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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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TLC fractionation and HRESIMS analysis identify emodin and coleon AL as bioactive constituents.Thin-layer chromatograms, HRESIMS spectra, and predicted structures of isolated, bioactive compounds. a, O. sinuatum; b, P. barbatus.
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pone-0014694-g003: TLC fractionation and HRESIMS analysis identify emodin and coleon AL as bioactive constituents.Thin-layer chromatograms, HRESIMS spectra, and predicted structures of isolated, bioactive compounds. a, O. sinuatum; b, P. barbatus.

Mentions: We next sought to isolate from O. sinuatum and P. barbatus extracts the principle components responsible for their anti-angiogenic effects. Both crude methanolic extracts were fractionated via thin-layer chromatography (TLC), using toluene/ethyl formate/formic acid (5:4:1) as the solvent. A single analytical-scale TLC plate (20×20 cm) was used to separate 10 mg of each extract, and was subsequently divided into 10-15 horizontal strips based on the presence of UV254 -absorbing and UV365-emitting components (Figs. 3 a, b). The silica was removed from these strips and extracted with methanol, after which the eluted constituents were subjected to bioactivity analysis in zebrafish, followed by high-resolution electrospray ionization mass spectroscopy (HRESIMS) for those exhibiting anti-angiogenic activity. For both O. sinuatum and P. barbatus, single TLC fractions were identified in this manner which phenocopied the anti-angiogenic activity shown by the crude extracts.


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)

TLC fractionation and HRESIMS analysis identify emodin and coleon AL as bioactive constituents.Thin-layer chromatograms, HRESIMS spectra, and predicted structures of isolated, bioactive compounds. a, O. sinuatum; b, P. barbatus.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0014694-g003: TLC fractionation and HRESIMS analysis identify emodin and coleon AL as bioactive constituents.Thin-layer chromatograms, HRESIMS spectra, and predicted structures of isolated, bioactive compounds. a, O. sinuatum; b, P. barbatus.
Mentions: We next sought to isolate from O. sinuatum and P. barbatus extracts the principle components responsible for their anti-angiogenic effects. Both crude methanolic extracts were fractionated via thin-layer chromatography (TLC), using toluene/ethyl formate/formic acid (5:4:1) as the solvent. A single analytical-scale TLC plate (20×20 cm) was used to separate 10 mg of each extract, and was subsequently divided into 10-15 horizontal strips based on the presence of UV254 -absorbing and UV365-emitting components (Figs. 3 a, b). The silica was removed from these strips and extracted with methanol, after which the eluted constituents were subjected to bioactivity analysis in zebrafish, followed by high-resolution electrospray ionization mass spectroscopy (HRESIMS) for those exhibiting anti-angiogenic activity. For both O. sinuatum and P. barbatus, single TLC fractions were identified in this manner which phenocopied the anti-angiogenic activity shown by the crude extracts.

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