Bioassay-guided isolation of norviburtinal from the root of Rehmannia glutinosa, exhibited angiogenesis effect in zebrafish embryo model

Abstract: Ethnopharmacological relevance: 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. Aim of the study: 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. Materials and 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 72h post-fertilization under fluorescence microscopy. Results: Our results indicated that RR aqueous crude extract (250μg/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 50μg/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. [Copyright &y& Elsevier]