Antiviral efficacy against hepatitis B virus replication of oleuropein isolated from Jasminum officinale L. var. grandiflorum

Journal of Ethnopharmacology 125 (2009) 265–268

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Antiviral efficacy against hepatitis B virus replication of oleuropein isolated from Jasminum officinale L. var. grandiflorum Guiqin Zhao a,b , Zhifeng Yin b , Junxing Dong a,∗ a b

Institute of Radiation Medicine, Academy of Military Medical Science, 27 Taiping Road, Haidian District, Beijing 100850, China Chengde Medical College, Chengde 067000, China

a r t i c l e

i n f o

Article history: Received 22 March 2009 Received in revised form 23 June 2009 Accepted 26 June 2009 Available online 4 July 2009 Keywords: Oleuropein Jasminum officinale L. var. grandiflorum Anti-HBV activity

a b s t r a c t Ethnopharmacological relevance: Jasminum officinale L. var. grandiflorum (JOG) is a folk medicine used for the treatment of hepatitis in south of China. Phytochemical studies showed that secoiridoid glycosides are the typical constituents of this plant. Aim of the study: The present study was undertaken to evaluate the effect of oleuropein (Ole) derived from the flowers of JOG on hepatitis B virus (HBV) replication in HepG2 2.2.15 cell line in vitro and duck hepatitis B virus (DHBV) replication in ducklings in vivo. Material and methods: The extracellular hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) concentrations in cell culture medium were determined by ELISA. DHBV in duck serum was analyzed by dot blot. Results: Ole blocks effectively HBsAg secretion in HepG2 2.2.15 cells in a dose-dependent manner (IC50 = 23.2 ␮g/ml). Ole (80 mg/kg, intraperitoneally, twice daily) also reduced viremia in DHBV-infected ducks. Conclusion: Ole therefore warrants further investigation as a potential therapeutic agent for HBV infection. © 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Plants of the genus Jasminum thunb. (Oleaceae) are widely distributed in the temperate and semitropical zone of Asia and Africa. The flowers of many Jasminum plants, such as Jasminum sambac, Jasminum polyanthum, Jasminum nudiflorum and Jasminum lang, are used as traditional or folk remedy in China for the treatment of arthritis, hepatitis, conjunctivitis, gastritis and diarrhea (Huang et al., 2001). Jasminum officinale L. var. grandiflorum (JOG) is one of the Jasminum plants used as a folk remedy for the treatment of hepatitis and duodenitis in south China (Nanjing, 2006). There are few reports about its chemical constituents and pharmacological properties. In our continued investigation on searching for safe and effective anti-HBV agents from traditional Chinese medicine, we found that the hydroalcoholic extraction of the flowers of JOG showed preferable antiviral efficacy against hepatitis B virus (HBV) replication in HepG2 2.2.15 cell line in vitro. Phytochemical studies on the extraction led to the isolation of eleven secoiridoid glycosides, including oleuropein (Ole; Zhao et al., 2007, 2008). HBV infection is still a major global health problem. It is responsible for the delayed sequel of cirrhosis and hepatocellu-

∗ Corresponding author. Tel.: +86 10 66931314; fax: +86 10 68164251. E-mail address: [email protected] (J. Dong). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.06.030

lar carcinoma (HCC) (Park et al., 2006). Approximately 2 billion people worldwide have been exposed to HBV, with approximately 360 million being chronically infected. Worldwide deaths from liver cancer caused by HBV infection probably exceed 1 million per year (Parkin et al., 1999). Beyond interferon-␣ (IFN-␣), only two drugs are currently approved for clinical use, e g. lamivudine and adefovir. Interferon-␣ is only partially effective for clinical use and is limited by its side effects. Lamivudine suppresses HBV through inhibition of reverse transcriptase, but the treatment often fails due to the emergence of mutations within the catalytic site of HBV DNA polymerase, which leads to drug-resistance in patients (Buti et al., 2001; Lai et al., 2003). Adefovir has been only recently introduced in hospitals in mainland China. It is highly desirable to prompt the search for new anti-HBV agents. Many screening efforts have been made to find antiviral agents from natural sources. Plants have long been used as remedies, and many are now being collected and examined in an attempt to identify possible sources of antivirals (Abad et al., 2000). In the last decades, as an alternative to conventional chemical agents, a large number of phytochemicals have been recognized as a way to control infections caused by viruses (Kalvatchev et al., 1997; Yamasaki et al., 1998; Abad et al., 1999a,b, 2000). Natural compounds, because of their structural diversity, provide a large opportunity for screening anti-HBV agents with novel structure and distinct mechanism of action.

