Suppressed effects of Phyllanthus urinaria L.ethyl acetate extract on hepatitis B virus both in vitro and in vivo

Background:Phyllanthus urinaria L.(P.urinaria)extract(PUE)has been used to inhibit hepatitis B virus(HBV).However,the underlying mechanism remains unclear.To investigate which PUE fractions and main components lead to against HBV and approach the relevant molecular mechanisms.Methods:P.urinaria was extracted with water,and then the decoction was extracted by petroleum ether,ethyl acetate,and n-butanol in turn.The HepG2.2.15 cell was treated with aqueous fraction,petroleum ether fraction,ethyl acetate fraction and n-butanol fraction,gallic acid(GA,C7H6O5)and corilagin(CL,C27H22O18),respectively.The medium was collected for hepatitis B surface antigen(HBsAg)and hepatitis B e antigen assays.Cell counting kit-8 method was used to identify cell proliferation.Also,the levels of cellular oxygen consumption,reactive oxygen species,and reduced glutathione were detected.The HBV modeling mice were treated with ethyl acetate fraction,entecavir and physiological saline,respectively.The serum was collected for HBsAg and inflammatory cytokines assays.Liver tissue metabolites were screened by LC-MS/MS method.Results:The ethyl acetate fraction(EAF)of P.urinaria could significantly inhibit HBV secretion in HepG2.2.15(P<0.05).Furthermore,two main constitutes in ethyl acetate fraction,GA and CL,could significantly inhibit HBV secretion and reduced cell proliferation(P<0.05).Also,GA and CL could increase cellular oxygen consumption,intracellular superoxide anions level,superoxide dismutase level and glutathione depletion.Compared with the Modeling group,EAF significantly decreased the expression levels of HBsAg,IL-1β,IFN-α(P<0.05).LC-MS/MS analysis results showed that EAF dramatically up-regulate hydroxyproline,maltotriose,betaine and down-regulate glutathione disulfide,taurocholate,taurochenodeoxycholate(P<0.05).Kyoto Encyclopedia of Genes and Genomes results show that the differential metabolites were mainly enriched in ATP-binding cassette transporters pathway.Conclusions:P.urinaria exhibits suppressed effects on HBV by modulating reactive oxygen species formation or metabolomics both in vitro and in vivo.These data indicate that P.urinaria may be an alternative therapeutic agent for the treatment of HBV-related hepatitis.

Network pharmacology and experimental validation to reveal the anti-hepatitis B virus pharmacological mechanism of Phyllanthus urinaria L.

Background:To explore the pharmacological mechanism of the anti-hepatitis B virus of Phyllanthus urinaria L.through network pharmacological analysis and experimental validation.Method:The active ingredient,target of action and target of action related to hepatitis B were clarified by searching the herb group identification,GeneCards and OMIM databases,and the protein interaction relationship was obtained by using the String database,and the protein interaction network map was constructed by using Cytoscape software.We also performed gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis of key targets of the anti-hepatitis B action of Phyllanthus urinaria L.and predicted the core targets and pathways of Phyllanthus urinaria L.anti-hepatitis B.The main targets predicted by network pharmacology were then validated by HepG2.2.15 cell experiments.Results:By searching active ingredient targets and hepatitis B disease targets,a total of 19 active ingredients and 64 related targets of action were retrieved from Phyllanthus urinaria L.,and a total of 51 common targets were obtained by mapping the obtained hepatitis B disease targets and drug targets.protein protein interaction network analysis indicated that targets including TNF,JUN,AKT1,IL-10,IL-1B,CAT,HMOX1,NFE2L2,and CASP3 and other targets may be the core targets.gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that the treatment of hepatitis B by Phyllanthus urinaria L.mainly included inflammation and oxidation-related processes,and the signaling pathways mainly included fluid shear stress and atherosclerosis,VEGF,and hepatocellular carcinoma.The results of the in vitro test showed that after the action of different concentrations of the extracts of the Phyllanthus urinaria L.in the safe concentration range on cells HepG2.2.15,HBsAg,HBeAg and hepatitis B virus DNA levels were significantly inhibited,and NFE2L2 and HMOX1 were affecting hepatitis B virus transcription and replication by regulating the oxidative stress response.Conclusion:Using an integrated network pharmacology approach,this study revealed the active components and potential targets of Phyllanthus urinaria L.for the treatment of the hepatitis B virus,providing a theoretical basis for the research and clinical application of Phyllanthus urinaria L..

