Mechanism of the purgative action of multiflorin A

Multiflorin A (1; kaempferol 3-O-β-D-(6-acetyl)-glucopyranosyl (1→4)-α-L-rhamnopyranoside), a kaempferol glycoside with an acetyl group in the sugar moiety, is the active principle of herbal medicines used for purging, such as the rose fruit (fruit of Rosa multiflora). In previous studies, we demonstrated that an acetyl group on the sugar moiety was responsible for the activity of 1and the active site of1 was the small intestine [1]. In the present study, we analyzed the purging related proteins of 1 in the small intestine mucosa by proteomic analysis. We repeated three purgative tests using 1 at intervals of ten days, and then selected three purgative-negative (PE-) mice that had no diarrhea in each test and three purgative-positive (PE+) mice that had diarrhea in each test. We then compared soluble protein profiles of the small intestine mucosae of both groups, and identified the differentially expressed proteins using 2-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and LC-MS/MS spectrometry. As a result, we identified seven proteins showing increased expression by the PE+ mice and six proteins showing increased expression by the PE- mice. We focused on calreticulin that had increased in the PE+ mice, and demonstrated in vivo that activated expression of 1 involves an acetyl transfer reaction. Calreticulin is a major intercellular calcium-binding protein, which acts also as an acetyltransferase, catalyzing the transfer of the acetyl group to a functional receptor protein, such as nitric oxide synthase, which are activated by acetylation. Curcumin, a histone acetyltransferase inhibitor significantly inhibits the purging activity. Valproic acid, a histone deacetylase inhibitor significantly enhanced the purging activity. These results suggest that the active mechanism of 1 involves an acetyl transfer reaction to an unkown functional receptor protein. We are currently performing studies to determine the functional receptor protein.