Studies On The Anti-HBV Effect And Chemical Constituents Of Geranium Carolinianum L.

Laoguancao,which is a traditional Chinese medicine,has driving away wind-dampness and opening channels and collaterals effects,and in clinic curing symptoms of wind-dampness flaccidity,spasm numbness and unconsciousness. Geranium carolinianum L.is used as Laoguacao in folk.It has been reported that an aqueous extract of this plant can improve the clinical symptoms of HBV infected patients.Therefore it was supposed that Geranium carolinianum L.might possess the activity to inhibit the replication of HBV and the expression of viral antigens. However,its direct anti-viral avtivity against HBV has not been reported before.In this study,the antiviral activity of the total flavonoids of Geranium carolinianum L.against HBV was investigated.To our knowledge,this is the first report of the anti-HBV effects of the total flavonoids extract from G carolinianum L.Our observations suggest that the total flavonoids have anti-HBV activity and the potential to be developed as an alternative or complementary anti-HBV agent.In order to identify the active ingredients,15 compounds were isolated and purified from total flavonoids fraction of G.carolinianum L.by chromatography on silica gel column, sephadex LH-20 chromatogram,including ellagic acid,geraniin,hyperin.Geraniin inhibited HBsAg and HBeAg secretion by more than 85.8%and 63.7%,respectively, at the non-cytotoxic concentration of 200μg/ml.The inhibitions of HBsAg and HBeAg secretion by geraniin were higher than the inhibition by the positive control Lamivudine,33.5%and 32.2%respectively,at the same concentration.This is the first report of the anti-HBV effects of geraniin and hyperin,the active substances derived from G.carolinianum L.The presence of the anti-HBV compounds may account for the effectiveness of this folk medicine in the treatment of HBV infections.In addition, relationship between structure and anti-HBV activity of isolated flavonoids was first reported.Part 1 Separation and confirmation of anti-HBV effective part of G.carolinianum L.1.It has been confirmed that G.carolinianum L.has anti-HBV activities from many herbs.The ethanol extract of Geranium carolinianum L.was subjected to sequential extractions with different organic solvents.The extracts were assayed for anti-HBV activities.The ethyl acetate fraction was found to contain the highest level of anti-HBV activity and further confirmed to have many flavones.2.Separation and purification of total flavonoids from G.carolinianum L.by macroporous adsorption resin.Through orthogonal experiments of normal reflux extraction,crude extract was obtained from G.carolinianum L.by reflux extraction using 50%ethanol for three times,1:8 of material to ethanol.It were compared that the ability of adsorption and separation total flavonoids in 5 different type of macroporous resin by means of static adsorption test and dynamic test,it showed that the total ability of low pole D-101 resin was superior to the other macroporous resin. All process conditions influencing on the process of separation and purification total flavonoids with D-101 resin were investigated in detail.The optimal technological conditions were:the concentration of the sample of G.carolinianum L., 1.0g/ml(amount of crude drug),the maximum adsorption capacity for total flavonoids, 21.23mg/g;the optimal column ratio of diameter to height,1:12;the flow velocity of adsorption,1 ml/min;the eluting reagent,70%ethanol(5 times of the volume of the resin);eluting velocity,1 ml/min.The technology of separating and purifying total flavonoids from G.carolinianum L.with D-101 was stable and effective.Part 2 In vitro and in vivo anti-hepatitis B virus activities of total flavonoids from Geranium carolinianum L.1.In pharmacological experiment,MTT assay was using to detect the toxicity of the prolifer of HepG₂2.2.15 cells treated with total flavonoids from G.carolinianum L.