) of5.4g, petroleum ether-ethyl acetate(1:1,V;V) extract(EA-2) of2.8g,60%ethanolextract(EA-3) of2.4g and ethyl acetate extract(EA-4) of2.5g.The anti-HBV experiments of four different polar fractions extracted from ethylacetate extract of Folium Artemisia Argyi in vitro. In order to detect the anti-hepatitisB virus effect of the four different polar fractions of ethyl acetate extracts, we useHepG2.2.15cells as cell model.1. MTT assay was used to evaluate the toxic effect of t EA-1, EA-2, EA-3andEA-4on HepG2.2.15cells. The results demonstrated that the toxic effect of EA-1,EA-2, EA-3and EA-4on HepG2.2.15cells were low. Among them, the TC50ofEA-1, EA-3were217.3μg mL-1,112.4μg mL-1, EA-2and EA-4were greater than200μg mL-1.2. ELISA method was used to observe the influence of EA-1, EA-2, EA-3andEA-4on HBeAg and HBsAg secreted by HepG2.2.15cells. The results demonstratedthat they all had a strong inhibition on the HepG2.2.15eell to exerete the HBsAg andHBeAg, the IC50of them respectively were2.63μg mL-1and7.33μg mL-1;7.57μg·mL-1and16.34μg mL-1;3.67μg mL-1and18.98μg mL-1;16.88μg mL-1and31.42μg mL-1.3. Real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) wasused for the determination of HBV DNA. The results showed that EA-1, EA-2, EA-3and EA-4all could inhibit the HBV DNA replication of HepG2.2.15cells andshowed a dose-dependent manner,whose effects was weaker than3TC.The IC50ofEA-1on HBV DNA was39.18μg mL-1. The ability of the other thee fractions toinhibit HBV DNA were weaker.Above all, we could evaluate the anti-HBV activity of EA-1, EA-2, EA-3andEA-4and screen the active fractions. In summary, as for EA-1, the therapeutic indexon the ninth day of HBsAg, HBeAg and cell supernatant HBV DNA were82.6,29.6and6.8; for EA-3, the therapeutic index on the ninth day of HBsAg and HBeAgwere30.6and5.9. Preliminary analyse of the chemical constituents of EA-1and EA-3.1. By using silica column chomatography and recrystallization, five mon-omer compounds were isolated from EA-1and EA-3, they were FAA20120831-A, FAA20130430A, FAA20130430B, FAA20130430C and FAA20130430D.Among which, the component of FAA20130430A and FAA20130430C are thesame; FAA20130430D is the same with FAA20120313F isolated in previousexperiment, and they were3’-methoxy cirsimaritin (5,4’-dihydroxy-6,7,3’-trimethoxy flavone).2. The purity of FAA20120831A, FAA20130430B and FAA20130430Cweredetermined by using TLC and HPLC methods. Results of TLC displayed thepurity of FAA20120831A, FAA20130430B and FAA20130430C were all greaterthan99%. Results of HPLC demonstrated that FAA20130430B had no impurity.As FAA20120831A and FAA20130430C had no ultraviolet-visible (UV) absorpt-ion, they could not be detected by HPLC.3. We identified FAA20120831A, FAA20130430B and FAA20130430C bymeans of1H-NMR,13C-NMR and2D NMR (DEPT, HMBC, HSQC,1H,1H-COSY)spectra and literature analysis, and they are respectively Dammaadienylaceta-te;5,7,4’-trihydroxy-3’,8-dimethoxyflavone and Isopropyl hexadecanoate. Dam-maradienylacetate and5,7,4’-trihydroxy-3’,8-dimethoxyflavone were firstly ext-racted from Folium Artemisiae Argyi.In this thesis, we firstly determine the anti-HBV activity of different polarfractions of Ethyl Acetate Extract Active fractions extracted from Folium ArtemisiaeArgyi and preliminarily analysis their chemical components. Dammaadienylacetate,and5,7,4’-trihydroxy-3’,8-dimethoxyflavone were firstly extracted from FoliumArtemisiae Argyi.,which layed foundation for the further research on new anti-HBVdrugs.