Study On Active Components And Mechanism Of Ligustrum Lucidum Inhibiting Hepatitis C Virus

Hepatitis C virus (HCV) is a member of the Flaviviridae family, it is an importantfactor in human liver disease as a serious threat to human health, which infects up to onehundred and seventy million people worldwide. Unfortunately, current therapy is onlycurative in approximately50%of HCV patients and has adversed side effects; In addition,according to the HCV vaccine is still blank. Therefore, the development of novel, effectiveand safety antivirals against HCV is a hotspot and frontier of HCV research and drugdevelopment at home and abroad.HCV is an enveloped positive-strand RNA virus possessing a single positive-strandRNA genome of approximately9.6kb. The genomic RNA consists of3regions: a5′-untranslated region (5′UTR), a3′-untranslated region (3′-UTR), and a single openreading frame (ORF). The ORF encodes a polyprotein of approximately3010amino acids,which is cleaved by cellular and viral proteases into HCV structural proteins (core, E1, E2)and non-structural proteins(P7, NS2, NS3, NS4A, NS4B, NS5A, NS5B). Thenon-structural protein NS5B provides RNA-dependent RNA polymerase (RdRp) activity. Itis a key enzyme for viral genome replication and thus represents an attractive target.The Chinese herb ligustrum lucidum (LL) has been used as a hepatoprotective agentfor centuries. We have previously reported that its aqueous extract directly inhibits HCVNS5B RNA-dependent RNA polymerase (RdRp) activity. The aim of this study was toanalyze the active components in the LL extract, detect their active components on HCVJFH1replication and inhibition of NS5B RdRp activity mechanism. The aqueous LLextract was subjected to further extraction by ethyl acetate and separation by thin layerchromatography (TLC), the inhibitory activity of separated fractions on HCV NS5B wasanalyzed by the inhibitory assay of NS5B activity and the active antiviral componentswithin the collected fractions were analyzed by High-Performance Liquid Chromatography(HPLC). The inhibitory effects of isolated LL fractions and their active compounds onHCV NS5B RdRp activity were examined by our established luciferase reporter assay andin vitro NS5B-catlyzed RNA synthesis assay. Real-time RT-PCR and Western blotexperiments are used to prove that replication of HCV JFH1can be reduced by LLextracts.The results showed that, The LL extract was separated into five fractions by TLC,only fractions1and2significantly inhibited JFH1replication and NS5B RdRp activity inHCV JFH1Cell infection model, fraction2possessed higher inhibitory activity thanfraction1. The HPLC analysis of these fractions combined with inhibitory assays indicatethat ursolic acid and oleanolic acid are major and broad-spectrum antiviral components offractions1and2, respectively. The antiviral effect of ursolic acid and oleanolic acid found that the selectivity index (SI) values of ursolic acid and oleanolic acid against JFH1replication are30.8and6.7, which are close to that of known HCV antiviral agent,ribavirin (6.7). Our results for the first time demonstrated that natural products oleanolicacid and ursolic acid can directly inhibiting HCV NS5B RdRp activity and reducing HCVJFH1replication, implying their potential usage as adjuvant HCV antivirals. It isanticipated that proper modifications of the compound may yield some ramifications withbetter antiviral activity against HCV that can be applied to clinic trials.