(TFGC) at varied concentration.The TC₅₀ is 152.13μg/ml.There was no significant difference of cell viability between TFGC-treated groups whose concentrations were below 100μg/mL2.In vitro:Treatment of HepG₂ 2.2.15 cells with TFGC at various concentrations (100μg/mL,50μg/mL,25μg/mL,12.5μg/mL and 6.25μg/mL for 10 days resulted in a significant reduction of Hepatitis B surface antigen(HBsAg) and Hepatitis B e antigen(HBeAg) secretion in HepG₂ 2.2.15 in a dose-dependent manner,with IC₅₀ values of 68.39μg/ml and 72.06μg/ml respectively.TFGC was more potent than Lamivudine(3TC) for inhibiting both HBsAg and HBeAg secretion.To further confirm the anti-HBV activity of TFGC in HepG₂ 2.2.15 cells,the HBV DNA levels were evaluated after TFGC treatment by Southern Blot.Consistent with the inhibitory effects on HBsAg and HBeAg secretion,Treatment of HepG₂ 2.2.15 cells with TFGC at various concentrations for 10 days resulted in the reduction of the intracellular HBV DNA levels in a dose-dependent manner.TFGC(100μg/ml) reduced intracellular HBV DNA level by 38.56%.By Flurescence Quantity PCR assay, the inhibit dosage curve of TFGC to intracellular HBV DNA of HepG₂2.2.25 showed "S" shape during 10 days monitering.The inhibition ratio is 35.9%.3.In vivo:The duck DHBV model represents a suitable and a widely used system for the study of in vivo activity of anti-HBV agents as well as their toxicity.We fed the ducks with TFGC at various concentrations(112 mg/kg,56 mg/kg,28 mg/kg)(i.g.) once daily for 10 days,no toxicity was observed.To examine the in vivo anti-HBV activity of TFGC,we first checked the plasma DHBV DNA levels of the infected ducks with and without treatment.TFGC reduced plasma and liver DHBV DNA levels in a dose-dependent manner.At dosage of 112 mg/kg,TFGC significantly inhibited DHBV DNA levels in the duck plasma.Densitometric quantitation of the dot blots revealed decrease in DHBV DNA levels(57.07%,69.00%and 57.84%for Day 5,10 of the TFGC treatment and 3 days alter the cessation of treatment respectively,n-6) in ducks treated with 112 mg/kg TFGC,as compared with the untreated ducks.Moreover, the rebound of the DHBV DNA levels in TFGC treated ducks was to a less extent as compared with the 3TC treated group.No significant differences were observed in the DHBV DNA levels of any of the controls.To further confirm the in vivo anti-HBV effect of TFGC in ducks,the DHBV DNA levels in the livers(obtained at days 5,10 of PPGC treatment and 3 days after the treatment was stopped) were examined by Southern hybridization analysis.Consistent with the inhibitory effect on the plasma DHBV DNA level,TFGC treatment dose-dependently reduced the DHBV DNA levels in the liver.Densitometric analysis of the autoradiographic signals indicated 26.96%,45.37%,and 29.12%inhibition (days 5,10 during the treatment and 3 days after the cessation of the treatment respectively) due to the TFGC treatment(112 mg/kg),whereas 3TC-treated(200 mg/kg) groups resulted in 43.98%,59.74%,and 40.06%inhibition at these three data points.To evaluate the pathological changes,liver sections from the above treatment groups were examined under the light microscope.In the control group,significant edemas could be observed in the endoplasmic reticulum,indicating the DHBV expression.Samples treated with TFGC,on the other hand,exhibited dose-dependent improvement in the edema formation.It is worth noting that TFGC at 112 mg/kg resulted more significant improvement than 3TC at 200 mg/kg.Part 3:Chemical constituents of total flavonoids and their anti-HBV activities15 compounds have been isolated from the total flavonoids of Geranium carolinianum L.Their structures were deduced on the basis of their spectral data and identified as hyperoside,hirsutrin,quercetin,quercetin-3-O-β-D-(6"-galloyl)galactoside],ellagic acid,gallic acid,kaempferol,myricetin,protocatechuic acid,corilagin,geraniin,kaempferol-7-O-rhamnoside,quercitrin,kaempferol-7-O-α-L-arabinofuranoside,Vitexin.Except hyperin,other compounds were isolated from this plant species for the first time.quercetin-3-O-β-D-(6"-galloyl)galactoside] and vitexin were isolated from the genus Geraniaceae for the first time.The structure activity relationship of structure and flavonoids anti-HBV effect were studied so as to find some regularities,which may provide the foundation for their exploitation and utility.Hydroxyl groups in the ring B were main active groups anti-HBV.The glucoside contributed to anti-HBV effects,which varied with sort of the glucoside.The phenolic hydroxyl compounds usually have anti-HBV activityThe HPLC was developed to determine the content of hyperin in total flavonoids of Geranium carolinianum L.The content of hyperin is more than 2.5%in the total flavonoids.