Characterization of Citrus aurantifolia bioactive compounds and their inhibition of human pancreatic cancer cells through apoptosis

In the current study, we have isolated and purified five putative compounds from lime (Citrus aurantifolia Swingle) seeds using Soxhlet extraction and chromatographic separation. The purity and structures of the isolated compounds were identified by TLC, HPLC and mass spectra as limonin, limonexic acid (LNA), isolimonexic acid (ILNA), beta-sitosterol glucoside (SG) and limonin glucoside (LG). These putative compounds exhibited significant inhibition of Panc-28 cells with IC50 values in the range of 18-42 mu M in MTT assay after incubation for 72 h, which was confirmed by viable cell count. Further, induction of apoptosis was confirmed through annexin-FITC staining of the cells and expression of apoptosis related proteins was also studied to understand the mechanism. Expression of apoptosis related protein showed apoptosis through induction of p53 by, p21 dependent manner and cytochrome-c mediated caspases driven intrinsic pathway. Expression of apoptosis favoring proteins in cells treated with isolated compounds were in the order of ILNA>LNA>SG>Limonin>LG based on the expression ratio of bax/bcl2. These results were further supported through fluorescent probed microscopic images. To the best of our knowledge, this is the first report on isolation and identification of LNA, SG and LG from lime and their mechanism of inhibition of human pancreatic cancer cells. (C) 2009 Elsevier B.V. All rights reserved.