RP-HPLC Separation of Isomeric Withanolides: Method Development, Validation and Application to In situ Rat Permeability Determination.

Withanolides are the group of active chemical constituents of Withania somnifera (L.) Dunal. Withaferin A, withanolide A and withanone presents three of the biologically most active constituents of this herb. These steroidal lactones are isomers of each other and thus, pose significant difficulty in their separation. In present study, a simple, specific and reliable RP-HPLC method has been developed and validated for their separation and simultaneous quantification. Separation was carried out on Lichrocart Purospher STAR RP-18e column (250 × 4.5 mm, 5 µm) using mobile phase, methanol and 0.01 M ammonium acetate buffer (pH 5) in the ratio 60:40, v/v. The calibration curves were linear (r2 > 0.99) for all the three compounds across concentration range of 1.56-50 µg/mL. The lower limit of quantification for all the analytes was 1.56 µg/mL. The intra-day and inter-day accuracy was between 88.65% and 110.66% and coefficient of variation was between 0.55 and 10.12. The analytes were stable under different storage conditions. The developed method was successfully applied to analyze the samples for simultaneous determination of permeability of the three withanolides in rats using in situ single-pass intestinal perfusion model. Withanolide A and withanone were found to be high permeability compounds while withaferin A could not be detected.

Synthesis and biological evaluation of some novel triazole hybrids of curcumin mimics and their selective anticancer activity against breast and prostate cancer cell lines.

The anti-cancer property of curcumin, an active component of turmeric, is limited due to its poor solubility, stability and bioavailability. To enhance its efficacy, we designed a novel series of twenty-four monocarbonyl curcumin analogue-1,2,3-triazole conjugates and evaluated their anti-cancer activity towards endocrine related cancers. The new compounds (17-40) were synthesized through CuAAC click reaction and SAR analysis carried out. Out of these all, compound 17 showed most significant anti-cancer activity against prostate cancer cells with IC50 values of 8.8µM and 9.5µM in PC-3 and DU-145 cells, respectively. Another compound 26 showed significant anti-cancer activity against breast cancer cells with IC50 of 6µM, 10µM and 6.4µM in MCF-7, MDA-MB-231 and 4T1 cells, respectively while maintaining low toxicity towards non-cancer originated cell line, HEK-293. Compounds 17 and 26 arrested cell cycle and induced mitochondria-mediated apoptosis in cancer cells. Further, both of these compounds significantly down-regulated cell proliferation marker (PCNA), inhibited activation of cell survival protein (Akt phosphorylation), upregulated pro-apoptotic protein (Bax) and down-regulated anti-apoptotic protein (Bcl-2) in their respective cell lines. In addition, in vitro stability, solubility and plasma binding studies of the compounds 17 and 26 showed them to be metabolically stable. Thus, this study identified two new curcumin monocarbonyl-1,2,3-triazole conjugate compounds with more potent activity than curcumin against breast and prostate cancers.