Ardipusilloside I induces apoptosis by regulating Bcl-2 family proteins in human mucoepidermoid carcinoma Mc3 cells.

BACKGROUND: Ardisia pusilla A. DC., family Myrsinaceae, is a traditional Chinese medicine named Jiu Jie Long with a variety of pharmacological functions including anti-cancer activities. In this study, we purified a natural triterpenoid saponin, ardipusilloside I, from Ardisia pusilla, and show that it exhibits inhibitory activities in human mucoepidermoid carcinoma Mc3 cells. We also investigated the underlying mechanisms of proliferation inhibition that ardipusilloside I exerts on Mc3 cells. METHODS: MTT test was used to detect cell proliferation. Cell apoptosis was detected by transmission electron microscopy, Hoechst-33342 staining, DNA fragmentation detection, and flow cytometry. We also used western blot analysis to detect the potential mechanisms of apoptosis. RESULTS: Ardipusilloside I affected the viability of Mc3 cells in a dose- and time-dependent manner. The IC50 of ardipusilloside I was approximately 9.98 µg/ml at 48 h of treatment. Characteristic morphological changes of apoptosis, including nuclear condensation, boundary aggregation and splitting, and DNA fragmentation, were seen after treatment with 10 µg/ml ardipusilloside I for 48 h. Western blots demonstrated that ardipusilloside I caused Mc3 cell death through the induction of apoptosis by downregulation of Bcl-2 protein levels and upregulation of Bax and caspase-3 protein levels. CONCLUSIONS: Our results revealed that ardipusilloside I could be a new active substance for mucoepidermoid carcinoma treatment. We demonstrated that the potential mechanism of inhibition might be through the induction of apoptosis by regulation of Bcl-2 family protein levels. This suggests a further rationale for the development of ardipusilloside I as an anti-cancer agent.

Determination of free and glucuronidated kaempferol in rat plasma by LC-MS/MS: application to pharmacokinetic study.

Flavanoid kaempferol is mainly present as glucuronides and sulfates in rat plasma, and small amounts of the intact aglycone are also detected. In the this study, a rapid, specific and sensitive liquid chromatography-electrospray ionization-tandem mass spectrometry method (HPLC-MS/MS) was developed and validated for determination of kaempferol and its major metabolite glucuronidated kaempferol in rat plasma. A liquid-liquid extraction with acetic ether was involved for the extraction of kaempferol and internal standard. Analytes were separated on a C18 column (150 mm x 2.1 mm, 4.5 microm, Waters Corp.) with isocratic elution at a flow-rate of 0.3 ml min(-1). The mobile phase was consisted of 0.5% formic acid and acetonitrile (50:50, v/v). The Quattro Premier HPLC-MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. The method was validated according to the FDA guidelines for validation of bioanalytical method. The validated method was successfully applied to the study of the pharmacokinetics in rats after oral administration of kaempferol with different doses.