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Zebrafish bioassay-guided natural product discovery: isolation of angiogenesis inhibitors from East African medicinal plants.

Crawford AD, Liekens S, Kamuhabwa AR, Maes J, Munck S, Busson R, Rozenski J, Esguerra CV, de Witte PA - PLoS ONE (2011)

Bottom Line: Natural products represent a significant reservoir of unexplored chemical diversity for early-stage drug discovery.Zebrafish bioassay-guided fractionation identified the active components of these plants as emodin, an inhibitor of the protein kinase CK2, and coleon A lactone, a rare abietane diterpenoid with no previously described bioactivity.These results suggest that the combination of zebrafish bioassays with analytical chromatography methods is an effective strategy for the rapid identification of bioactive natural products.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, University of Leuven, Leuven, Belgium. alexander.crawford@pharm.kuleuven.be

ABSTRACT
Natural products represent a significant reservoir of unexplored chemical diversity for early-stage drug discovery. The identification of lead compounds of natural origin would benefit from therapeutically relevant bioassays capable of facilitating the isolation of bioactive molecules from multi-constituent extracts. Towards this end, we developed an in vivo bioassay-guided isolation approach for natural product discovery that combines bioactivity screening in zebrafish embryos with rapid fractionation by analytical thin-layer chromatography (TLC) and initial structural elucidation by high-resolution electrospray mass spectrometry (HRESIMS). Bioactivity screening of East African medicinal plant extracts using fli-1:EGFP transgenic zebrafish embryos identified Oxygonum sinuatum and Plectranthus barbatus as inhibiting vascular development. Zebrafish bioassay-guided fractionation identified the active components of these plants as emodin, an inhibitor of the protein kinase CK2, and coleon A lactone, a rare abietane diterpenoid with no previously described bioactivity. Both emodin and coleon A lactone inhibited mammalian endothelial cell proliferation, migration, and tube formation in vitro, as well as angiogenesis in the chick chorioallantoic membrane (CAM) assay. These results suggest that the combination of zebrafish bioassays with analytical chromatography methods is an effective strategy for the rapid identification of bioactive natural products.

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Crude methanolic extracts and isolated compounds inhibit vascular outgrowth in fli-1:EGFP transgenic embryos.All embryos are 40 hpf, with anterior to the left, scale bar  =  100 µm. a, b, DMSO-treated control; c, embryo treated with 200 µg/ml O. sinuatum extract; d, embryo treated with 10 µg/ml P. barbatus extract; e, embryo treated with 8 µM emodin; f, embryo treated with 2 µM coleon AL.
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pone-0014694-g001: Crude methanolic extracts and isolated compounds inhibit vascular outgrowth in fli-1:EGFP transgenic embryos.All embryos are 40 hpf, with anterior to the left, scale bar  =  100 µm. a, b, DMSO-treated control; c, embryo treated with 200 µg/ml O. sinuatum extract; d, embryo treated with 10 µg/ml P. barbatus extract; e, embryo treated with 8 µM emodin; f, embryo treated with 2 µM coleon AL.

Mentions: We chose an angiogenesis assay based on the evaluation of intersegmental vessel (ISV) outgrowth in fli-1:EGFP transgenic embryos [19], which exhibit vasculature-specific expression of enhanced green fluorescent protein (EGFP) in the trunk and tail during embryonic and larval development (Figs. 1a, b). With respect to natural product research, fli-1:EGFP zebrafish have been used to characterize the angiogenic activity of Angelica sinensis (dong quai) [24], as well as the anti-angiogenic activity of solenopsin, an alkaloid isolated from Solenopsis invicta (fire ants) [25]. Similar transgenic lines, with fluorescent reporter proteins expressed under the control of the endothelial cell-specific flk-1/VEGFR2 promoter, have recently enabled an ENU mutagenesis screen to identify genetic determinants of vascular development [26] and a small-molecule screen to identify novel angiogenesis inhibitors [27].