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We report here the strong inhibitory effect of Ole derived from JOG on the expression of HBsAg, in HepG2 2.2.15 cells without an effect on the viability of the cells. More importantly, we attempt to demonstrate that Ole showed remarkably potent antiviral activity against DHBV replication in vivo in the absence of any obvious signs of toxicity. This work provides essential information that Ole deserves further investigation as a potential alternative or complementary anti-HBV agent, and will enhance the understanding on the enthnopharmacology of JOG and other plants that contain Ole. 2. Materials and methods 2.1. Cell culture

2.4. Cytotoxicity assay by MTT The cytotoxicity of Ole was analyzed by the MTT assay [3-(4,5dimethylthiazol)-2,5-diphenyl tetrazolium bromide]. HepG2 2.2.15 cells were cultivated at 2 × 104 cells/well into 96-wellmicroplate in 100 ␮l culture medium and grown for 48 h to reach approximate confluence conditions. Cells were treated or not treated with Ole in duplication every 4 days for 8 days, and then 10 ␮l of MTT (5 g/ml) was added to each well and further incubated in CO2 incubator at 37 ◦ C for 4 h. The optical density (OD) at 560 nm was obtained. Data were calculated as a percentage of negative control cells that were not treated with Ole (Rubinstein et al., 1990). The concentrations of Ole were 6.3, 12.5, 25.0, 50.0 and 100.0 ␮g/ml, respectively. 2.5. Experimental inoculation of ducklings

HepG2 2.2.15 cells, a HBV-transfect human HepG2 cell line (Sells et al., 1987), were provided by Chinese Academy of Medical Sciences. The HepG2 2.2.15 cells were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibico, USA) supplemented with 10% (v/v) fetal calf serum (Gibico, USA) and antibiotics (100 units/ml penicillin and 100 ␮g/ml streptomycin) at 37 ◦ C in a humidified incubator at 5% CO2 (Guo et al., 2007), and 380 ␮g/ml G418 in the medium was added to select the HepG2 2.2.15 cell line (Shin et al., 2005). 2.2. Treatment of Ole and lamivudine Ole was extracted from the dried bud of JOG which was collected from Guangdong province of China in January 2005 and identified by Mr. Bin LI, Academy of Military Medical Science of China. A plant sample has been conserved in the specimen room of Academy of Military Medical Science of China. The structure of Ole (Fig. 1) was elucidated by spectroscopic and chemical means (Zhao et al., 2007). Lamivudine (3TC) as a positive control was purchased from GlaxoSmithKline (Suzhou, China). Cells at 2 × 104 cells/well (96-wellmicroplates, Falcon, Oxnard, CA, USA) were treated with Ole at 37 ◦ C in duplication every 4 days for 8 days. The HepG2 2.2.15 cells as control were washed twice with phosphate-buffered saline (PBS) and re-feed with culture medium every 4 days for 8 days. Then the corresponding suspension was collected for analysis of the levels of HBsAg and HBeAg (Zhou et al., 2007). The concentrations of Ole were 1.6, 3.2, 6.3, 12.5, 25.0 and 50.0 ␮g/ml, respectively. 2.3. Determination of HBsAg and HBeAg After incubation with various concentrations of Ole at 37 ◦ C in 5% CO2 for 8 days, conditioned culture medium was collected. The concentration of HBsAg or HBeAg was detected by an enzyme-linked immunosorbent assay (ELISA) kit (Kewei diagnostic reagent Co., Beijing, China) and quantified relative to a standard curve of serial dilutions of recombinant HBsAg or HBeAg (Zhou et al., 2007).

