Quinone Reductase Specific Activity Research Articles

Activity-guided isolation of phase II enzyme inducers from buckwheat flour methanolic extracts.

Buckwheat is an important alternative crop and a raw material for functional food formulation. Phase II detoxification proteins/enzymes provide cytoprotective roles against oxidative stress and inflammation originating from various stressors. We aimed to identify and characterize potential phase II enzyme inducers from methanolic extracts of buckwheat flour, using an activity-guided fractionation based on the induction of quinone reductase (QR) (EC 1.6.5.2) in Hepa 1c1c7 cells. We isolated the QR inducers N-trans-feruloyltyramine (I), syringic acid (II), quercetin (III) and myricetin (IV). The relative QR-inducing ability, as well as the concentration required to double QR specific activity (CD values, in parentheses), decreased in the order: quercetin (3.0 µmol L-1 ) > N-trans-feruloyltyramine (24 µmol L-1 ) > myricetin (58 µmol L-1 ) > syringic acid (5.4 mmol L-1 ). Quercetin and N-trans-feruloyltyramine exhibited the greatest extent of QR induction of an approximately four-fold maximum induction and these compounds also exhibited the greatest values for the ratio of IC50 (i.e. level to reduce viability by 50%): CD values of 11 and > 8.3, respectively, among the four QR inducers isolated. Isobologram analyses for binary combinations of compounds I-IV revealed primarily antagonistic interactions for QR induction. These findings add to our understanding of the nutraceutical potential of buckwheat as a chemoprophylactic dietary component. © 2018 Society of Chemical Industry.

Isolation and identification of cytoprotective agents from nonpolar extracts of buckwheat flour

Buckwheat is a raw material used in formulating many foods, and some evidence exists that it can contribute health benefits beyond simple nutritive value. The objective of this study was to isolate and identify potential cytoprotective agents from buckwheat crude extracts using a bioassay-guided strategy. Crude extracts were obtained by successively extracting with n-hexane and ethyl acetate, followed by solvent partitioning. The active fraction(s) were then screened by a quinone reductase (QR) induction bioassay in vitro, and subjected to sequential fractionation with flash column chromatography and preparative thin-layer chromatography. Consequently, three pure compounds were isolated, identified and evaluated for bioactivity. Ferulic acid ethyl ester (1) was the most potent isolate (reported for the first time in buckwheat), doubling QR specific activity (CD value) at 2.1μM, whereas furaneol (2) was a moderate QR inducer with a CD value of 185μM. Protocatechuic acid (3) was least ineffective at inducing QR with a CD value of 2.0mM. Binary mixtures of the three isolated components acted borderline additively/antagonistically in the QR bioassay.

Antioxidant and quinone reductase inducing activities of ethanolic fractions from purple maize

Ethanolic extracts of three different varieties of purple maize (Zea mays L.) were examined for scavenging activities toward nitric oxide (NO) and superoxide (·O2−) and the ability to induce quinone reductase (QR). The crude extracts showed a dose-dependent antioxidant activity in the following order: generic purple > Oaxaca 332 > Veracruz 42. The extract of the generic purple variety exhibited the highest ability to induce QR activity and antioxidant activity was split into several fractions that showed antioxidant activities and capacity for doubling QR specific activity. Fraction III doubled QR at a content of 105 μg/mL (CD value). Semi-preparative LC and UV spectral and MS analyses of fraction III indicated the presence of glucosidic forms of cyanidin, peonidin, pelargonidin and their respective acylated counterparts.

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.

