Purification of citrus limonoids and their differential inhibitory effects on human cytochrome P450 enzymes

Abstract

Recent studies demonstrated that citrus limonoids and flavonoids possess numerous health promoting properties. In the present study, glucosides of limonoids and flavonoids were purified from citrus molasses and limonoid aglycones from citrus seeds. Glucosides were separated on styrene (divinylbenzene), Q-sepharose resins with increasing concentration of sodium chloride. A pH-dependent cold precipitation was carried out for the isolation of naringin in large quantity. Major aglycones such as limonin and nomilin were isolated from seeds by direct crystallization and minor limonoids were purified by vacuum liquid chromatography. The structures of the isolated compounds were confirmed by NMR spectra. Individual limonoids were tested for O-dealkylase and hydroxylase activities of human cytochrome P450 (CYP) isoenzymes such as CYP1A2, CYP1B1, CYP3A4 and CYP19, using ethoxyresorufin, methoxyresorufin and dibenzylfluorescein as substrates. Partial to high inhibition of CYPs was observed in dose-dependent assays. Significant (P < 0.001) reductions in enzyme activities were observed with purified compounds above 2 µmol. Kinetic analyses indicated that limonin glucoside inhibited CYP19 competitively (IC 50 , 7.1 µ mol L −1 ), whereas Nomilinic acid glucoside inhibited it noncompetitively (IC 50 , 9.4 µ mol −1 ). Nomilinic acid glucoside was the most potent limonoid, with an overall IC 50 of <10 µ mol, for all the enzymes tested. The differential inhibition of CYPs can be ascribed to structural variations of the limonoid nucleus. Limonoid inhibition of key CYPs involved in carcinogenesis supports growing evidence that citrus limonoids act as anticancer agents.

The Chemistry and Pharmacology of Citrus Limonoids

“…No surprise, then, if the two CLs present similar activities and potencies of action [3,47,51,58,77]. Paralleling what has been observed for 1 and its glucoside 11, the glucoside of 4 (12) sharply reduced cancer cell viability as compared to aglycone [58] thus corroborating the hypothesis that glucosides may be better active apoptosis-inducing agents [132].…”

“…The unbalanced solubility profile of the latter may explain the relatively low number of studies on their respective biological activities which are hampered by difficult isolation from natural sources and poor pharmacokinetics. Analgesic [45], anticancer [46][47][48][49][50][51][52][53][54][55][56][57][58][59], anti-inflammatory [45,[60][61][62], antibacterial [63,64], antioxidant [4,65,66], antiviral [67], larvicidal [68,69] Anticancer [46,47,58,59,70], anti-hyperglycemic [71], anti-inflammatory [25,62], antioxidant [4,65,66], antiviral [67], larvicidal [68,69] …”

“…Thus, Poulose et al [47] have evaluated the effects of limonoid aglycones and glucosides on the activity of human CYP isoenzymes including CYP1A2, CYP1B1, CYP19, and CYP3A4. Individual limonoids were tested for O-dealkylase and hydroxylase activities of the human CYP isoenzymes using ethoxyresorufin, methoxyresorufin and dibenzylfluorescein as substrates.…”

Limonin 7-methoxime interferes with Escherichia coli biofilm formation and attachment in type 1 pili and antigen 43 dependent manner

“…1) were determined by spectrometric studies. Chemical shifts of limonin, LG, DFL and LM were recorded and compared with previously reported data (Mandadi et al, 2007;Perez et al, 2009;Poulose, Jayaprakasha, Mayer, Girennavar, & Patil, 2007). The 1 H and 13 C spectra of L7OX with assignments of various signals are presented in Table 3.…”

Degreening and postharvest storage influences ‘Star Ruby’ grapefruit (Citrus paradisi Macf.) bioactive compounds

“…Butylated hydroxytoluene (BHT), lycopene, b-carotene, narirutin, naringin, didymin, poncirin, limonin, nomilin, and DHB were procured from Sigma Aldrich Co. (St. Louis, MO, USA). Compounds namely DNAG and 5G7MC were purified in the lab according our published methods (Girennavar, Jayaprakasha, Jadegoud, Nagana Gowda, & Patil, 2007;Poulose, Jayaprakasha, Mayer, Girennavar, & Patil, 2007).…”