Study On Anti-hepatitis B Virus Activity And The Component Analysis Of Folium Artemisia Argyi Ethyl Acetate Extrate

Folium Artemisia Argyi is the dry leaf of Artemisia argyi Lévl. et Vant,it is distributed all over the country and it is rich in resources..As the traditional Chinese medicine,it is leaves used as a medicine. Ancient books recorded as "medicine to stop the bleeding," it could disperse cold and relieve pain,warm meridians to stop bleeding,which was commonly used in gynecology. Modern experimental studies show that that Folium Artemisia Argyi has antibacterial, antiviral, and asthma,cholagogue function, inhibit platelet aggregation, and other pharmacological effects, There are also a variety of traditional Chinese medicine prescriptions or western medicine containing the effective constituent of Folium Artemisia Argyi,used for the treatment of cardiovascular system, digestive system, gastrointestinal system, as well as a variety of diseases such as uterine. The study found that main chemical components of Folium Artemisia Argyi including essential oil, coumarin,flavonoids, sterols and terpenoids, trace elements, etc.In the early stage of the research in vitro and vivo,we found that the volatile oil and ethyl acetate extract of Folium Artemisia Argyi have anti HBV activity for the first time,then researched the effective part,then researched it in the manufacture of anti-HBV drugs,this research was authorized 2 national invention patents. In order to further explore the ethyl acetate extract(EA)of anti-HBV effective parts, clear the material basis of efficacy, this topic will be on the basis of previous research studies, further to analysis the chemical composition of EA,and then studying on anti HBV activity of these monomer compounds.1、Preparation of different polar parts of EA. Crushingthe right amount of Folium Artemisia Argyi.First, extracting the volatile oil by steam distillation,collecting oil and filtering residue,drying residue.We extracted filter residues fortwice following by petroleum ether(30~60℃) and ethyl acetate,then we obtained ethyl acetate extract(EA),we charged for 10 kg, and then got EA for 206.873 g.Then we extracted EA for twice following by petroleum ether(60~90℃), petroleum ether-ethyl acetate(1: 1), 60% ethanol, the extracts are EA-1(96.821g), EA-2(66.121g),EA-3(12.520g) and after extracting by 60% ethanol EA-4(5.911g).2、Separation and purification of EA-1、EA-2、EA-3 and EA-4. We separated and purified 12 compounds from these four parts by silica gel column chromatography、recrystallization 、 preparative thin layer chromatography, etc. Compounds are analyzed by the system pre-experiment preliminary: compounds 1,2,3,5 were steroidal, triterpenoids, compounds 4,6,7,8,9,10 were flavonoids, compounds 11 and12 were coumarin.3、Structure identification of Compound 1~12.Through the TLC high and low concentration、three kinds of solvent system and different color conditions to identify these 12 compounds : each compound purity greater than 99%.Analysing and identifying by TLC, 1H-NMR, 13C-NMR, and MS :compound 1 and compound 5were the same compound hentriacontane; compound 2 and compound 3 were the same compound stigmasterol;compound 4,7,8 and 10 were the same compound 5,4’-dihydroxy-6,7,3’- trimethoxy flavone; compound 9 was 5,7,4’-trihydroxy-3 ’,8-dimethoxy flavone; compound 11 and compound 12 were obtained by preparative thin layer method from the treatment ofⅠ-F、Ⅱ-E、Ⅱ-G and Ⅲ-C; compound 11 was an umbrella shaped flowers lactone, that is 7-hydroxycoumarin; compound 12 was scopoletin, that is 7- hydroxyl 6 methoxy coumarin.Compound 6 due to lack of related two-dimensional maps, some of the peaks can not be attributed, therefore unable to determine its structure.4 、 The different compound experiment against HBV in vitro.We chosed HepG2.2.15 as cells model.There were seven sample, respectively, compound 2(stigmasterol),compound 4(5,4’- dihydroxy-6,7,3’- trimethoxy flavone),compound 6,compound 9(5,7,4’-trihydroxy-3 ’, 8-dimethoxy flavone), compound11(7-hydroxycoumarin), compound 12(7-hydroxyl-6-methoxy-coumarin)and compound FAA20120831A(dammaradienylacetate). We detected the toxic effects of different components of HepG2.2.15 cells by MTT method and detected the effects ofHBsAg and HBeAg secretion in HepG2.2.15 cells supernatant by ELISA method.The results showed:each compound have certain toxicity on the HepG2.2.15 cells,the half toxic concentration(TC50) of compound 2 was 117.7μg·mL-1,the TC50 of compound 4 was 51.3μg·mL-1,the TC50 of compound 6 was 33.0μg·mL-1, the TC50 of compound 9 was 14.8μg·mL-1, the TC50 of compound 11 was 30.4μg·mL-1, the TC50 of compound 12 was 65.0μg·mL-1, the TC50 of FAA20120831 A was115.3μg·mL-1. Each compound had inhibitory effect on HBsAg and HBe Ag,besides,they presented time and dose-dependent, the half inhibitory concentration(IC50) of compound 4 to HBeAg was 11.5μg·mL-1, the IC50 of compound 11 to HBsAg was14.5μg·mL-1;For 9 days, the therapeutic index(TI) of compound 4 to HBeAg was4.46, the TI of compound 11 to HBsAg was 2.10. The experimental results show that,each compound have better activity against HBV.In this topic,We further to research the constituents of argyi EA parts for its anti HBV active.And then we isolated and purified for twelve compounds from it,and then identified the structural with them.Then seven tested samples were screened in vitro activity. The results showed that all the compounds had good activity against HBV, which provide a basis for activity screening against HBV in vivo and anti HBV effective parts of the new drug.