Isolation and effects of citrus limonoids on cytochrome p450 inhibition, apoptotic induction and cytotoxicity on human cancer cells.

This dissertation illustrates an efficient purification method for citrus limonoids and flavonoids, while examining their effects on cytochrome P450 inhibition and apoptotic induction on human neuroblastoma (SH-SY5Y) and colonic adenocarcinoma (Caco-2) cells. The first study developed a bulk purification method for limonoids, from seeds and molasses of citrus fruits, using a combination of chromatographic techniques. This also resulted in an efficient purification method for naringin and hesperidin from citrus byproducts. The second study investigated the inhibitory effects of purified limonoids and flavonoids on the enzymatic activities of different isoforms of human cytochrome P450. O-Dealkylase and hydroxylase activities of CYP1A2, CYP1B1, CYP3A4 and CYP19, using specific substrates such as ethoxyresorufin (ethoxyresorufin O-dealkylase, EROD), methoxyresorufin (methoxyresorufin O-dealkylase, MROD), and dibenzylfluorescein (DBF), were found to be significantly (P < 0.001) reduced at micromolar levels. A kinetic analysis showed competitive and non-competitive modes of inhibition by limonoids, on CYP19 hydroxylase activity. The results corroborate the active role of limonoids in the redox cycling mechanisms. The third study examined the antioxidant and apoptotic inducing ability of limonoid glucosides on human neuroblastoma cells. Four limonoid glucosides, LG (17beta-D glucopyranoside limonin), OG (obacunone 17beta-D glucopyranoside), NAG (nomilinic acid 17beta-D glucopyranoside), and DNAG (deacetylnomilinic acid 17beta-D glucopyranoside), have shown superoxide scavenging at millimolar levels. Micromolar amounts of LG and OG induced rapid necrosis of SH-SY5Y cells. Cytotoxicity was correlated (P = 0.046) to a concentration and timedependent increase in caspase 3/7 activity. Analyses of DNA content during the S phase of the cell cycle indicated reductions of 86.6% for LG and 82.3% for OG as compared to untreated. The results validate the antineoplastic distinctiveness of limonoid glucosides. In the fourth study, cytotoxic effects of limonoid aglycones and glucosides were assessed on human SH-SY5Y neuroblastoma and colon carcinoma (CaCo-2) cell lines and compared with the non-cancerous Chinese hamster ovary (CHO) cells. Significant (P < 0.001) cytotoxic effects were observed only on cancerous cells, over 24 to 36 h. The study revealed a marked increase in the DNA content of aneuploidic cells, which results in cell cycle arrest. The results confirm that glycosides are the most active apoptotic inducing form.