ISOLATION AND IDENTIFICATION OF VITAMIN-D3, 25-HYDROXYVITAMIN D3, 1,25-DIHYDROXYVITAMIN D3 AND 1,24,25-TRIHYDROXYVITAMIN D3 IN SOLANUM-MALACOXYLON INCUBATED WITH RUMINAL FLUID

It has been shown that Solanum malacoxylon contains 1alpha25-dihydroxyvitamin D3-glycoside. The presence of vitamin D3 and 25-hydroxyvitamin D3 has also been suggested. In the present study vitamin D3 and three of its metabolites, including 1alpha,25-dihydroxyvitamin D3, were detected in plant leaf extracts preincubated with ruminal fluid (SMRF). Extraction of SMRF with non-polar organic solvents and purification of the lipid extract by TLC followed by HPLC yielded nine ultraviolet-absorbing (264 nm) peaks. Four of them co-migrated on a Zorbax-Sil HPLC column with synthetic standards of vitamin D3, 25-hydroxyvitamin D3, 1alpha,25-dihydroxyvitamin D3 and 1,24R,25-trihydroxyvitamin D3, respectively. These compounds were unequivocally identified by means of mass spectrometry. The results confirm that Solanum malacoxylon contains, in addition to 1alpha,25-dihydroxyvitamin D3, vitamin D3, 25-hydroxyvitamin D3 and possibly other as yet unidentified derivatives. As 1,24,25-trihydroxyvitamin D3 is absent in plant extracts not incubated with ruminal fluid, the data also indicate that rumen microbes may convert 1alpha,25-dihydroxyvitamin D3 into 1,24,25-trihydroxyvitamin D3.