Anti-hepatitis B Virus Effect And Possible Mechanism Of Action Of Edelweiss(Leontopodium Leontopodioides)

Hepatitis B which is caused by hepatitis B virus(HBV) is a kind of social infectious disease that exerts a threat to public health. The number of people with HBV in china is as high as 10 percent of total population. However, no effective drug and therapeutic methods can cure hepatitis B so far. It is urgent affairs to further intensify research and development of anti-HBV new drug. Although some antiviral agents such as interferon and nucleosides have been approved for the treatment of chronic HBV infection, HBV patients have few choices because of severe side effects and drug resistance. Edelweiss [Leontopodium leontopodioides(Willd.) Beauv] is a resourceful and eutherapeutic herb in folk from compositae. It is widely used to treat all kinds of the inflammatory diseases such as hepatitis in folk. Therefore, it is of significance to carry out modern study of Edelweiss.The first part of this paper was based on the previous research. The active components of Edelweiss were extracted by methanol and then purified by gel /silica gel column chromatography. The extract was analyzed using a HPLC system. In the second part, the anti-hepatitis B activities of this extract in vitro and in vivo were studied using HBV transgenic cells and mice, respectively. Meanwhile, the different expressions of heme oxygenase-1(HO-1), as an anti-HBV potential target, were compared and analyzed in three liver cells. Furthermore, the effect of the extract on the expression of HO-1 was also investigated. 1 Preparation of Edelweiss extract and its anti-HBV activity 1.1 Preparation of Edelweiss extractThe dry edelweiss was soaked with methanol at room temperature. The extracts were combined and concentrated under reduced pressure, yielding a dark-brown concrete, which was dissolved in hot water. This solution was extracted using the different polar solvent, respectively. The n-butanol fraction was further purified by gel and reverse phase silica gel column chromatography. The purified Edelweiss extract was analyzed using HPLC assay and its total phenolic acid content was determined using the ferric-ferricyanide method. The results of quantification indicated that the total phenolic acid content of the extract was 10.27%.1.2 Anti- HBV effect of Edelweiss extractAnti-HBV assay in vitro: To measure the effect of Edelweiss extract on the expression of HBV antigens and HBV DNA, Hep G2.2.15 cells were treated with various concentrations of the test compound in the 96-well plates. The medium with the compound was replaced every 3 days. On the third day, the replaced medium was assayed for HBs Ag and HBe Ag. On the sixth day, the replaced medium was measured for HBs Ag/HBe Ag by ELISA assay and HBVDNA by fluorescence quantitation PCR assay. Lamivudine was used as the reference drug. The results showed that the extract significantly inhibited HBs Ag and HBe Ag expressions at the third and sixth days, whereas its effect on HBV DNA replication is not marked. Anti-HBV assay in vivo: HBV transgenic mouse was employed to evaluate the anti-HBV activity of Edelweiss extract in vivo. The different doses of edelweiss extract were administered orally to the different groups of mice once per day for 30 days. The serum GOT, GPT, HBs Ag and HBe Ag levels, the liver HBV-DNA levels and the liver pathological changes were determined, respectively. The results suggested that the extract significantly reduced the serum GOT/GPT and HBs Ag/ HBe Ag levels. Meanwhile, the serum HBV-DNA level was declined at a dose of 100 mg/kg, while there is no marked influence in liver HBV-DNA levels and the liver histopathology change.2 Anti-HBV mechanism of Edelweiss extract 2.1 Different expressions of HO-1 in three liver cells linesThe expression levels of HO-1 m RNA was detected by RT-PCR in HL7702、Hep G2、Hep G2.2.15 cells lines. The results showed that the the m RNA levels of HO-1 in Hep G2 cells were significantly higher than that of HL7702 cells. The difference of HO-1 expressions in Hep G2.2.15 cells and HL7702 cells was not significant.2.2 Effect of Edelweiss extract on the expression of HO-1Hep G2.2.15 cells were employed to evaluate the effect of edelweiss extract on HO-1 gene expression. HO-1 m RNA level was detected with RT-PCR assay on the sixth day. The results indicated that edelweiss extract significantly increased the expression level of HO-1 m RNA. It suggested that the anti-HBV effect of edelweiss extract may be related to the expression of HO-1 gene.In summary, the study determined the different polar components of Edelweiss and verified the in vitro and in vivo anti-hepatitis B effects of Edelweiss extract. Additionally, the upregulation of HO-1 may contribute to the anti-HBV effect of this extract. These data provide a scientific explanation for the folkloric uses of Edelweiss(Leontopodium leontopodioides) in the treatment of hepatitis.