Studies On The Chemical Constituents And Anti-HBV Activity In Vitro Of Viola Diffusa Ging

Hepatitis B virus(HBV)is the main etiology of acute and chronic hepatitis,cirrhosis,liver cancer and some other diseases.Chronic HBV infection finally can develop to liver cirrhosis,liver cell carcinoma.In our country,nearly 2 billions of people have infected with HBV,about 250 thousand people died of chronic liver disease(hepatocellular carcinoma and cirrhosis of the liver)which related with hepatitis B,and the incidence rate is rising unceasingly each year.Interferon therapy and nucleoside drugs are the main treatment methods for HBV infections,but the treatment effects are very limited due to the scavenge difficulty and drug resistance.Traditional Chinese medicine(TCM),which has been widely used for thousands of years to treat chronic hepatitis B,may provide an alternative source for the discovery of potent anti-HBV agents.1 backgroundViolaceae Viola plants with about 500species,widely distributed in tropical,temperate,and subtropical.There are about 111 species in China.Most Violaceae Viola plants have the effects of heat-clearing,detoxicating,cooling the blood and detumescence by the aerial parts.They are widely applied to furunculosis,swelling,superficial infection,carbuncle of the back,erysipelas,and all kinds of inflammation in folk,also be used in the treatment of wind heat cough,lung consumption,asthma,rhinitis,keratitis,pharyngitis,mastitis,rheumatism.Viola diffusa Ging,also known as Qixinglian,Huanguaxiang,Pufujin,Dibaicao or Tianjiecai,a perennial herb belongs to Viola genus of Violaceae,is widely distributed in south of the Yangtze river.Viola diffusa Ging is cold and slightly bitter in nature,and has effects on heat-clearing and edema-reducing,pus-discharging and detoxicating,promoting tissue regeneration and bonesetting.People have used its aerial parts satisfactorily as medicine for the treatment of hepatitis,carbuncle toxin,wind-heat cough,eyelid inflammation,gingivitis,scald,snakebite."Fujian Drug records" says Viola difusa Ging drug has obvious effect in the treatment of hepatitis.Traditional Chinese Medicine Department in NO.4 hospital of Hangzhou use Viola diffusa Ging Mistura in treatment of acute jaundice hepatitis,and jaundice index returned to normal for only 3 days.People usually mash fresh Viola diffusa Ging for topical application to treat cut hemorrhage.According to"Fujlan plant records",flavonoids,polysaccharides and mucilaginous are the mainly chemical compositions of Viola diffusa Ging.Modern pharmacological experiment proves that Viola diffusa Ging has anti-inflammatory,antioxidant,and antitumor activities.The resource of Viola diffusa Ging is abundant,the treatment effects for hepatitis is definite according to folk medicine and clinical experience,but there has been little reports about the chemical constituents and bioactivities of Viola diffusa Ging up to now.In view of this,the research on the chemical constituents and anti-HBV activity in vitro of Viola diffusa is carried out in this thesis,which will provide a scientific and reasonable basis for the further research,development and utilization of Viola diffusa.2 ObjectivesIn this thesis,we studied systematly on the chemical constituents and anti-HBV activity in vitro of Viola diffusa Ging,and established the methods for the determination of total flavonoids and triterpenoids in Viola diffusa Ging.The research work will provide a scientific basis for confirming the active components in Viola diffusa,establishing the quality standards and searching a novel anti-HBV drug or leading compound.3 Methods3.1 Phytochemical research:The dried aerial parts of Viola diffusa(10kg),collected from Yongchun County Fujian,was refluxed with hot 95%ethanol and concentrated under reduced pressure to give a deep brown extractum.The extractum was partitioned successively with petroleum ether,ethyl acetate and n-butanol.Petroleum ether Part(18 g),Ethyl acetate Part(75 g)and n-Butanol Part(168 g)were directly treated by column chromatography packed with D-101 Macroporous resin,silica gel,Sephadex LH-20,and preparative thin-layer chromatography.The compounds were isolated and their chemical structures were elucidated by of their physico-chemical properties and modern spectroscopic analysis,including 1H-NMR?13C-NMR?DEPT?HMBC?HMQC?MS?HR-MS?NOESY.,3.2 in vitro Anti-HBV experiment:Compound 1,2,3,4,5,6,7,8,9,12,13 were prepared into six concentrations(100,50,25,12.5,6.25,3.125?mol/L)respectively,and their toxic effects on HepG2.2.15 cell lines were investigated by MTT colorimetric method.The anti-HBV effects in vitro of these compounds were also evaluated by detecting the contents of HBsAg,HBeAg in supernatant fluid of HepG2.2.15 cell lines by ELISA method.SPSS 13.0 software was used for statistical analysis.The experimental data were represented as x±s(mean±SD).For OD value of the same compound in different concentrate,one-way ANOVA was applied after testing of homogeneity of variance.If variance was of homogeneity,LSD method for pairwise comparison between groups was adopted.When variance was of heterogeneity,Dunnett's