title: UFLC/MS-IT-TOF guided isolation of anti-HBV active chlorogenic acid analogues from Artemisia capillaris as a traditional Chinese herb for the treatment of hepatitis.
abstract: BACKGROUND
  Hepatitis B induced by HBV is a serious health problem. Artemisia capillaris (Yin-Chen) has long been used to treat hepatitis in traditional Chinese medicine. Coumarins, flavonoids and organic acids were revealed as its hepatoprotective and choleretic components, but its anti-HBV active components remain unknown. This current study focused on its anti-HBV active constituents by various chromatographic methods.
  
  
  METHODS
  LC/MS and bioassay-guided fractionation on the active extract of Artemisia capillaris led to the isolation of nine chlorogenic acid analogues. Structures of the isolates were elucidated by MS/MS and NMR techniques. Anti-HBV assay was performed on HepG 2.2.15 cell line in vitro: reduction of HBsAg and HBeAg secretions was measured by an ELISA method; inhibition of HBV DNA replication was monitored by real-time quantitative PCR and cellular toxicity was assessed by a MTT method.
  
  
  RESULTS
  The 90% ethanol extract of Artemisia capillaris (Fr. AC) showed significantly inhibitory activity on HBV DNA replication with an IC₅₀ value of 76.1 ± 3.9 μg/mL and low cytotoxic effects (SI>20.1). To clarify its active constituents, the extract was further separated into 3 sub-fractions (AC-1, AC-2 and AC-3), of which Fr. AC-2 was the most active fraction against HBeAg secretion and HBV DNA replication with IC50 values of 44.2 ± 2.8 and 23.2 ± 1.9 μg/mL. Nine chlorogenic acid analogues were detected from the active part (Fr. AC-2) by a LC/MS technique and further separated by a HPLC method. The isolates were determined as chlorogenic acid (1), cryptochlorogenic acid (2), neochlorogenic acid (3), 3,5-dicaffeoylquinic acid (4), 4,5-dicaffeoylquinic acid (5), 3,4-dicaffeoylquinic acid (6), chlorogenic acid methyl ester (7), cryptochlorogenic acid methyl ester (8), neochlorogenic acid methyl ester (9). Compounds 1-6 possessed potent activity against HBV DNA replication with IC50 values in the range of 5.5 ± 0.9-13.7 ± 1.3 μM. Di-caffeoyl analogues (4-6) also exhibited activity against the secretions of HBsAg and HBeAg. Esterified analogues (7-9) showed dramatically decreased anti-HBV activity, indicating that carboxyl group is closely associated to the anti-HBV activity.
  
  
  CONCLUSIONS
  This investigation was focused on the active fractions of Artemisia capillaris and their active compositions, which showed that Fr. AC-2 was the main active section of Artemisia capillaris and chlorogenic acid analogues were the main constituents contributing to its anti-HBV activity. These results support the ethnopharmacological use of Artemisia capillaris as anti-HBV agents.
authors.affiliation: State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China.
authors.forename: Chang-An
authors.initials: CA
authors.lastname: Geng
authors.affiliation: State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China.
authors.forename: Yun-Bao
authors.initials: YB
authors.lastname: Ma
authors.affiliation: State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China.
authors.forename: Xiao-Yan
authors.initials: XY
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China.
authors.forename: Hao
authors.initials: H
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China.
authors.forename: Tuan-Wu
authors.initials: TW
authors.lastname: Cao
authors.affiliation: State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China.
authors.forename: Kang
authors.initials: K
authors.lastname: He
authors.affiliation: State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China.
authors.forename: Hao
authors.initials: H
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China.
authors.forename: Xue-Mei
authors.initials: XM
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China. Electronic address: chenjj@mail.kib.ac.cn.
authors.forename: Ji-Jun
authors.initials: JJ
authors.lastname: Chen
mesh_terms: D019306:Artemisia; D002726:Chlorogenic Acid / Q000031:analogs & derivatives*; D002853:Chromatography, Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D056945:Hep G2 Cells; D006509:Hepatitis B / Q000188:drug therapy*; D006514:Hepatitis B Surface Antigens / Q000187:drug effects; D006513:Hepatitis B e Antigens / Q000187:drug effects; D006801:Humans; D008279:Magnetic Resonance Imaging; D008516:Medicine, Chinese Traditional; D010936:Plant Extracts / Q000737:chemistry* / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D053719:Tandem Mass Spectrometry
chemical_list: D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens; D010936:Plant Extracts; D002726:Chlorogenic Acid
keywords: 3,4-dicaffeoylquinic acid (PubChem CID 5459216/5281780); 3,5-dicaffeoylquinic acid (PubChem CID: 6474310); Anti-HBV activity; Artemisia capillaris; Chlorogenic acid (PubChem CID: 1794427); Chlorogenic acid analogues; Chlorogenic acid methyl ester (PubChem CID: 6476139); Cryptochlorogenic acid (PubChem CID: 9798666); Neochlorogenic acid (PubChem CID: 5280633); UFLC/MS-IT-TOF