Isolation and identification of phase II enzyme-inducing agents from nonpolar extracts of green onion (Allium spp.)

The objective of the study was to isolate and identify potential cancer preventive constituents from green onion based on the ability to induce quinone reductase (QR, a representative phase II enzyme) in murine hepatoma cells (Hepa 1c1c7). Crude nonpolar solvent extracts were prepared from freeze-dried green onion by sequential refluxing with hexane and then ethyl acetate, followed by liquid-liquid extraction. Active fractions were subjected to the Hepa 1c1c7 bioassay-guided steps of flash chromatography, thin layer chromatography (TLC), and high-pressure preparative liquid chromatography (HPLC) to afford pure isolates capable of inducing QR. Multiple fractions were active in inducing QR. Five pure compounds were isolated from active fractions and identified using spectroscopic methods; these were p-hydroxyphenethyl trans-ferulate (1), 5,6-dimethyl-2-pyridinecarboxylic acid (2), ferulic acid (3), 1-(6-hydroxy-[3]pyridyl)-propan-1-one (4), and N-trans-feruloyl 3-O-methyldopamine (5). p-Hydroxyphenethyl trans-ferulate (1) doubled QR specific activity in Hepa 1c1c7 cells at a level of 2.1 microg/mL (6.6 microM).     

Grapefruit and oroblanco enhance hepatic detoxification enzymes in rats: possible role in protection against chemical carcinogenesis

Citrus fruits are considered to be functional foods that promote good health. This study was carried out to assess the effect of oroblanco and grapefruit consumption on hepatic detoxification enzymes. Male Sprague-Dawley rats were provided with either regular drinking water (control) or experimental treatments of oroblanco juice, grapefruit juice, or a sugar mix for 6 weeks. After 1 week of treatment, half the animals in each group were injected with the procarcinogen 1,2-dimethylhydrazine. Grapefruit juice significantly increased activity and expression of the hepatic phase I enzyme, cytochrome P450 CYP1A1, with a marked trend toward enhanced NAD(P)H:quinone reductase (QR) activity. Oroblanco juice significantly increased glutathione S-transferase phase II enzyme activity along with CYP1A1 expression and a notable trend toward increased activity of both CYP1A1 and QR. These results suggest that these citrus fruits are bifunctional inducers, modulating both phase I and phase II drug-metabolizing enzymes to enhance hepatic detoxification.     

Induction of phase II detoxification enzymes in rats by plant-derived isothiocyanates: comparison of allyl isothiocyanate with sulforaphane and related compounds

Plants of the family Brassicaceae contain high levels of glucosinolates. The latter compounds are degraded to isothiocyanates, some of which have been shown to be potent inducers of phase II detoxification enzymes in vitro. In the present study, the ability of six plant-derived isothiocyanates (allyl isothiocyanate, iberverin, erucin, sulforaphane, iberin, and cheirolin) to increase tissue levels of the phase II detoxification enzymes quinone reductase (QR) and glutathione S-transferase (GST) in a variety of rat tissues has been compared. At the low dose level employed (40 micromol/kg/day), cheirolin was without effect in any tissue. All of the other isothiocyanates, however, increased GST and QR activities in the duodenum, forestomach, and/or the urinary bladder of the animals, with the greatest effects being seen in the urinary bladder. With the exception of cheirolin, little difference was observed in the inductive activity of the various isothiocyanates. Phase II enzymes are known to protect against chemical carcinogenesis, and the selectivity of isothiocyanates in inducing such enzymes in the bladder is of interest in view of recent epidemiological studies showing a decreased incidence of cancer of this organ in individuals with a high dietary intake of Brassica vegetables.     

Effect of anthocyanin fractions from selected cultivars of Georgia-grown blueberries on apoptosis and phase II enzymes