Sera from duck that are congenitally infected with DHBV are the sources of DHBV used for inoculation (Zoulim et al., 1996). Duck serum positive for duck HBsAg (DHBsAg) at 1:1000 of dilution was used for infection. Ducklings at 1 day of age (Nanjing Qianjin Fowl Inc., Nanjing) were intravenously infected with DHBV at a dose of 0.2 ml DHBV (+) serum per duckling (containing 5.7 × 106 cpm of DHBV DNA equivalents). Seven days later, Ole was administered (20, 40, or 80 mg/kg, intraperitoneally, twice daily) for 10 days. Ole was solubilized in isotonic saline solution and administered orally in a liquid diet. The isotonic saline liquid diet was also administered to the animals as negative control. Lamivudine (50 mg/kg) was used as positive control. DHBV DNA was measured at days 0(T0), 5(T5), 10(T10), and 3 after cessation of treatment at 10 days (day p3) by dot blot. Throughout the experiments, animals were processed according to Regulations for the Administration of Affairs Concerning Experimental Animals (1988, China) and Implementing Regulations of the Administration on Medical Experiments on animals (1998, China). 2.6. Dot blot analysis for DHBV DNA For the measurement of DHBV DNA in the serum, a previously reported method was used with modifications (Yao et al., 2001). Briefly, 50 ␮l of duckling serum was directly spotted on the nitrocellulose membrane, and DHBV DNA was detected with 5 -[alpha-32 P] deoxycytidine labeled full-length DHBV genomic DNA. Incorporation of radioactivity was determined by Molecular Dynamics storage phosphor screen cassette (Amersham Bioscience). The phosphor screen was scanned with a Typhoon 9410 scanner (Amersham Bioscience) and quantified by using the ImageQuant (Molecular Dynamics) software. 2.7. Statistics To compare the values (means ± S.D.) before and after treatment, a paired student t-test was employed. Differences among study groups were tested by unpaired student t-test for equal or unequal variances depending on a preliminary F-test for the homogeneity of variance. The threshold of significance was set at p = 0.05 (Zhou et al., 2007). 3. Results 3.1. Cytotoxic effect of Ole on HepG2 2.2.15 cell viability

Fig. 1. Chemical structure of oleuropein.

The results from the MTT test showed that there was no significant difference of cell viability between Ole-treated groups whose concentrations were below 50 ␮g/ml and control group. TC50 of Ole, defined as the concentration that inhibited 50% cellular growth in comparison to untreated controls, was over 100 ␮g/ml in HepG2

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Fig. 2. Dot blots of DHBV DNA in duck serum in the oleuropein (Ole) treatment study. Serum DHBV DNA was quantified by the dot blot hybridization method using cloned DHBV DNA as the control and analyzed with a Bio-image analyzer. Lamivudine was used as positive control. Data was expressed as mean ± S.D. (n = 6); and were statistically analyzed using Dunnett’s multiple comparison test. The Ole treatment (80 mg/kg) significantly inhibited DHBV DNA in ducks (p < 0.05 for T5; p < 0.01 for T10). There were no significant differences between the untreated controls on days T0, T5, T10, and p3 (p > 0.05).

2.2.15 cells. The cytotoxicity of Ole was measured to determine the treatment concentrations of Ole in the HepG2 2.2.15 cell culture system . 3.2. Antiviral effect of Ole on HepG2 2.2.15 cell Treating HepG2 2.2.15 cells with Ole significantly inhibited the expression of HBsAg in a dose-dependent manner (p < 0.05), while the amount of HBeAg remains constant. The IC50 value of Ole for HBsAg was 23.2 ␮g/ml. 3.3. Inhibition of HBV replication on DHBV-infected ducklings Serum samples were taken at days 0, 5, 10 and 3 after the cessation of Ole treatment (n = 6), respectively. Serum DHBV DNA was analyzed by dot hybridization (Fig. 2). DHBV DNA levels in the Oletreated groups at dosage of 80 mg/kg were significantly lower than those of untreated controls at days 5 and 10, respectively (p < 0.05 for day 5; p < 0.01 for day 10). The inhibition declined to a nonsignificant level when the dose of Ole was reduced to 40 mg/kg. The body weights of the ducklings treated with Ole were without significant difference compared to that of the untreated ducklings (p = 0.38). 4. Discussion Many of natural products extracted from Chinese traditional or folk medicines exhibit a variety of biological activities including anti-HBV activity. For example, the polyphenolic extraction from Geranium carolinianum L. (PPGC) showed significantly anti-

HBV activities on HBV replication both in vitro and in vivo. PPGC effectively suppressed the secretion of the HBV antigens in a dosedependent manner with IC50 values of 46.85 mg/ml for HBsAg and 65.60 mg/ml for HBeAg. In the DHBV infected ducks, after PPGC was dosed intragastricly once a day for 10 days, the plasma DHBV DNA level was reduced, with an ED50 value of 47.54 mg/kg (Li et al., 2008). For another example, wogonin and ellagic acid, extracted from Scutellaria radix and Phyllanthus urinaria respectively, possessed potent anti-HBV activity. Wogonin effectively suppressed the secretion of the HBV antigens and reduced HBV DNA level in a dose-dependent manner (Guo et al., 2007) while ellagic acid blocked effectively HBeAg secretion in HepG2 2.2.15 cells (Shin et al., 2005). Both wogonin and ellagic acid are under early development as anti-HBV drugs candidate. In our primary study, we selected some kinds of traditional Chinese medicine, which have long been used in the folk treatment of chronic hepatitis in China, for screening novel anti-HBV agents. Among them, the hydroalcoholic extract of JOG suppressed the expression of HBsAg in HepG2 2.2.15 system, which is widely used as a useful “in vitro” model for evaluation of novel anti-HBV drugs (Romero et al., 2005). In this report, we attempted to demonstrate the efficient anti-HBV activity of Ole which was extracted from JOG both in vitro and in vivo. Two lines of evidence support this conclusion. First, Ole blocks effectively HBsAg secretion with an IC50 value of 23.2 ␮g/ml in HepG2 2.2.15 cells. Second, Ole (80 mg/kg, twice daily) reduced viremia in DHBV-infected ducks. The mechanism of how Ole suppresses HBsAg gene expression in the HepG2 2.2.15 cells is not yet clear. Ole may directly travel into the cell and alter the transcriptional machinery of HBsAg gene as glucocorticoid does (Kaspa et al., 1986). Alternatively, Ole may target the cell

