Yee-Ping Ho

Simultaneous determination of seven major isosteroidal alkaloids in bulbs of Fritillaria by gas chromatography.

The present paper describes the development of a most simple, sensitive, and specific gas chromatographic method to date, for the direct determination of seven major bioactive isosteroidal alkaloids, namely ebeiedine, ebeiedinone, ebeienine, hupehenine, isoverticine, verticine, verticinone and imperialine, in Fritillaria species, a commonly used antitussive traditional Chinese medicinal (TCM) herb. In the present study, a commercially available Supelco SAC-5 capillary column (30 m x 0.25 mm, 0.25 microm) specifically designed for the analysis of steroids was utilized for the direct determination of Fritillaria alkaloids. Calibration curves were obtained by spiking authentic compounds and the internal standard (solanidine) into herbal samples prior to extraction. Extraction was conducted simply by shaking the pre-alkalized diethyl ether solution (5.0 ml) containing dried herb (0.1 g) for 2 h. All calibration curves showed good linear regressions (r2>0.995) within test ranges. The assay was reproducible and accurate with the overall intra- and inter-day variation and accuracy of less than 10% and more than 90%, respectively. The developed GC method was successfully utilized to analyze seven major bioactive alkaloids in seven Fritillaria species, and the results demonstrate that this direct GC analytical method is suitable for the quality control of this commonly used antitussive TCM herb.

Determination of the release of hydrolyzed demethylcantharidin from novel traditional Chinese medicine–platinum compounds with anticancer activity by gas chromatography

The present paper describes the development of a simple, accurate and reproducible gas chromatographic method for the determination of hydrolyzed demethylcantharidin release from a novel series of traditional Chinese medicine (TCM)-platinum compounds possessing potent anticancer and protein phosphatase 2A (PP2A)-inhibition properties. The salient features of the validated assay were a limit of detection (LOD) of 2 microg/mL, a limit of quantitation (LOQ) of 6 microg/mL, an intra- and inter-day precision of less than 11%, and an accuracy of more than 92%. The developed GC-flame ionization detection (FID) method was successfully utilized for the analysis of hydrolyzed demethylcantharidin, the TCM component that is slowly released from the novel compounds over 24 h, leading to PP2A inhibition. Further structural confirmation was achieved by GC-MS. The GC method is suitable for further mechanistic, pharmacokinetic and metabolic studies of the TCM-Pt compounds that might prove to be new anticancer agents with novel mechanisms of cytotoxic action.

East-west fusion-necrosis and apoptosis acting in concert by demethylcantharidin-integrated platinum complexes.

The different types of cell death occurring in HCT116 colorectal cancer cell after the treatment of cisplatin, carboplatin, oxaliplatin, DMC, Pt(NH3)2(demethylcantharidin:DMC) and Pt(R,R-1,2-diaminocyclohexane: DACH)(DMC) were examined. Pt(NH3)2(DMC) and Pt(R,R-DACH)(DMC) are the two DMC-integrated platinum complexes:Pt(R,R-DACH)(DMC) with the same Pt moiety as oxaliplatin and Pt(NH3)2(DMC) akin to carboplatin. Using the light scattering properties of cells combined with propidium iodide (PI) red fluorescence to distinguish between early apoptotic and necrotic cells, the results confirmed that apoptosis, which triggered by cisplatin, carboplatin and oxaliplatin, was the major type of cell death, while the major DMC-induced cell death type was necrosis. The increase in the necrotic cell population was observed after Pt((NH3)2(DMC) treatment when compared with carboplatin; therefore, the DMC ligand in Pt(NH3)2(DMC) contributing to cell death was demonstrated. However, the DMC ligand in Pt(R,RDACH)( DMC) failed to elevate the necrotic cell population significantly in contrast to oxaliplatin, thus Pt(R,R-DACH) in Pt(R,RDACH)( DMC) dominantly contributed to cell death. The IC50 value (antiproliferative activity) reflects the net effect of drugs on cell proliferation resulting from inhibition of cell growth and division, and induction of cell death. The sub-G1 populations representing the sum of the amounts of late apoptotic cells and necrotic cells after the treatment of cispatin, carboplatin, oxaliplatin, DMC, Pt(NH3)2(DMC) and Pt(R,R-DACH)(DMC) were found to be not correlated with the corresponding IC50 values;therefore, the rate of cell growth and division inhibition rather than the rate of induction of cell death dictated to the IC50 values. This combined analysis of IC50 values and the sub-G1 population data also reveals that the platinum compounds containing R,R-DACH are most efficient in inhibiting cell growth and division, while carboplatin induces cell death most rapidly. When the Pt-DNA adducts are believed to be major cytotoxic species, the combined analysis of the IC50 values and the sub-G1 population data infers that the R,R-DACH-Pt-1,2-d(GpG) intrastrand cross-links caused by oxaliplatin treatment are more effective in inducing cell growth and division inhibition, while the (NH3)2Pt-1,3- d(GpXpG) intrastrand cross-links caused by carboplatin treatment can trigger cell death more rapidly. The rate of cell growth and division inhibition and the cell death rate induced by the main cisplatin-DNA adducts:(NH3)2Pt-1,2-d(GpG) intrastrand cross-links lie in between.