title: Bioassay-guided isolation of norviburtinal from the root of Rehmannia glutinosa, exhibited angiogenesis effect in zebrafish embryo model.
abstract: BACKGROUND
  The root of Rehmannia glutinosa (RR) is commonly used as a wound-healing agent in various traditional Chinese herbal formulae; while angiogenesis is one of the crucial aspects in wound-healing.
  
  
  OBJECTIVE
  The objective of the present study was to investigate the angiogenesis effects of RR aqueous crude extract and its active component(s) using zebrafish model.
  
  
  METHODS
  The in vivo angiogenesis effect was studied using transgenic TG(fli1:EGFP)(y1)/+(AB) zebrafish embryos by observing the capillary sprouts formation in sub-intestinal vessel (SIV) of zebrafish embryos after 72 h post-fertilization under fluorescence microscopy.
  
  
  RESULTS
  Our results indicated that RR aqueous crude extract (250 μg/ml) exhibited significant angiogenesis effect, with an increase in capillary sprouts formation in SIV. Following sequential solvent partition of the RR aqueous crude extract with dichloromethane, ethyl acetate and n-butanol successively, the dichloromethane fraction (DCM) was found to have the most sprouts formation in the SIV region. Subjected to column chromatography, DCM fraction was further fractionated into six sub-fractions and among these tested, the sub-fraction C2 exhibited the most potent angiogenesis effect. The major component, C2A, was isolated and identified as norviburtinal using nuclear magnetic resonance (NMR) and mass spectrometry (MS). The compound norviburtinal (at 50 μg/ml) was shown to possess significant angiogenesis effect in zebrafish model (p < 0.001).
  
  
  CONCLUSIONS
  Norviburtinal was, for the first time, found in the extract of RR and possessed novel angiogenesis effect. Bioassay-guided fractionation suggested that norviburtinal was not the only active component responsible for the angiogenesis effect of RR.
authors.affiliation: Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong.
authors.forename: Cheuk-Lun
authors.initials: CL
authors.lastname: Liu
authors.forename: Ling
authors.initials: L
authors.lastname: Cheng
authors.forename: Hin-Fai
authors.initials: HF
authors.lastname: Kwok
authors.forename: Chun-Hay
authors.initials: CH
authors.lastname: Ko
authors.forename: Tai-Wai
authors.initials: TW
authors.lastname: Lau
authors.forename: Chi-Man
authors.initials: CM
authors.lastname: Koon
authors.forename: Ming
authors.initials: M
authors.lastname: Zhao
authors.forename: Ching-Po
authors.initials: CP
authors.lastname: Lau
authors.forename: Kit-Man
authors.initials: KM
authors.lastname: Lau
authors.forename: Chun-Wai
authors.initials: CW
authors.lastname: Wong
authors.forename: Ping-Chung
authors.initials: PC
authors.lastname: Leung
authors.forename: Kwok-Pui
authors.initials: KP
authors.lastname: Fung
authors.forename: Clara Bik-San
authors.initials: CB
authors.lastname: Lau
mesh_terms: D020001:1-Butanol / Q000737:chemistry; D000085:Acetates / Q000737:chemistry; D043925:Angiogenesis Inducing Agents / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D000818:Animals; D030801:Animals, Genetically Modified; D001681:Biological Assay; D005591:Chemical Fractionation; D002845:Chromatography; D046149:Embryo Culture Techniques; D004625:Embryo, Nonmammalian / Q000098:blood supply / Q000187:drug effects*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008752:Methylene Chloride / Q000737:chemistry; D008856:Microscopy, Fluorescence; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D010936:Plant Extracts / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D018517:Plant Roots; D010946:Plants, Medicinal; D032267:Rehmannia / Q000737:chemistry; D012997:Solvents / Q000737:chemistry; D013729:Terpenes / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics
chemical_list: D000085:Acetates; D043925:Angiogenesis Inducing Agents; D010936:Plant Extracts; D012997:Solvents; D013729:Terpenes; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C485184:enhanced green fluorescent protein; C423729:norviburtinal; D049452:Green Fluorescent Proteins; D008752:Methylene Chloride; C007650:ethyl acetate; D020001:1-Butanol