In this paper, we report chemical structures of five compounds including four novel polyhydroxylated cardiac steroids in the eggs of a toad, Bufo marinus. These cardiac steroids were purified by high-performance liquid chromatography, and their structures were determined to be 11 alpha,19-dihydroxy-telocinobufagin (I), 11 alpha-hydroxytelocinobufagin (II), 11 alpha,19-dihydroxymarinobufagin (III), 11 alpha-hydroxymarinobufagin (IV) and 19-hydroxytelocinobufagin (V) on the basis of spectral data of nuclear magnetic resonance and mass spectroscopy. All the five compounds showed biological activity, as tested by inhibition of Na+,K(+)-ATPase activity and of [3H]ouabain binding to the receptor on Na+,K(+)-ATPase. This is the first finding of bufadienolides as cardiac steroids in animal eggs.
Matrix metalloproteinases (MMPs), especially membrane-type 1 matrix metalloproteinase (MT1-MMP), which generates an active form of MMP-2 from proMMP-2, are deeply involved in angiogenesis as well as in tumor cell migration and metastasis. To obtain a specific inhibitor for MT1-MMP, we screened a number of natural and synthetic compounds using recombinant human MMP-2, MMP-7, and soluble MT1-MMP in a fluorogenic peptide cleavage assay. (-)-Epigallocatechin 3-O-gallate (EGCG) followed by (-)-epigallocatechin 3,5-di-O-gallate and epitheaflagallin 3-O-gallate, was found to have potent and distinct inhibitory activity against MT1-MMP. Therefore, we investigated the effect of EGCG on the suppression of MMP-2 activation as determined by gelatin zymography, and observed that the active form of MMP-2 in the conditioned medium of human umbilical vein endothelial cells was decreased in the presence of EGCG. The results suggest the possibility that tea polyphenols suppress tumor growth through the suppression of angiogenesis.
Three major groups of endogenous digitalis-like substances (EDLS) have been identified in the plasma of the toad, Bufo marinus. One group of compounds, present in fresh plasma, is composed of chromatographically homogeneous polar conjugates, principally bufadienolide 3-sulfates, which exhibit relatively weak Na(+)-K(+)-adenosinetriphosphatase (ATPase) inhibitory activity. A second and larger group of compounds, also found in fresh plasma, includes chromatographically heterogeneous conjugates, which are effective inhibitors of Na(+)-K(+)-ATPase; these compounds possess properties similar to those of bufotoxins. The third group of EDLS consists of free unconjugated bufadienolides, which are also effective Na(+)-K(+)-ATPase inhibitors. These unconjugated bufadienolides are present in relatively low concentrations in fresh toad plasma, but appreciable quantities are enzymatically generated from conjugates (believed to consist principally of bufotoxins) during the in vitro incubation of plasma. We suggest that the extent to which circulating polar EDLS are enzymatically deconjugated in vivo may be important in the regulation of the digitalis-sensitive Na(+)-K(+)-ATPase of toad brain, the only known digitalis-sensitive Na(+)-K(+)-ATPase in the toad.
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