Zebrafish bioassay-guided natural product discovery: isolation of angiogenesis inhibitors from East African medicinal plants.

Crawford AD, Liekens S, Kamuhabwa AR, Maes J, Munck S, Busson R, Rozenski J, Esguerra CV, de Witte PA - PLoS ONE (2011)

Crude methanolic extracts and isolated compounds inhibit vascular outgrowth in fli-1:EGFP transgenic embryos.All embryos are 40 hpf, with anterior to the left, scale bar  =  100 µm. a, b, DMSO-treated control; c, embryo treated with 200 µg/ml O. sinuatum extract; d, embryo treated with 10 µg/ml P. barbatus extract; e, embryo treated with 8 µM emodin; f, embryo treated with 2 µM coleon AL.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040759&req=5

pone-0014694-g001: Crude methanolic extracts and isolated compounds inhibit vascular outgrowth in fli-1:EGFP transgenic embryos.All embryos are 40 hpf, with anterior to the left, scale bar  =  100 µm. a, b, DMSO-treated control; c, embryo treated with 200 µg/ml O. sinuatum extract; d, embryo treated with 10 µg/ml P. barbatus extract; e, embryo treated with 8 µM emodin; f, embryo treated with 2 µM coleon AL.
Mentions: We chose an angiogenesis assay based on the evaluation of intersegmental vessel (ISV) outgrowth in fli-1:EGFP transgenic embryos [19], which exhibit vasculature-specific expression of enhanced green fluorescent protein (EGFP) in the trunk and tail during embryonic and larval development (Figs. 1a, b). With respect to natural product research, fli-1:EGFP zebrafish have been used to characterize the angiogenic activity of Angelica sinensis (dong quai) [24], as well as the anti-angiogenic activity of solenopsin, an alkaloid isolated from Solenopsis invicta (fire ants) [25]. Similar transgenic lines, with fluorescent reporter proteins expressed under the control of the endothelial cell-specific flk-1/VEGFR2 promoter, have recently enabled an ENU mutagenesis screen to identify genetic determinants of vascular development [26] and a small-molecule screen to identify novel angiogenesis inhibitors [27].

Bottom Line: Natural products represent a significant reservoir of unexplored chemical diversity for early-stage drug discovery.Zebrafish bioassay-guided fractionation identified the active components of these plants as emodin, an inhibitor of the protein kinase CK2, and coleon A lactone, a rare abietane diterpenoid with no previously described bioactivity.These results suggest that the combination of zebrafish bioassays with analytical chromatography methods is an effective strategy for the rapid identification of bioactive natural products.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical Sciences, University of Leuven, Leuven, Belgium. alexander.crawford@pharm.kuleuven.be

ABSTRACT
Natural products represent a significant reservoir of unexplored chemical diversity for early-stage drug discovery. The identification of lead compounds of natural origin would benefit from therapeutically relevant bioassays capable of facilitating the isolation of bioactive molecules from multi-constituent extracts. Towards this end, we developed an in vivo bioassay-guided isolation approach for natural product discovery that combines bioactivity screening in zebrafish embryos with rapid fractionation by analytical thin-layer chromatography (TLC) and initial structural elucidation by high-resolution electrospray mass spectrometry (HRESIMS). Bioactivity screening of East African medicinal plant extracts using fli-1:EGFP transgenic zebrafish embryos identified Oxygonum sinuatum and Plectranthus barbatus as inhibiting vascular development. Zebrafish bioassay-guided fractionation identified the active components of these plants as emodin, an inhibitor of the protein kinase CK2, and coleon A lactone, a rare abietane diterpenoid with no previously described bioactivity. Both emodin and coleon A lactone inhibited mammalian endothelial cell proliferation, migration, and tube formation in vitro, as well as angiogenesis in the chick chorioallantoic membrane (CAM) assay. These results suggest that the combination of zebrafish bioassays with analytical chromatography methods is an effective strategy for the rapid identification of bioactive natural products.

Show MeSH
Related in: MedlinePlus