In recent years, considerable attention has been paid to anthocyanins due to their abilities to inhibit oxidative stress and cell proliferation. The regulations of apoptosis and the phase II enzymes glutathione-S-transferase (GST) and quinone reductase (QR) are other potential mechanisms through which flavonoids such as anthocyanins may prevent cancer. Our study confirmed that anthocyanin fractions from high bush blueberry cultivars increased apoptosis using two different methods: DNA fragmentation and caspase-3 activity. The effect of anthocyanins on the activity of the detoxifying enzymes GST and QR was also determined. Major anthocyanins identified were delphinidin, cyanidin, peonidin, petunidin, and malvidin. In Tifblue and Powderblue cultivars, DNA fragmentation increased at anthocyanin concentrations from 50 to 150 microg/mL, but cells treated with the anthocyanin fraction of Brightblue and Brightwell showed a prominent ladder at 50-100 microg/mL when compared to cells treated with 150 microg/mL. There was a significant difference in the caspase-3 activity (P < 0.05) between the control cells and the cells treated with anthocyanins from all of the cultivars. The response correlated positively with dose. The QR activity was lower in all cells treated with an anthocyanin fraction from Tifblue, Powderblue, Brightblue, and Brightwell cultivars than in control cells (P < 0.05). The activity decreased gradually when treated with increased concentrations of anthocyanin fractions (50-150 microg/mL) in the Tifblue and Powderblue cultivars. The GST activity was lower (P < 0.05) in cells treated with anthocyanin fractions from all of the cultivars and at all concentrations. These results indicated that apoptosis was confirmed in HT-29 cells when treated with anthocyanins from blueberry cultivars at 50-150 microg/mL concentrations, but these same concentrations decrease QR and GST activities rather than induce them.     

β-carboline derivatives and diphenols from soy sauce are in vitro quinone reductase (QR) inducers

A murine hepatoma (Hepa 1c1c7) cellular bioassay was used to guide the isolation of phase II enzyme inducers from fermented soy sauce, using quinone reductase (QR) as a biomarker. A crude ethyl acetate extract, accounting for 8.7% of nonsalt soluble solids of soy sauce, was found to double relative QR specific activity at 25 μg/mL (concentration required to double was defined as a "CD value"). Further silica gel column fractionation yielded 17 fractions, 16 of which exhibited CD values for QR induction of <100 μg/mL. The four most potent fractions were subfractionated by column and preparative thin layer chromatography, leading to the isolation and identification of two phenolic compounds (catechol and daidzein) and two β-carbolines (flazin and perlolyrin), with respective CD values of 8, 35, 42, and 2 μM. Western blots confirmed that the increases in QR activity corresponded to dose-dependent increases in cellular levels of NAD[P]H:quinone oxidoreductase 1 protein by these four QR inducers. To the authors' knowledge, this is the first report on the ability of β-carboline-derived alkaloids to induce phase II enzymes.     

In vitro activity of almond skin polyphenols for scavenging free radicals and inducing quinone reductase

Observational studies and clinical trials suggest nut intake, including almonds, is associated with an enhancement in antioxidant defense and a reduction in the risk of cancer and cardiovascular disease. Almond skins are rich in polyphenols (ASP) that may contribute to these putative benefits. To assess their potential mechanisms of action, we tested the in vitro effect of ASP extracted with methanol (M) or a gastrointestinal juice mimic (GI) alone or in combination with vitamins C (VC) or E (VE) (1-10 micromol/L) on scavenging free radicals and inducing quinone reductase (QR). Flavonoid profiles from ASP-M and -GI extracts were different from one another. ASP-GI was more potent in scavenging HOCl and ONOO (-) radicals than ASP-M. In contrast, ASP-M increased and ASP-GI decreased QR activity in Hepa1c1c7 cells. Adding VC or VE to ASP produced a combination- and dose-dependent action on radical scavenging and QR induction. In comparison to their independent actions, ASP-M plus VC were less potent in scavenging DPPH, HOCl, ONOO (-), and O 2 (-) (*). However, the interaction between ASP-GI plus VC promoted their radical scavenging activity. Combining ASP-M plus VC resulted in a synergistic interaction, inducing QR activity, but ASP-GI plus VC had an antagonistic effect. On the basis of their total phenolic content, the measures of total antioxidant activity of ASP-M and -GI were comparable. Thus, in vitro, ASP act as antioxidants and induce QR activity, but these actions are dependent upon their dose, method of extraction, and interaction with antioxidant vitamins.     

