Inhibition of Hepatitis B Virus Replication by Rheum palmatum L. Ethanol Extract in a Stable HBV-pro

VIROLOGICA SINICA Feb 2007, 22(1): 14-20CLC number: R373 Document code:A Article ID: 1003-5125(2007)01-0014-07Inhibition of Hepatitis B Virus Replication by Rheum palmatum L. EthanolExtract in a Stable HBv-producing Cell LineYan sUN, Li-jun Ll2. 3, Jing Lr, Zhi LI**(1. College ofLife Sciences, Shaanxi Normal University, Xi'an, Shannxi, 710062; 2. Bioengineering Institute, JimeiUniversity, Xiamen, Fujian, 361021; 3. Department of Applied Biology and Chemical Technology, The Hong KongPolytechnic University, Hung Hom, Hong Kong: 4. Department of Infection Disease, Xiangfan Central HospitalXiangfan, Hubei, 441021; 5. The Institute of Molecular Biology, The University of Hong Kong.Abstract: Hepatitis B virus(HBV)infection is a severe health problem in the world. However, there isstill not a satisfactory therapeutic strategy for the HBV infection. To search for new anti-HBV agents withhigher efficacy and less side-effects, the inhibitory activities of traditional Chinese medicine rheumpalmatum L ethanol extract(RPE)against HBV replication were investigated in this study. Quantitativereal-time polymerase chain reaction( PCR) was employed to analyze the inhibitory activity of RPe againstHBV-DNA replication in a stable HBV-producing cell line Hep AD38; the expression levels of HBVsurface antigen (HBsAg) and e antigen (HBeAg) were also determined by enzyme linked immunosorbentassay (ELISA)after RPE treatment. RPE could dose-dependently inhibit the production of HBV-DNAand HBsAg. The concentration of 50% inhibition(ICso)was calculated at 209.63, 252.53 ug/mL, respetively. However, its inhibitory activity against HBeAg expression was slight even at high concentrationsRPE had a weak cytotoxic effect on HepAD38 cells( CCso=1 640 Hg/mL) and the selectivity index(si)was calculated at 7.82: Compared with two anthraquinone derivatives emodin and rhein, rPe showedhigher ability of anti- HBV and weaker cytotoxicity. So Rheum palmatum L. might possess other functional agents which could effectively inhibit hBv-DNA replication and HBsAg expression. Furtherpurification of the active agents, identification and modification of their structures to improve the efficacyand decrease the cytotoxicity are requiredKey words: Hepatitis B virus(HBV); Antiviral; Rheum palmatum L ethanol extract (RPE); HepAD38Hepatitis B is an infectious disease of the liver caused 350 million people are chronic carriers of HBV( 23, 2by Hepatitis B virus(HBV). Globally, about two billion 19, 5). These carriers are at high risk for developmentpeople have been infected by HBV and approximately of中国煤化 Eluding liver cirrhosis,CNMHGReceived:200605-31, Accepted:2006-07-06Equally contributing authorC呀芳毁author.le82930845.862353-mail:lizhi@snnu.edu.cnSUN et alInhibition of Hepatitis B Virus Replication by RPE in a Stable HBV-producing Cell Lineliver failure and hepatocellular carcinoma(HcC)and the commercial anthraquinone derivatives emodin andmore than one million carriers die from these diseases rhein which are derived from rheumeach year(23, 2). In China, the prevalence of HBV infection is very severe, with an estimated 120 millie1. Materials and methodschronically infected carriers, up to 12 million suffering 1.1 Preparation of herbal plant extract and anthrafrom chronic hepatitis B(5)uinone solutionsInterferon and lamivudine are the mainly acceptable The herbal plant Rheum palmatum L was purchasedantiviral drugs for inhibition of HBV replication( 14, 8). from a herbal materials supply house and verified.OneBoth of drugs can induce virological and biochemical kilogram of dried, ground rhizome was macerated inremission in the treatment of chromic hepatitis B, but 1 000 mL 95% ethanol. After incubation overnight, thetheir clinical chromic hepatitis B, but their clinical sample was subjected to ultrasonication for 30benefit is somewhat limited due to viral mutation, cost, minutes, followed by centrifugation. The residue wasfrequent adverse effects(7)and the likelihood of re- extracted twice, then the supernatant solutions werelapse after the termination of treatment. Moreover, only mixed, filtered, concentrated and lyophilized to give aabout 20% of patients benefit from combination therapy dark brown powder. The dried extract was re-resolvedwith interferon and lamivudine( 13, 9). So, the need to in dulbecco,s modified Eagle's medium(DmEmidentify effective anti- HBV agents is still urgently Gibco BRL) with 0.2%dimethyl sulfoxide(DMSOrequired. The herbal plant Rheum palmatum L is a tradi- Sigma, St. Louis, MO) to the appropriate concentra-tional Chinese medicine which is widely distributedtions. Emodin(1, 3, 8-trihydroxy-6-methylanthraquinonemainland China and which has a long history to treating CisHnoOs)and rhein (1, 8-dihydroxy-9, 10-anthraquino-gastroenteritic and viral diseases(4). Rheum palmatum ne-3-carboxylic acid, Cus HsO,)(Fig. 1. ) were purchasedL. contains several anthraquinone derivatives, which from Sigma Chemical Company and dissolved inhave showed activities against some viruses including ves DMEM with 0. 