6]-Dehydroshogaol, a minor component in ginger rhizome, exhibits quinone reductase inducing and anti-inflammatory activities that rival those of curcumin

Quinone reductase (QR) inducing activity, a biomarker for Phase II enzyme induction, was detected in a crude ginger rhizome ethanolic extract using a murine hepatoma cell (Hepa 1c1c7) bioassay. An ethyl acetate-partitioned fraction became enriched in QR inducing activity with a CD value (concentration required to double QR specific activity) of 7 μg/ml and was further separated by silica flash chromatography. The four major fractions recovered exhibited CD values ranging 10–20 μg/mL for QR inducing activity. 1H NMR analysis revealed an abundance of gingerols in the first 3 fractions, and this led to a comparative assessment of QR inducing and anti-inflammatory activities among [6]-gingerol and structurally related vanilloids from ginger and other spices. [6]-Dehydroshogaol (DHSG), a minor component of fresh ginger, had QR inducing activity (CD = 8.4 μM) of comparable potency to the well known cancer preventive agent, curcumin (CD = 4.3 μM). In contrast, [6]-gingerol the major pungent component of ginger showed less potent QR inducing activity (CD = 55 μM) and capsaicin exhibited the weakest inducing activity (CD = 103 μM). [6]-Shogaol, [6]-DHSG and curcumin were also potent inhibitors of nitric oxide (NO) synthesis, inhibiting 50% NO evolution in activated macrophages at levels of 10–12 μM (IC 50 values). Comparatively, IC 50 values for [6]-gingerol and capsaicin were about an order of magnitude greater (less potent) at 90 μM and 169 μM, respectively. This appears to be the first report of biological activities of [6]-DHSG, and Phase II enzyme-inducing ability of shogoal and gingerol.

Dietary supplementation of ferulic acid and ferulic acid ethyl ester induces quinone reductase and glutathione- S-transferase in rats

Ferulic acid ethyl ester (FAEE) and ferulic acid (FA) were fed to rats to determine in vivo efficacy in elevating selected phase II enzyme activities and antioxidant capacity. Male Sprague–Dawley rats were fed the AIN-93M control diet, or the control diet supplemented with 1% FA, 1% FAEE, or 0.1% FAEE for 2 weeks. Quinone reductase (QR), glutathione- S-transferase (GST), thioredoxin reductase (TxR) activities, and oxidised and reduced glutathione were determined for brain, lung, liver, kidney, intestine and colon tissues. Both FA and FAEE (1%) supplementation increased QR and GST specific activities in kidney and colon tissues by 23–38% relative to the control diet. FAEE (1%) supplementation also induced QR specific activity, by 1.46- and 1.27-fold over the control diet, in intestinal and lung tissue of animals, whereas FA did not. No effect of diet was observed on liver cytochrome P450-1A1 activity. These results demonstrate that dietary FA and FAEE induce QR activity in the colon, small intestine, lung, and kidney, and improve glutathione antioxidant status in the colon and intestine. Therefore, dietary FA and FAEE may elevate defences to oxidative- and xenobiotic-induced stress in vivo.

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.

Limited contribution of isoflavones to hepatocellular phase II enzyme-inducing activity of soybean ( Glycine max) extracts

Soy flour was successively extracted with ethyl acetate, ethanol, and water and tested for quinone reductase (QR) induction in the Hepa 1c1c7 cell line. The ethanol extract was found to be the most potent, with a CD value (concentration required to double QR specific activity) of 460 μg/ml. Phase separation of the ethanol extract and a phospholipid removal procedure yielded a fraction that doubled QR at 80 μg/ml. Reverse phase medium pressure chromatography of the purified ethanol extract yielded 13 fractions. Constituents of selected fractions were identified using mass spectrometry and NMR analysis. Fractions 2, 8, and 10 contained 47% of the recovered mass and were the least active of the isolates. These three fractions contained isoflavone glycosides. The latest eluting fractions 11–13 were found to be the most potent, and contained the majority of recovered inducing units (IUs), a measure of potency.

Quinone reductase inducing and antioxidant activities of aqueous isolates of green bean (Phaseolus vulgaris L.)