Antioxidant functions of selected allium thiosulfinates and S-alk(en)yl-L-cysteine sulfoxides

Pure thiosulfinates, R-S(O)S-R (2), where R = Me (2a), Pr (2b), or All (2c), at levels up to 4 mM were not capable of scavenging hydrogen peroxide or superoxide anion. Relative to standard antioxidants (ascorbic acid, n-propyl gallate, butylated hydroxytoluene, Trolox, and reduced glutathione), these thiosulfinates were 1-3 orders of magnitude less efficient at reducing 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, 0.5-2 orders of magnitude less efficient at quenching singlet oxygen, and about equally effective at scavenging hydroxyl radical. Generally, AllS(O)SAll (2c) was the most effective and PrS(O)SPr (2b) was the least effective thiosulfinate in these assays, except that MeS(O)SMe (2a) exhibited no quenching effect toward singlet oxygen. These thiosulfinates were also incapable at levels up to 0.1 mM (where they were toxic) of in vitro induction of quinone reductase (QR) in murine hepatoma (hepa 1c1c7) cells. However, S-1-propenyl-L-cysteine sulfoxide (isoalliin, 1a) and cycloalliin (3) induced QR in this system at 2 mM and 1 mM, respectively, although doubling of QR required levels of 10-15 mM.     

Polyphenolic compounds, antioxidant capacity, and quinone reductase activity of an aqueous extract of Ardisia compressa in comparison to mate (Ilex paraguariensis) and green (Camellia sinensis) teas

Aqueous extracts of the leaves of Ardisia compressa (AC) have been used in folk medicine to treat various liver disorders including liver cancer. The objective of this study was to partially characterize and determine the total polyphenol content, antioxidant capacity, and quinone reductase activity of A. compressa tea in comparison to mate (Ilex paraguariensis, MT) and green (Camellia sinensis,GT) teas. Total polyphenol content, antioxidant capacity, and phase II enzyme induction capacity were measured by the modified Folin-Ciocalteu, ORAC, and quinone reductase (QR) assays, respectively. The major polyphenols in AC were not catechins. HPLC retention times and standard spikes of AC indicated the presence of gallic acid, epicatechin gallate, ardisin and kaempferol. Using catechin as standard, the total polyphenol value of AC (36.8 +/- 1.1 mg/mg DL) was significantly lower than GT (137.2 +/- 5.8 mg equivalent of (+)-catechin/mg dried leaves, DL) and MT (82.1 +/- 3.8 mg/mg DL) (P < 0.001). Antioxidant capacity (AC, 333; GT, 1346; MT, 1239 mmol Trolox equivalents/g DL) correlated with total polyphenol values (r(2) = 0.86, P < 0.01). AC (4.5-12.5 microg/mL) induced QR enzyme, in Hepa1c1c7 cells, up to 15%. MT and GT showed no induction at the concentrations tested (0.5-10.5 and 0.5-12.5 mg/mL, respectively). These results suggest that AC has a different mechanism of protection against cytotoxicity that is not related to its antioxidant capacity. Further studies are needed to determine such mechanisms and to explore its potential as a chemopreventive or therapeutic agent.     

Phenolic derivatives from soy flour ethanol extract are potent in vitro quinone reductase (QR) inducing agents

The fractionation of soy flour directed by a cellular bioassay for induction of phase 2 detoxification enzymes was used to identify quinone reductase (QR) inducing agents. A phospholipid-depleted, 80% methanol-partitioned isolate from a crude ethanol extract of soy flour was resolved using normal phase medium-pressure liquid chromatography (MPLC). Early eluting fractions were found to be the most potent QR inducing agents among the separated fractions. Fraction 2 was the most potent, doubling QR at <2 mug/mL. Further fractionation of this isolate led to the identification of several constituents. Fatty acids and sn-1 and sn-2 monoacylglycerols were identified, but were not highly potent QR inducers. Benzofuran-3-carbaldehyde, 4-hydroxybenzaldeyde, 4-ethoxybenzoic acid, 4-ethoxycinnamic acid, benzofuran-2-carboxylic ethyl ester, and ferulic acid ethyl ester (FAEE) were also identified as QR inducing constituents of this fraction. FAEE was the most potent of the identified constituents, doubling QR specific activity at 3.2 muM in the cellular bioassay.     