T3 method for pairwise comparisons between groups was employed.Significant level was set at ? = 0.05 and P<0.05 was considered statistically significant.3.3 Content determination:Oleanolic acid and rutin were used as the reference substances respectively,5%vanillin-acetic acid-perchloric acid and 5%NaNO2-10%AI(NO3)3-4%NaOH as the developer,and the contents of total triterpenoids and total flavonoids in Viola diffusa Ging were detected at 555 nm and 408 nm respectively by ultraviolet spectrophotometry.4 Results4.1 Nineteen compounds were isolated from the Viola diffusa Ging extractum and thirteen compounds were elucidated.These compounds are listed as follows:violaic A(1),violaic B(2),violalide(3),epifriedelano(1 4),friedelin(5);clerosterol(6),clerosterol galactoside(7),decorinone(8),decortinol(9),isodecortinol(10),stigmast-25-ene-3?,5?,6?-triol(11),cerevisterol(12),palmitic acid(13).Among them,compound 1,2,3,11 are new compounds,and compound 4,7,8,9,10,12 are isolated from this genus for the first time.4.2 Compound 1,2,3,9,12 of all different concentration groups could inhibit the secretion of HBsAg(IC50 separately for 26.2 ?mol/L?33.7 ?mol/L?104.0?mol/L?62.0 ?mol/L?112.8 ?mol/L)and HBeAg(IC50 separately for 8.0 ?mol/L?15.2 pmol/L?21.6 ?mol/L?49.6 ?mol/L?35.0?mol/L).Compound 4,6,7,8 selectively inhibited HBeAg secretion,and the inhibition rate of Compound 8 amounted to 40.7%at the concentration of 12.5?mol/L.Compound 13 selectively inhibited HBsAg secretion,and the inhibition rate was 53.1%in 100 ?mol/L.Compound 5 did not show any anti-HBV effects in the range of the observation concentration.4.3 Treatment of HepG2.2.15 cells with drug concentrations(100,50,25,12.5,6.25,3.125 ?mol/L)resulted in the reduction of the survival rate in a dose dependent manner.TC50 of Compound 3,4,5,6,8,9,13 was more than 100 ?mol/L,and thus showed the low cell toxicity;TC50 of compound 7,12 was 77.9?mol/L,89.6?mol/L respectively and exhibited mild cell toxicity.Compound 1,which the cell survival rate was very low and TC50 was 30.4mol/L at the concentration of 50 ?mol/L showed the highest toxicity.Compound 2 in100 ?mol/L,cell survival rate was 47.0%,TC50 was 84.6mol/L,also showed some toxicity.DMSO had no significant effect on the experimental results.It concluded that Compound 1 showed the highest toxicity,followed by compound 2,7,12,Compound 3 4,5,6,8 was less toxic,and Compound 9,13 was the smallest.4.4 TI of Compound 2,3,9 were more than 2,and belonged to the effective,low toxic drugs;Compound 1 and 12 belonged to the less effective,toxic drugs because TI of the two compounds to HBsAg was 1.16 and 0.8 in spite of TI to HBeAg was more than 2.Compound 4,6,7,8 belonged to the effective,low-toxic drugs,while Compound 4,6,and 8 selectively acted on HBeAg and Compound 7 acted on HBsAg.4.5 Among Compound 1?5 with friedelane type structure,Compound 1,2,3 showed anti-HBV activity both on HBsAg and HBeAg,and Compound 1,2 showed significant effects.Compound 4 selectively inhibited HBeAg,and Compound 5 had no anti-HBV activity.Analysis the structure-activity relationship of the five compounds,it suggested that seven lactone in A ring of triterpenoids may be an important unit for the anti-HBV activity.Additionally among all the compounds,the substituted group of C-3 was 3-OH or 3-O-glycoside,had inhibition effects on HBsAg and/or HBeAg except Compound 5 with C-3 ketone group,and it deduced that C3-OH or 3-O-glycoside was oxided to ketone group,the activity of the compounds lost,thus C-3 substituted group may influence the anti-HBV activity to some extent.4.6 The established methods for determination of flavonoids and triterpenoids in Viola diffusa Ging were accurate,simple and rapid.The total triterpenoids showed a good linear relationship at the range of 0.004?0.036mg/mL,and the average recovery was 101.3%,RSD was 4.42%(n = 9);the total flavonoids showed a good linear relationship at the range of 0.01003?0.06018mg/mL,and the average recovery was 108.0%,RSD was 4.92%(n = 9).The contents of total triterpenoids and total flavonoids in Viola diffusa Ging were 1.14mg/g and 55.25mg/g respectively..5 ConclusionsIn this thesis,19 compounds were isolated from the 95%ethanol extracts of Viola diffusa Ging,13 of them were eluciated,including 5 friedelane type triterpenoids,7 stigmastane compounds and 1 fatty acid.Among them,compound 1,2,3,11 are new compounds,and compound 4,7,8,9,10,12 were isolated from this genus for the first time.Evaluated by TI,Compound 2,3,9 belonged to effective,low toxic drugs;compound 1,12 belonged to less effective,toxic drugs;Compound 4,6,7 and 8 selectively inhibited HBeAg and Compound 4,6 8 belonged to effective,low-toxic drugs,Compounds 7 belonged to less effective,toxic drugs;Compound 13 selectively inhibited HBsAg and belonged to effective,low-toxic drugs.The established methods for determination of total flavonoids and triterpenoids in Viola diffusa Ging were accurate,simple and rapid.The studies of Viola diffusa Ging on the chemical constituents and anti-HBV activity in vitro provides a scientific basis to searching a novel anti-HBV drug or leading compound.