Abstract Aqueous extracts of green bean ( Phaseolus vulgaris L.) were examined for antioxidant functions in terms of total reducing power (TRP), peroxyl radical quenching (PRAC), total antioxidant activity (TAA), and the ability to induce quinone reductase (QR) as a biomarker for phase II detoxification enzymes in Murine hepatoma (Hepa) cells. In terms of Trolox equivalents (TE), crude aqueous extracts exhibited 71 μmol TE/g dry weight (gdw) for TRP, 29 μmol TE/gdw for PRAC, and 8.5 μmol TE/gdw for TAA. Aqueous extractable QR-inducing activity was fractionated by Sephadex LH-20 chromatography with several resulting fractions capable of doubling QR specific activity in the Hepa bioassay. Of 10 recovered fractions, the first and most polar fraction doubled QR at a level of 39 μg/ml (CD value), while the ninth fraction exhibited a similar CD value of 35 μg/ml. Semi-preparative LC and UV spectral analysis of fraction 9 indicated the presence of quercetin and kaempferol glycosides, and simple phenolics among the enriched QR-inducing isolates.

QSAR studies on 1,2-dithiole-3-thiones: modeling of lipophilicity, quinone reductase specific activity, and production of growth hormone

Studies on modeling of lipophilicity (log P) quinone reductase specific activity (logCDQR) and production of growth hormone (logCDGH) of 1,2-dithiole-3-thiones have been carried out using distance-based topological indices. The regression analysis of the data has shown that the set of compounds exhibit ‘familial’ relationships in that excellent results are obtained by dividing the data set into two or more classes (families).

Induction of NADPH:Quinone Reductase by Dietary Phytoestrogens in Colonic Colo205 Cells

Phytoestrogens are a group of naturally occurring diphenolic compounds present in legumes, whole grains, fruits, and vegetables. High consumption of phytoestrogen-rich foods has been linked to a reduced incidence of cancers at many sites. A potential mechanism of dietary anticarcinogenesis involves the induction of detoxifying phase II enzymes such as NADPH:quinone reductase (QR). This study, therefore, examined the ability of six prominent phytoestrogens to affect cellular expression of QR in colonic cells. Colo205 cells were cocultured with various concentrations (0.001 to 10.0 μM) of each phytoestrogen, and then were assessed for cytosolic QR activity, cell growth, and QR mRNA expression. A maximum of 6- to 8-fold induction of QR activity was observed for both enterolactone and genistein, although at high concentrations they showed an adverse effect upon cell growth. The concentrations required to double the specific activity of QR for enterolactone and genistein were about 0.04 and 0.14 μM, respectively. A 2- to 3-fold increase of QR specific activity was found with either biochanin A (1.1 μM) or coumestrol (12.0 μM) treatments. No significant effects were found for daidzein or formononetin treatments. QR induction was further confirmed by using reverse transcription-polymerase chain reaction (RT-PCR) techniques to measure mRNA expression. A significant correlation between the expression of QR mRNA and the corresponding QR activity was observed ( r = 0.76, P < 0.001). The results demonstrated that certain dietary phytoestrogens are capable of QR induction in Colo205 cells by promoting QR mRNA expression, and suggest a novel mechanism by which dietary phytoestrogens may be implicated in colorectal cancer chemoprevention.

Isolation of bioactive constituents from Vaccinium myrtillus (bilberry) fruits and cell cultures

Tissues from bilberry ( Vaccinium myrtillus L.) fruits and callus cultures were extracted and fractionated to investigate phytochemical bioactivity. Major fractions containing flavonoids (proanthocyanidins and anthocyanin pigments) and a hexane extract containing largely chlorophylls, carotenoids, sterols and lipids were isolated, and further screened for anticancer activity by in vitro testing of their ability to induce the phase II xenobiotic detoxification enzyme quinone reductase (QR) in murine hepatoma cells. Although flavonoid fractions were inactive in the assay, a clear, significant induction of QR activity resulted from testing the hexane extract. The concentration of the extract required to double QR specific activity was 9 μg/150 μl per well, which indicates that one or more components possess substantial QR-inducing capacity, comparable to known QR inducers such as furanone and catechol. β-Sitosterol and the carotenoids lutein and zeaxanthin were identified as components of the active hexane extract, which also exhibited a growth suppressing effect against two human breast cancer cell lines MCF-7 and BT-20.