Pinostrobin from honey and Thai ginger (Boesenbergia pandurata): a potent flavonoid inducer of mammalian phase 2 chemoprotective and antioxidant enzymes

Over 60 different samples comprising 35 distinct honeys were evaluated for their ability to induce mammalian phase 2 detoxication enzymes using a microtiter plate assay of quinone reductase (QR) induction with murine hepatoma cells in microtiter plates. This assay has been used extensively to identify and isolate a variety of natural and synthetic inducers from plants. All 35 honeys examined induced elevations of mammalian QR activity ranging from 153 to 2155 units/g with a mean of 630 and a median of 417 units/g. The concentrations for doubling the QR activity (CD) of certain of the prominent flavonoids found in honey were also assessed (pinostrobin, 0.5 microM; pinocembrin, 110 microM; chrysin, 25 microM) and compared to those of related, more commonly described flavonoids such as quercetin (2.7 microM) and myricetin (58 microM). On the basis of the extremely high QR inducing potency of one of these compounds, pinostrobin (5-hydroxy-7-methoxyflavanone), a bioassay-guided search was conducted which revealed a dietary source of pinostrobin, Boesenbergia pandurata (fingerroot), with extraordinarily high ability to induce mammalian phase 2 detoxication enzymes. Although the QR inducing activity of buckwheat honeys was 2155 +/- 951 units/g (n = 8 samples), which is less than 10% of the average values obtained from fresh broccoli, the potency of fingerroot rhizomes (ca. 110,000 units/g) is even higher than that of broccoli and the potencies of fingerroot oil and powdered rhizome (ca. 500,000 units/g) rival that of broccoli sprouts.     

Effect of diallyl sulfide on in vitro and in vivo Nrf2-mediated pulmonic antioxidant enzyme expression via activation ERK/p38 signaling pathway

Increasing oxidative stress is intimately involved in the pathogenesis of lung failure. Nuclear factor-erythroid 2 related factor 2 (Nrf2) is a key element in redox homeostasis. Nrf2 regulates antioxidant-associated genes that are often the target of phytochemicals in chemoprevention. This study evaluated the effect of diallyl sulfide (DAS), which is present in garlic, on the expression of antioxidant enzymes in the rat lung and the Nrf2 modulation in MRC-5 lung cells. DAS increased the activities of glutathione S-transferase, glutathione reductase, and catalase as well as the GSH/GSSG ratio compared with the lung of untreated control rats (p < 0.05). The pulmonic superoxide dismutase, glutathione peroxidase, NAD(P)H:quinone oxidoreductase 1, and catalase mRNA levels were also significantly increased (p < 0.05) after DAS treatment. Following DAS treatment, DAS level was measured in the plasma after 7 days of oral administration, and the C(max) value was 15 ± 4.2 μM. The total amount of pulmonic Nrf2 and the nuclear translocation of Nrf2 were elevated in DAS-treated rats, clarifying the effect of DAS on the modulation of antioxidant enzymes. Furthermore, DAS could induce nuclear translocation of Nrf2 via ERK/p38 signaling pathway in lung MRC-5 cells. This study demonstrates that DAS administration can significantly induce the activity of antioxidant enzymes in rat lungs and suggests a possible use for DAS as a dietary preventive agent against oxidative stress-induced lung injury.     

Direct antioxidant activity of purified glucoerucin, the dietary secondary metabolite contained in rocket (Eruca sativa Mill.) seeds and sprouts

Rocket (Eruca sativa Mill. or Eruca vesicaria L.) is widely distributed all over the world and is usually consumed fresh (leafs or sprouts) for its typical spicy taste. Nevertheless, it is mentioned in traditional pharmacopoeia and ancient literature for several therapeutic properties, and it does contain a number of health promoting agents including carotenoids, vitamin C, fibers, flavonoids, and glucosinolates (GLs). The latter phytochemicals have recently gained attention as being the precursors of isothiocyanates (ITCs), which are released by myrosinase hydrolysis during cutting, chewing, or processing of the vegetable. ITCs are recognized as potent inducers of phase II enzymes (e.g., glutathione transferases, NAD(P)H:quinone reductase, epoxide hydrolase, etc.), which are important in the detoxification of electrophiles and protection against oxidative stress. The major GL found in rocket seeds is glucoerucin, GER (108 +/- 5 micromol g(-)(1) d.w.) that represents 95% of total GLs. The content is largely conserved in sprouts (79% of total GLs), and GER is still present to some extent in adult leaves. Unlike other GLs (e.g., glucoraphanin, the bio-precursor of sulforaphane), GER possesses good direct as well as indirect antioxidant activity. GER (and its metabolite erucin, ERN) effectively decomposes hydrogen peroxide and alkyl hydroperoxides with second-order rate constants of k(2) = 6.9 +/- 0.1 x 10(-)(2) M(-)(1) s(-)(1) and 4.5 +/- 0.2 x 10(-)(3) M(-)(1) s(-) , respectively, in water at 37 degrees C, thereby acting as a peroxide-scavenging preventive antioxidant. Interestingly, upon removal of H(2)O(2) or hydroperoxides, ERN is converted into sulforaphane, the most effective inducer of phase II enzymes among ITCs. On the other hand, ERN (and conceivably GER), like other ITCs, does not possess any chain-breaking antioxidant activity, being unable to protect styrene from its thermally (37 degrees C) initiated autoxidation in the presence of AMVN. The mechanism and relevance of the antioxidant activity of GER and ERN are discussed.     

