Urinary metabolites of cinobufagin in rats and their antiproliferative activities.

Cinobufagin was one of the important cardenolidal steroids and a major component of Chan'Su, a famous traditional Chinese medicine. The urinary metabolites of cinobufagin after single oral doses of 25 mg kg⁻¹ in rats were investigated. Eleven metabolites were isolated and purified by liquid-liquid extraction, open-column chromatography, medium-pressure liquid chromatography, as well as semi-preparative high-performance liquid chromatography. Their structures were elucidated by chemical and various spectroscopic methods, which were identified as desacetylcinobufagin (M-1), 3-oxo-desacetylcinobufagin (M-2), 3-oxo-cinobufagin (M-3), 3-epi-desacetylcinobufagin (M-4), 3-epi-12ß-hydroxyl desacetylcinobufagin (M-5), 5ß-hydroxyl cinobufagin (M-6), 5ß-hydroxyl desacetylcinobufagin (M-7), 12ß-hydroxyl cinobufagin (M-8), 1ß,12ß-dihydroxyl cinobufagin (M-9), 12ß-hydroxyl desacetylcinobufagin (M-10) and 1ß,12ß-dihydroxyl desacetylcinobufagin (M-11), respectively. Among them, M-1 was the main urinary metabolite of cinobufagin with a yield of 17.7%. Most metabolites were hydroxylated products of cinobufagin at C-1ß, 5ß and 12ß positions, as well as deacetylated products at C-16. Except M-1, M-4 and M-7, the other eight metabolites were novel in vivo metabolites of cinobufagin. Some metabolites showed potential cytotoxicity against human hepatoma cells (HepG2) and human leukaemia (K562, HL-60) cells; however, their cytotoxicities generally decreased after metabolic conversion.