Characterization of quinone reductase, glutathione and glutathione s-transferase in human myeloid cell lines: Induction by 1,2-dithiole-3-thione and effects on hydroquinone- induced cytotoxicity

In this study, we have characterized quinone reductase (QR), glutathione (GSH), glutathione S-transferase (GST) and their induction by a chemoprotector, 1,2-dithiole-3-thione (D3T), in the human myeloid cell lines ML-1 and HL-60. In addition, we also examined the toxicity of hydroquinone (HQ), a benzene metabolite, to these two cell lines. Both of the cell lines contain a basal level of cellular GSH, which is similar in the two cell lines. Although ML-1 cells contain much higher QR specific activity than HL-60 cells, which are relatively QR deficient, the GST specific activity of ML-1 cells is 1.8 times less than that of HL-60 cells. Immunoblot experiments showed that the GST in these two cell lines is GST π. In addition, HL-60 cells exhibit 4.5 times more myeloperoxidase specific activity than ML-1 cells. Inclusion of D3T in the cultures could induce significant increase in cellular GSH content and QR activity, but not GST activity in either cell line. Treatment with HQ caused both inhibition of cell proliferation and loss of cell viability in these two myeloid cell lines. HQ treatment also resulted in a significant depletion of cellular GSH, which preceded the loss of cell viability. Pretreatment of Both cell lines with buthionine sulfoximine, an inhibitor of GSH biosynthesis, markedly increased HQ-induced toxicity. In contrast, the presence of dicumarol, a QR inhibitor, failed to potentiate HQ-induced toxicity in ML-1 cells. On the other hand, pretreatment of these two myeloid cell lines with D3T significantly protected against HQ-induced inhibition of cell proliferation and cell death. Therefore, the above results suggest that GSH but not QR is an important factor involved in the toxicodynamics of HQ in these myeloid cells.

Elevation of quinone reductase activity by anticarcinogenic antioxidants

NAD(P)H:quinone reductase exhibitis broad specificity in the reduction of endogenous and exogenous quinones and quinone imines, such as those derived from polycyclic aromatic carcinogens, phenolic steroids, vitamin K, and numerous therapeutic drugs. This enzyme is found in several cell compartment and is widely distributed among tissues. In contrast to several other flavoprotein dehydrogenases, quinone reductase catalyzes obligatorily two electron reductions. Extensive studies by Huggins and by others have shown that the quinone reductase in liver and some other tissues of rats is inducible by various polycyclic hydrocarbons and aromatic amines, as well as by certain azo dyes. Huggins perceived that the relative effectiveness of such compounds in inducing quinone reductase correlated with their abilities to protect against toxicity and carcinogenesis. Certain antioxidants are also known to protect against the tumorigenic and toxic effects of carcinogens. Studies on the mechanisms underlying the protective effects of BHA, BHT, ethoxyquin, and disulfiram have revealed that these compounds alter the activity profiles of several enzymes which metabolize carcinogenic and toxic compounds. We have observed that quinone reductase specific activity is increased markedly in mouse liver and several extrahepatic tissues in response to dietary BHA, ethoxyquin, and disulfiram, whereas BHT has been shown by others to enhance this enzymatic activity in rat liver. These findings confirm and extend the correlation between the ability to elevate quinone reductase activity and to confer protection against carcinogenesis and toxicity. The broad specificity of quinone reductase, its apparent inability to catalyze one electron reductions of quinones, its widespread distribution, and its inducibility by a variety of structurally dissimilar protective compounds, suggest that quinone reductase may play a significant local protective role in varous regions of the cell.