José Luis Poveda Pérez

Limonin methoxylation influences the induction of glutathione S-transferase and quinone reductase.

Previous studies have indicated the chemopreventive potential of citrus limonoids due to the induction of phase II detoxifying enzymes. In the present study, three citrus limonoids were purified and identified from sour orange seeds as limonin, limonin glucoside (LG), and deacetylnomilinic acid glucoside (DNAG). In addition, limonin was modified to defuran limonin and limonin 7-methoxime. The structures of these compounds were confirmed by NMR studies. These five compounds were used to investigate the influence of phase II enzymes in female A/J mice. Our results indicated the highest induction of glutathione S-transferase (GST) activity against 1-chloro-2,4-dinitrobenzene (CDNB) by DNAG (67%) in lung homogenates followed by limonin-7-methoxime (32%) in treated liver homogenates. Interestingly, limonin-7-methoxime showed the highest GST activity (270%) in liver against 4-nitroquinoline 1-oxide (4NQO), while the same compound in the stomach induced GST by 51% compared to the control. The DNAG treated group induced 55% in stomach homogenates. Another phase II enzyme, quinone reductase (QR), was significantly induced by limonin-7-methoxime by 65 and 32% in liver and lung homogenates, respectively. Defuran limonin induced QR in lung homogenates by 45%. Our results indicated that modification of limonin has differential induction of phase II enzymes. These findings are indicative of a possible mechanism for the prevention of cancer by aiding in the detoxification of xenobiotics.

In vivo induction of phase II detoxifying enzymes, glutathione transferase and quinone reductase by citrus triterpenoids.

BackgroundSeveral cell culture and animal studies demonstrated that citrus bioactive compounds have protective effects against certain types of cancer. Among several classes of citrus bioactive compounds, limonoids were reported to prevent different types of cancer. Furthermore, the structures of citrus limonoids were reported to influence the activity of phase II detoxifying enzymes. The purpose of the study was to evaluate how variations in the structures of citrus limonoids (namely nomilin, deacetyl nomilin, and isoobacunoic acid) and a mixture of limonoids would influence phase II enzyme activity in excised tissues from a mouse model.MethodsIn the current study, defatted sour orange seed powder was extracted with ethyl acetate and subjected to silica gel chromatography. The HPLC, NMR and mass spectra were used to elucidate the purity and structure of compounds. Female A/J mice were treated with three limonoids and a mixture in order to evaluate their effect on phase II enzymes in four different tissues. Assays for glutathione S-transferase and NAD(P)H: quinone reductase (QR) were used to evaluate induction of phase II enzymatic activity.ResultsThe highest induction of GST against 1-chloro-2,4-dinitrobenzene (CDNB) was observed in stomach (whole), 58% by nomilin, followed by 25% isoobacunoic acid and 19% deacetyl nomilin. Deacetyl nomilin in intestine (small) as well as liver significantly reduced GST activity against CDNB. Additionally isoobacunoic acid and the limonoid mixture in liver demonstrated a significant reduction of GST activity against CDNB. Nomilin significantly induced GST activity against 4-nitroquinoline 1-oxide (4NQO), intestine (280%) and stomach (75%) while deacetyl nomilin showed significant induction only in intestine (73%). Induction of GST activity was also observed in intestine (93%) and stomach (45%) treated with the limonoid mixture. Finally, a significant induction of NAD(P)H: quinone reductase (QR) activity was observed by the limonoid mixture in stomach (200%). In addition, the deacetyl nomilin treatment group displayed an increase in QR activity in liver (183%) and intestine (22%).ConclusionThe results of the present study suggests that, dietary intake of citrus limonoids may provide a protective effect against the onset of various cancers by inducing the activity of certain phase II detoxifying enzymes in specific organs.

Development of a method for the quantification of D-glucaric acid in different varieties of grapefruits by high-performance liquid chromatography and mass spectra

Grapefruits were found to contain D-glucaric acid, which has anticancer properties. In the present investigation, a method has been developed for the quantification of D-glucaric acid in grapefruit by high-performance liquid chromatography (HPLC) using a simple isocratic mobile phase with detection at 210 nm. Grapefruit samples were homogenized, centrifuged and filtered through a 0.45 microm membrane and injected into a HPLC system. The developed method was used for the quantification of D-glucaric acid in nine widely used grapefruit varieties. Furthermore, the identity of D-glucaric acid was confirmed by mass spectra. Seasonal variation of D-glucaric acid within the individual varieties were also measured in fruits harvested during November, February and May. The overall trend of D-glucaric acid level was increased from early to late season fruits. The developed method has a sensitivity of D-glucaric acid as low as 0.05 microg with an accuracy and precision >95%. This method was found to be simple, fast, accurate and reproducible. Moreover, the identity of D-glucaric acid was confirmed by mass spectra. Additionally, the labor intensity and cost of sample preparation were greatly reduced as compared to reported methods. This is the first report on quantification of D-glucaric acid in different varieties of grapefruits from three harvesting sessions.