Structural and functional characterization of pronyl-lysine,a novel protein modification in bread crust melanoidins showing in vitro antioxidative and phase I/II enzyme modulating activity

Application of an in vitro antioxidant assay to solvent fractions isolated from bread crust, bread crumb, and flour, respectively, revealed the highest antioxidative potential for the dark brown, ethanol solubles of the crust, whereas corresponding crumb and flour fractions showed only minor activities. To investigate whether these browning products may also act as antioxidants in biological systems, their modulating activity on detoxification enzymes was investigated as a functional parameter in intestinal Caco-2 cells. The bread crust and, in particular, the intensely brown, ethanolic crust fraction induced a significantly elevated glutathione S-transferase (GST) activity and a decreased phase I NADPH-cytochrome c reductase (CCR) activity compared to crumb-exposed cells. Antioxidant screening of Maillard-type model mixtures, followed by structure determination, revealed the pyrrolinone reductones 1 and 2 as the key antioxidants formed from the hexose-derived acetylformoin and N(alpha)-acetyl-L-lysine methyl ester or glycine methyl ester, chosen as model substances to mimic nonenzymatic browning reactions with the lysine side chain or the N terminus of proteins, respectively. Quantitation of protein-bound pyrrolinone reductonyl-lysine, abbreviated pronyl-lysine, revealed high amounts in the bread crust (62.2 mg/kg), low amounts in the crumb (8.0 mg/kg), and the absence of this compound in untreated flour. Exposing Caco-2 cells for 48 h to either synthetically pronylated albumin or purified pronyl-glycine (3) significantly increased phase II GST activity by 12 or 34%, respectively, thus demonstrating for the first time that "pronylated" proteins as part of bread crust melanoidins act as monofunctional inducers of GST, serving as a functional parameter of an antioxidant, chemopreventive activity in vitro.     

Constituents of Musa x paradisiaca cultivar with the potential to induce the phase II enzyme,quinone reductase

A new bicyclic diarylheptanoid, rel-(3S,4aR,10bR)-8-hydroxy-3-(4-hydroxyphenyl)-9-methoxy-4a,5,6,10b-tetrahydro-3H-naphtho[2,1-b]pyran (1), as well as four known compounds, 1,2-dihydro-1,2,3-trihydroxy-9-(4-methoxyphenyl)phenalene (2), hydroxyanigorufone (3), 2-(4-hydroxyphenyl)naphthalic anhydride (4), and 1,7-bis(4-hydroxyphenyl)hepta-4(E),6(E)-dien-3-one (5), were isolated from an ethyl acetate-soluble fraction of the methanol extract of the fruits of Musa x paradisiaca cultivar, using a bioassay based on the induction of quinone reductase (QR) in cultured Hepa1c1c7 mouse hepatoma cells to monitor chromatographic fractionation. The structure and relative stereochemistry of compound 1 were elucidated unambiguously by one- and two-dimensional NMR experiments ((1)H NMR, (13)C NMR, DEPT, COSY, HMQC, HMBC, and NOESY) and single-crystal X-ray diffraction analysis. Isolates 1-5 were evaluated for their potential cancer chemopreventive properties utilizing an in vitro assay to determine quinone reductase induction and a mouse mammary organ culture assay.     

