title: Investigation of the absorbed and metabolized components of Danshen from Fuzheng Huayu recipe and study on the anti-hepatic fibrosis effects of these components.
abstract: BACKGROUND
  Fuzheng Huayu recipe (FZHY) was formulated on the basis of Chinese medicine theory in treating liver fibrosis. It has a significant efficacy against liver fibrosis caused by chronic hepatitis B, with the action mechanisms of inhibition of hepatic stellate cell activation, protection of hepatocyte oxidative injury and regulations of hepatic matrix remodeling etc.
  
  
  OBJECTIVE
  To identify the absorbed components and metabolites of Danshen in FZHY in rat serum, and find their active components for anti-liver fibrosis.
  
  
  METHODS
  A valid high performance liquid chromatography-electrospray ionization ion trap mass spectrometry (HPLC-ESI/MS(n)) method was established to investigate the absorbed and metabolized compounds of Danshen in FZHY in rat serum after oral administration. Mass spectra were acquired in both negative and positive modes. Otherwise, to evaluate the anti-hepatic fibrosis efficacies of absorbed and metabolized compounds, the LX-2 cell line of hepatic stellate cell (HSC), which was crucial cellular basis of fibrogenesis, was cultured and incubated with absorbed compounds, the cytotoxicity was determined with the cellomics Multiparameter Cytotoxicity Kit 1 by High Content Screening (HCS), the cell proliferation was assayed with EdU-DNA incorporation, and the cell activation was analyzed through α-smooth muscle actin (α-SMA) expression with high content screening technology.
  
  
  RESULTS
  More than 11 compounds and 2 metabolites from Danshen were identified in the serum after oral administration of FZHY by comparing their mass spectra and retention behavior with reference compounds or literature data. Among these compounds, there were no obvious changes in nuclear morphology, membrane permeability with blow 96 μM of six polar compounds treatment in comparison with control cells, respectively. And the salvianolic acid B (6 μM, 48 μM), caffeic acid (6 μM, 48 μM) and rosmarinic acid (48 μM) could obviously inhibit LX-2 cells proliferation, down-regulate α-SMA expression.
  
  
  CONCLUSIONS
  The results proved that the established method could be applied to analyze the absorbed into blood compounds of Danshen after oral administration FZHY. These absorbed compounds included 11 compounds and 2 metabolites of Danshen. Among them, the salvianolic acid B, caffeic acid and rosmarinic acid were the effective components of FZHY to anti-hepatic fibrosis effects.
authors.affiliation: Institute of Liver Diseases, Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine, 528 Zhangheng Road, Shanghai 201203, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Yang
authors.forename: Dan-Ping
authors.initials: DP
authors.lastname: Shen
authors.forename: Qin-Lan
authors.initials: QL
authors.lastname: Wang
authors.forename: Yan-Yan
authors.initials: YY
authors.lastname: Tao
authors.forename: Cheng-Hai
authors.initials: CH
authors.lastname: Liu
mesh_terms: D000818:Animals; D001572:Benzofurans / Q000494:pharmacology; D002109:Caffeic Acids / Q000494:pharmacology; D002463:Cell Membrane Permeability / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D002934:Cinnamates / Q000494:pharmacology; D053630:Depsides / Q000494:pharmacology; D004365:Drugs, Chinese Herbal / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*; D006801:Humans; D008103:Liver Cirrhosis / Q000188:drug therapy* / Q000378:metabolism; D008297:Male; D010618:Phenanthrolines / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D027561:Salvia miltiorrhiza / Q000737:chemistry*; D000097123:Rosmarinic Acid
chemical_list: D001572:Benzofurans; D002109:Caffeic Acids; D002934:Cinnamates; D053630:Depsides; D004365:Drugs, Chinese Herbal; D010618:Phenanthrolines; D000097123:Rosmarinic Acid; C532829:fuzheng huayu; C028972:dan-shen root extract; C076944:salvianolic acid B; C040048:caffeic acid