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membrane, delivering an inhibitory signal to the nucleus to suppress HBsAg gene expression as insulin does (Ting et al., 1989; Chou et al., 1989). The mechanism of how Ole reduced viremia in DHBVinfected ducks is not yet clear. How Ole exerts its anti-HBV effect needs to be clarified further in our lab. Ole is a unique secoiridoid glycoside present in nature. Studies have shown that Ole possesses a wide range of pharmacologic and health promoting properties including anti-inflammatory (Diaz et al., 2000), anti-rheumatic (Visioli et al., 1998), diuretic (Ribeiro Rde et al., 1986), anti-atherogenic (Visioli and Galli, 2001), antipyretic (Visioli et al., 1995) and antiviral (Ma et al., 2001; Micol et al., 2005) effects. The results reported in this paper provide the first experimental evidence of notable anti-HBV activity of Ole both in vitro and in vivo. It will greatly enhance our understanding on the enthnopharmacology of JOG and other plants that contain Ole. 5. Conclusion In summary, Ole extracted from the flowers of JOG demonstrated indubitable anti-HBV activity in HepG2 2.2.15 cells test in vitro and DHBV-infected ducklings test in vivo. Ole blocks effectively HBsAg secretion in HepG2 2.2.15 cells in a dose-dependent manner with IC50 value of 23.2 ␮g/ml, without an effect on the viability of the cells. Ole (80 mg/kg, intraperitoneally, twice daily) also remarkably reduced DHBV replication in DHBV-infected ducklings in vivo in the absence of any obvious signs of toxicity. Our study suggests that Ole might play an important role in the treatment of hepatitis with JOG and thus this compound is worthy to be further investigated. Acknowledgements We are grateful to Prof. Zhuang Li (Chinese Academy of Medical Sciences) for providing excellent technical assistance, and Mr. Tianjie Jin (Chengde Medical College) for critical reading of this manuscript. References Abad, M.J., Bermejo, P., Gonzales, E., Iglwsias, I., Irurzun, A., Carrasco, L., 1999a. Antiviral activity of Bolivian plant extracts. General Pharmacology 32, 499–503. Abad, M.J., Bermejo, P., Sanchez Palomino, S., Chiriboga, X., Carrasco, L., 1999b. Antiviral activity of some South American medicinal plants. Phytotherapy Research 13, 142–146. Abad, M.J., Guerra, J.A., Bermejo, P., Irurzun, A., Carrasco, L., 2000. Search for antiviral activity in higher plant extracts. Phytotherapy Research 14, 604–607. Buti, M., Sanchez, F., Cotrina, M., Jardi, R., Rodriguez, F., Esteban, R., Guardia, J., 2001. Quantitative hepatitis B virus DNA testing for the early prediction of the maintenance of response during lamivudine therapy in patients with chronic hepatitis B. The Journal of Infectious Diseases 183, 1277–1280. Chou, C.K., Su, T.S., Chang, C., Hu, C.P., Huang, M.Y., 1989. Insulin suppresses hepatitis B surface antigen expression in human hepatoma cells. The Journal of Biological Chemistry 264, 15304–15308. Diaz, A.M., Abad, M.J., Fernandez, L., Recuero, C., Villaescusa, L., Silvan, A.M., Bermejo, P., 2000. In vitro anti-inflammatory activity of iridoids and triterpenoid compounds isolated from Phillyrea latifolia L. Biological & Pharmaceutical Bulletin 23, 1307–1313. Guo, Q.L., Zhao, L., You, Q.D., Yang, Y., Gu, H.Y., Song, G.L., Lu, N., Xin, J., 2007. Antihepatitis B virus activity of wogonin in vitro and in vivo. Antiviral Research 74, 16–24.

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