Screening of Anti-Newcastle Disease Virus Ingredients of Phyllanthus urinaria

[ Objective ] The aim of the study was to screen the antiviral ingredients of Chinese herbal medicine Phyllanthus urinaria in order to devel- op new drugs for the treatment of viral diseases of poultry. [ Method ] The whole grass of phyllanthus was respectively decocted in 75% ethanol （E） and pure water （PF）, and then the active ingredients were separately extracted in petroleum ether （PE）, ethyl acetate （EA）, and n-butyl alcohol （BU）. The extracts were inoculated on chicken embryo fibroblast （CEF） to observe their inhibitory effect on cytopathic effect （CPE） of Newcastle disease virus （NDV）, inoculated into chicken embryos to observe the changes of the hemagglutination titer of NDV, and inoculated in chickens to determine the mortality and evaluate their effect of immune protection. [ Result] The E -PE and E -BU extracts, especially E -BU extract, inhibi- ted CPE of NDV on CEF and significantly inhibited the proliferation of NDV in chicken embryo （P〈0.05） ; the survival rate of E-PE and E-BU group was extremely significantly higher than that of ribavirin group and the normal saline control （ P 〈0.01 ）, with E - PE group more higher. [ Condusion] The water extract of phyllanthus is less effective; E -PE extract and E -BU extract should effectively inhibit the CPE and proliferation in chicken embryo of NDV and also have better immune protection against NDV.

Neuroprotective effect of Phyllanthus urinaria extract on ischemic brain injury in rats

Objective:To investigate the neuroprotective effect of Phyllanthus urinaria on ischemic stroke and its primary mechanism.Methods:Adult SD rats were selected as the research object,and the right middle cerebral artery infarction rat model was established by the modified suture method(MCAO).Observe the neurological deficit score at 24h,48h and 72h after the model is successfully prepared,then TTC staining method to detect the area of cerebral infarction,the content of superoxide dismutase(SOD),nitric oxide(NO)and endothelial NOS(eNOS)in brain tissue;Immunofluorescence method was used to detect the expression of Caspase-3 positive cells in brain tissue;Western blot method was used to detect the expression of PI3K and AKT protein in brain tissue.72 experimental animals were randomly divided into 4 groups,sham operation group,model group(MCAO),extract of Phyllanthus urinaria low dose group(PuL5 g/kg),high dose group(PuL10 g/kg),and make MCAO model after 7 days of continuous administration,and continue to infuse the medicine once/d until the material is obtained.Results:No neurological deficits in the sham operation group,The 24h,48h and 72h of the modelling showed that the neurological impairment of the two doses of extract of Phyllanthus urinaria and the MCAO group was more severe than that of the sham operation group(P<0.01),however,with the prolongation of the modeling time,the neurological function scores of the two doses of extract of Phyllanthus urinaria were lower than those of the MCAO group,the most significant at 72h(P<0.01);The infarct size of the rats in the two dose groups of extract of Phyllanthus urinaria was lower than that of the MCAO group(P<0.01),and there was no dose dependence between the two groups,the content of SOD in the MCAO group was reduced,and the content of NO and eNOS was increased than the sham operation group(P<0.05),Compared with the MCAO group,the two administration groups significantly increased the content of SOD,decreased the content of NO and eNOS(P<0.05);Although the expression of Caspase-3 positive cells in the two administration groups was higher than that in the sham operation group,it was significantly lower than that in the MCAO group(Plow<0.05,Phigh<0.01);The expression of PI3K and AKT protein in the brain tissue of the MCAO group was significantly lower than that of the sham operation group(P<0.05),but the expression of PI3K and AKT protein in brain tissue of extract of Phyllanthus urinaria in high and low dose groups was significantly higher than that in MCAO group(P<0.05).Conclusion:Extract of Phyllanthus urinaria can improve neurological damage and cerebral infarction area in rats,and reduce the expression of Caspase-3 positive cells in MCAO rats,and then increase the expression levels of PI3K and AKT proteins to protect ischemic brain injury.

Extract from Phyllanthus urinaria L.Inhibits Hepatitis B Virus Replication and Expression in Hepatitis B Virus Transfection Model in Vitro

Objective： To explore the effects of the extract from Phyllanthus urinaria L. on hepatitis B virus （HBV） replication and expression in HBV transient transfection model in vitro. Methods： The eukaryotic expression plasmid pHBVI.1, which contains l.l-fold-overlength genome of HBV, was transfected into the human hepatoma cell line, HepG2, to establish and assess the HBV transient transfection model. The extract from Phyllanthus urinaria L. was prepared in different concentrations and methyl thiazolyl tetrazolium was used to detect the maximum nontoxic concentration of the drug. The extract from Phyllanthus urinaria L. were added into the transfected cell, at the concentrations of 0.8, 0.2 and 0.05 g/L, respectively. Four days after drug application, enzyme-linked immuno sorbent assay was used to detect the concentration of HBsAg in the supernatants, Southern blot was applied to analyze HBV DNA level, and Western blot was used to detect the expression of HBcAg in cells. Results： After the transfection of plasmid pHBVI.1 into HepG2 cells, the concentration of HBsAg in supernatants was increased obviously as compared with that of the normal cells （P〈0.05）, and all expected HBV replicative intermediates were confirmed by Southern blot analysis, which ensured the successful establishment of the HBV transient transfection model. After the application of drugs at the concentrations of 0.8 and 0.2 g/L, the level of HBsAg was obviously decreased in the supernatants, as compared with that of the virus group （P〈0.05）; Southern blot showed that the level of HBV rc DNA, ds DNA, ss DNA was obviously reduced compared with that of the virus group （P〈0.01）; Western blot revealed that the expression of HBcAg in the drug group was obviously inhibited, as compared with that of the virus group （P〈0.01）. Conclusions： The extract from Phyllanthus urinaria L. obviously inhibited replication and expression of HBV in HBV transfected cell lines in vitro, thus exerting distinctive anti-HBV effects.