Suppressive effect of the ethanolic extract of adlay bran on cytochrome P-450 enzymes in rat liver and lungs

Adlay ( Coix lachryma-jobi L. var. ma-yuen Stapf) is a grass crop and is reported to protect against various diseases such as cancer. To investigate the effect of the ethanolic extract of adlay bran (ABE) on drug-metabolizing enzymes and glutathione-related antioxidant enzymes in rats, three groups of eight male Sprague-Dawley rats each were fed a control diet or a diet containing 5 or 10% ABE for 4 weeks. Significant decreases in microsomal cytochrome P-450 (CYP) 1A1-catalyzed ethoxyresorufin O-deethylation, CYP2C-catalyzed diclofenac 4-hydroxylation, CYP2D-catalyzed dextromethorphan O-demethylation, and CYP3A-catalyzed testosterone 6β-hydroxylation in the liver and CYP1A1-catalyzed ethoxyresorufin O-deethylation in the lungs of rats fed ABE were observed. Immunoblot analyses also showed decreases of CYP1A1, 1A2, 2C6, 2C11, 2D1, 2E1, 3A1, and 3A2 in the liver and CYP1A1 in the lungs. Furthermore, rats fed the 10% ABE diet had a higher glutathione content and glutathione peroxidase, glutathione reductase, and glutathione S-transferase activities in the lungs, but such an increase was not noted in the liver. Inhibition of various CYP-catalyzed enzyme reactions by ABE in rat and human liver microsomes had also been shown. The results of this study indicate that ABE feeding may suppress CYP enzyme activities and CYP protein expression in the liver and lungs of rats. Moreover, the increase of the antioxidant potential by ABE is tissue-specific.     

Comparison of the bioactivity of two glucoraphanin hydrolysis products found in broccoli, sulforaphane and sulforaphane nitrile.

Epidemiological and laboratory studies suggest that dietary broccoli may prevent or delay a variety of cancers. Broccoli and other crucifers contain a relatively unique family of secondary metabolites called glucosinolates. Glucoraphanin, the major glucosinolate in broccoli, is hydrolyzed by an endogenous plant myrosinase to form either the potent anticarcinogen sulforaphane (SF) or sulforaphane nitrile (SF nitrile). The bioactivities of SF and SF nitrile were compared in rats and in mouse hepatoma cells. Male, 4-week-old, Fischer 344 rats were administered SF or SF nitrile (200, 500, or 1000 micromol/kg) by gavage daily for 5 days. Hepatic, colonic mucosal, and pancreatic quinone reductase and glutathione S-transferase activities were induced by high doses of SF, but not by SF nitrile. When Hepa 1c1c7 cells were exposed to increasing levels of each compound for 24 h, quinone reductase showed a 3-fold maximal induction over control at 2.5 microM SF and a 3.5-fold maximal induction over control at 2000 microM SF nitrile, the highest concentration tested. These results demonstrate that SF nitrile is substantially less potent than SF as an inducing agent of phase II detoxification enzymes. Therefore, glucoraphanin hydrolysis directed toward the production of SF rather than SF nitrile could increase the potential chemoprotective effects of broccoli.     

Oligophosphopeptides derived from egg yolk phosvitin up-regulate gamma-glutamylcysteine synthetase and antioxidant enzymes against oxidative stress in Caco-2 cells

Previously, we have found phosphopeptides (PPPs) from hen egg yolk phosvitin possess a potent antioxidative activity against oxidative stress in human intestinal epithelial cells, Caco-2. However, their biological activity at the cellular level has not yet fully understood. The objective of this study is to evaluate the regulation of glutathione (GSH) biosynthesis-associated and antioxidant enzymes against oxidative stress in Caco-2 cells using an in vitro model. Treatment of 1 mM H2O2-induced Caco-2 cells with PPPs increased cellular GSH levels, concomitant with a significant increase in gamma-glutamylcysteine synthetase (gamma-GCS) activity and the expression of gamma-GCS heavy subunit mRNA. Furthermore, intracellular glutathione reductase, glutathione S-transferase, and catalase activities were elevated by PPPs. In addition, PPPs with high content of phosphorus showed higher induction of these enzyme activities than PPPs without phosphorus. These data indicate that oligophosphopeptides from hen egg yolk phosvitin can up-regulate cellular GSH biosynthesis-associated enzymes activity and antioxidative activities, which play key roles against tissue oxidative stress in the human intestinal epithelial cells.