2% DMSO to the appropriate concentraicular stomatitis virus, herpes simplex virus types l and tions. The resulting solutions were used in the following2, parainfluenza, vaccinia virus, human cytomegalovi- analysisrus and poliovirus(22, 1, 3, 20). It was also reported that 1.2 Cells cultureRheum palmatum L. volatile oil could inhibit HBVThe Hepad3 8 is a human hepatocellular carantigens(HBsAg and HBeAg) expression(24). There- cinoma derivative cell line which can reproduce hepa-fore, it is supposed that Rheum palmatum L. might titis B viruses under control of tetracycline( 11). Hep-possess active agents which may inhibit many virusesHOHincluding HBV. In this study, we evaluated the cytotoxicity and the inhibitory activities of Rheum palmatum L.中国煤化工ethanol extract(rPE)against HBV-DNA replication andCNMHGHBV antigens expression in a stable HBV-producingFig 1 Chemical structure of emodin(R=OH, R-CH,)and rheincell line HepAD38, and compared these activities with (R= H, RCOOH)16VIROLOGICA SINICAVol 22 No. 1AD38 cells were maintained in DMEM supplemented were collected after 8 days treatment and separated bywith 10%FBS, 1% penicillin/streptomycin, 1% gluta- centrifugation at 10 000 x g at 4C for 5 minutes. Anmine, 100 ug/mL kanamycin, 400 ug/mL G418 and 0.3 aliquot of the culture medium was used for estimationug/mL tetracycline at 37C in an atmosphere of 5% COz of HBsAg and HBeAg. The remaining medium wasand 100% humidity. When the assay was started, cells processed to obtain HBV virions by using the polyethyvere washed with pre-warmed phosphate-buffered lene glycol (PEG) precipitation method (6). The viralsaline(PBS)and fed with medium without tetracycline. DNA recovered from the secreted HBV particles wasHBV replication in HepAD38 cells was induced by quantified by real-time PCR using a commercial real-withdrawal of tetracycline from the culture mediumtime PCR HBV diagnostic kit(PG Biotech, Shenzhen,1.3 Cell viability assayChina)and the amplification was performed accordingPrior to the investigation of anti-HBV effects, the cell to the manufacturers instructiongrowth inhibiting effects of the Rheum palmatum L. 1.5 Measurement of HBsAg and HBeAgethanol extract(RPE)and the anthraquinone derivatives After treatment of RPE and anthraquinone solutions,were determined with the mTT assay(15). Briefly the culture medium was collected and clarified as describedcells were plated in 96-well tissue culture plates at a above The levels of HBsAg and HBeAg were deter-density of 1 x 10 cells/well in DMEM culture medium mined by the enzyme linked immunosorbent assayand allowed to incubate for 48 hours. RPE and anthra- (ELISA) kits and performed according to the protocolquinone solutions were added to various final concen- provided with the kits(Abbott Murex, Wiesbaden, Gertrations in triplicates and DMEM with 0. 2% DMSo many)was used as control. After incubation, 20uL 3-(4, 5-di- 1.6 Statistical analysismethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide The results were calculated as the percentage of con-(MTT) solution(5 mg/mL) was added into each well trols and expressed as the mean SE of independent ex-and further incubated at 37 C for another 4 hours. Then, periments. Data were analyzed by the Paired-samples t150 uL DMSO was added and the solution was shaken test using the Spss 12.0 software program( Spss Inc,for 10 minutes to dissolve the mTt formazan crystal. Chicago, IL). Statistical significance was defined asThe absorbance was measured by a microplate reader P<0.05 compared with control valuessystems, Foster City, CA)at 570 nm with a 650 nm2. Results1. 4 Quantification of extracellular HBV-DNA2. 1 Effects of RPE, emodin and rhein on cell via-Hepad38 cells were inoculated at a density ofbility1x105 cells/well in 24-well tissue culture plates. TheThe cell growth inhibitorv effects of the rPe(rheumRPE and the anthraquinone solutions were added to the中国煤化工modin and rhein wereCNMHGmediums after 2 days incubation. Then, the cells were measured to determine une ant-hbV treatment concentgrown in the presence of these drugs for 8 days with the rations in the cell culture system. HepAD38cellsmedium changed every two days. The culture mediums were incubated with various concentrations of emodinSUN et alInhibition of Hepatitis B Virus Replication by RPE in a Stable HBV-producing Cell LineBCEmodinRhein8100810080备6254g/mL-F6.25 !g/mL6258mL[-+-25ug/mL20=·50Fig. 2. Cytotoxic effect ofRPE (A), emodin(B)and rhein(C)on HepAD38 cells assessed by MTT. A time and dose-dependent cytotoxiceffect was observed. Representative data from three independent experiments are shown120100■ Rheum palmatum 1口emoethanol extract(RPE)8