Efficacy of Early Treatment on 52 Patients with Preneoplastic Hepatitis B Virus-Associated Hepatocellular Carcinoma by Compound Phyllanthus Urinaria L.

Objective:To observe the change in the number of antibodies of preneoplastic hepatocellular carcinoma(HCC) using early treatment by Compound Phyllanthus Urinaria L.(CPUL) on patients with preneoplastic hepatitis B virus(HBV)-associated HCC.Methods:A total of 102 cirrhosis patients with regenerative or dysplastic nodules whose sera were tested positive for at least one of these six proteins(five up-regulated genes URG4,URG7,URG11,URG12 and URG19,and one down-regulated gene DRG2) were assigned randomly to two groups using continual random codes by SPSS software.Fifty-two patients were in the treatment group and 50 patients were in the control group.CPUL was used in the treatment group for 3 years,while the control group did not receive any treatment.The changes in HBV-DNA level,number of antibodies,and hepatocarcinogenesis occurred were observed.Patients who did not develop HCC were followed up for another 2 years.Results:HBV-DNA levels decreased >2log in 22.2%(10/45) of patients in the treatment group in contrast to only 5.0%(2/40) of patients in the control group(P=0.0228).The number of antibodies that were tested positive in the treatment group(1.08± 1.01)was significantly lower compared with the control group(2.11 ±1.12) after 24 months of drug treatment(P<0.01).Both the positive rates of anti-URG11(33/52) and anti-URG19(31/52) were over 60%at baseline in the two groups,and were decreased to 48.1%(25/52) and 46.2%(24/52) respectively at 36 months of drug treatment,while the rates increased to 68.0%(34/50) and 66.0%(33/50) respectively(P=0.0417,P=0.0436) in the control group.The positive rate of anti-DRG2 was increased to 55.8%(29/52) at 36 months of drug treatment,while in the control group was decreased to 36.0%(18/50,P=0.0452).Among the 102 patients who developed HCC,2 were in the treatment group and 9 were in the control group,meaning that a significant difference between the two groups(P=0.0212).In11 patients who developed HCC,anti-URG11 and anti-URG19 were always positive,while anti-DRG2 was negative.Patients newly developing HCC were 6(20.0%) in the control group,and only one(2.5%) in the treatment group(P=0.0441) during 2-year follow-up after the end of the treatment.Conclusions:Anti-URG11,anti-URG19 and antiDRG2 could be used as early markers in the prediction of the therapeutic efficacy of CPUL in treating preneoplastic HCC.CPUL is useful in preventing or delaying the development of HBV-associated cirrhosis to HCC.

Triterpenoids from Whole Plants of Phyllanthus urinaria

Objective To study the chemical constituents of whole plant of Phyllanthus urinaria and their biological activity.Methods The chemical constituents were isolated and purified by repeated column chromatography over silica gel,Rp-C18（reverse phase）,MCI,and Sephadex LH-20,as well as semi-preparative HPLC.NMR spectroscopic analyses were used for the structure identification.In this case,the inhibitory rate of NO production of the isolated triterpenoids was evaluated.Results Seven triterpenoids,identified as28-norlup-20（29）-ene-3,17β-diol（1）,betulin（2）,β-betulinic acid（3）,3-oxofriedelan-28-oic acid（4）,oleanolic acid（5）,3R-E-coumaroyltaraxerol（6）,and 3R-Zcoumaroyltaraxerol（7）,were isolated and identified from the whole plants of P.urinaria.Compounds 1-5 exerted inhibitory effects against NO production in LPS-induced RAW264.7 mouse macrophages with the inhibitory rate of NO production ranging from 4.0%to 27.8%at the concentration of 25 μmol/L.Conclusion To the best of our knowledge,this is the first report of compounds 1-4,6,and 7 from the family Euphorbiaceae.Compounds 1-5 exhibited inhibitory effects against NO production in LPS-induced RAW264.7 mouse macrophages.