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Xanthones from the tropical fruit mangosteen (Garcinia mangostana) display anti-inflammatory and anti-oxidative activities. Here, we isolate and identify potential inducers of the aryl hydrocarbon receptor (AhR) and nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathways from mangosteen using a bioassay-guided strategy. Mangosteen fruit pericarp extracts were subjected to sequential solvent extractions, followed by chromatography coupled with NMR spectroscopy and mass spectrometric analyses for identification and isolation of pure compounds. Isolation of active fractions led to seven prenylated xanthones that were identified and subsequently evaluated for bioactivity. In vitro luciferase reporter cellular assays using H1L6.1c3 (AhR induction) and HepG2-ARE (Nrf2 induction) were used to identify AhR and Nrf2 activators. All seven prenylated xanthones displayed AhR inducing activity, whereas only five xanthones activated Nrf2. Garcinone D (GarD) significantly upregulated AhR/Cyp1a1 and Nrf2/HO-1 protein expression and enhanced zonula occludens-1 and occludin protein levels in HT-29 cells. In addition, GarD inhibited oxidative stress-induced intestinal epithelial barrier dysfunction by enhancing tight junction (TJ) proteins and inhibition of reactive oxygen species production. Inhibition of AhR by pretreating cells with an AhR antagonist revealed that the AhR pathway is required for the improved epithelial barrier functions of GarD. These results highlight a dual mechanism by GarD that confers protection against intestinal epithelial barrier dysfunction.

The onion-derived metabolite, S-1-propenylmercaptocysteine (CySSPe), protects against oxidative stress and exhibits anti-inflammatory effects by modulating cellular redox homeostasis. We sought to establish whether CySSPe activates nuclear factor erythroid 2-related factor 2 (Nrf2) and whether activation of Nrf2 by CySSPe involves modification of the Kelch-like ECH-associated protein-1 (Keap1) to manifest these effects. We found that CySSPe stabilized Nrf2 protein and facilitated nuclear translocation to induce expression of antioxidant enzymes, including NQO1, HO-1, and GCL. Moreover, CySSPe attenuated tert-butyl hydroperoxide-induced cytotoxicity and dose-dependently inhibited reactive oxygen species production. Silencing experiments using Nrf2-siRNA confirmed that CySSPe conferred protection against oxidative stress by activating Nrf2. CySSPe enhanced cellular pool of reduced glutathione (GSH) and improved GSH:GSSG ratio. Pretreatment of cells with l-buthionine-S,R-sulfoximine (BSO) confirmed that CySSPe increases de novo synthesis of GSH by upregulating expression of the GSH-synthesizing enzyme GCL. Treatment of cells with CySSPe elevated hydrogen sulfide (H2S) production. Inhibition of H2S-synthesizing enzymes, cystathionine-gamma-lyase (CSE) and cystathionine-beta-synthase (CBS), by pretreating cells with propargylglycine (PAG) and oxyaminoacetic acid (AOAA) revealed that H2S production was partially dependent on a CSE/CBS-catalyzed β-elimination reaction with CySSPe that likely produced 1-propenyl persulfide (RSSH). Depleting cells of their GSH pool by exposure to BSO and diethylmaleate attenuated H2S production, suggesting a GSH-dependent formation of H2S, likely via the reduction of RSSH by GSH. Finally, treatment of cells with CySSPe persulfidated Keap1, which may be the mechanism involved for the stabilization of Nrf2 by CySSPe. Taken together, our results showed that attenuation of oxidative stress by CySSPe is associated with its ability to produce H2S or RSSH, which persulfidates Keap1 and activates Nrf2 signaling. This study provides insights on the potential of CySSPe as an onion-derived dietary agent that modulates redox homeostasis and combats oxidative stress.

The Allium vegetable-derived metabolite, S-alk(en)ylmercaptocysteine (CySSR), has been reported to modulate oxidative stress and inflammatory responses. However, the underlying mechanisms of action and structure-activity relationships are not completely understood. We investigated the mechanistic basis of the protective effects of CySSR on pro-inflammatory responses involving redox/oxidative stress induced by E. coli lipopolysaccharide (LPS) using RAW 264.7 cells. CySSR (R = allyl, “A” or 1-propenyl, “Pe”) pre-treatments conferred concentration-dependent reductions in cytokines (TNF-α, IL-1β and IL-6), NO production and iNOS (inducible nitric synthase) overexpression, and attenuated oxidant production in LPS-stimulated RAW 264.7 cells where viability remained > 90%. These protective effects were manifested through inhibited activation of the nuclear factor-kappa B (NF-κB) signaling pathway via suppression of the IκB kinases (IKK) phosphorylation possibly by transforming growth factor β-activated kinase 1 or a kinase further upstream the canonical NF-κB signaling pathway. The attenuation of LPS-induced inflammation by CySSRs was associated with enhanced levels of cellular cysteine (CySH) and glutathione (GSH) mediated by cellular import/reduction of CySSR and the induction of glutamate cysteine ligase (GCL), one of > 200 nuclear factor erythroid 2-related factor 2 (Nrf2) regulated proteins. The reduction of anti-inflammatory effect of CySSR following pretreatment of cells with L-buthionine-S,R-sulfoximine (BSO) implicates GSH having a major role in reducing inflammation, likely in the context of other Nrf2-regulated antioxidant enzymes that scavenge H2O2 and peroxides using GSH as co-substrate. The anti-inflammatory effect of CySSPe was significantly greater than CySSA for almost all indicators measured, and cell metabolites of CySSRs may have a role in attenuating NF-κB signaling.

Summary Methanolic extracts of bran from black glutinous rice cultivars were screened for potential health benefits using cellular bioassays for quinone reductase (QR) induction in murine hepatoma cells and inhibition of NO production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. Fractionation of the crude extract by semi-preparative HPLC afforded respective fractions enriched in phenolic acids, anthocyanins and proanthocyanidins. The relative potency of QR induction was phenolic acids > proanthocyanidins > anthocyanins, and the activity of the crude extract was conserved among the aggregate of these fractions. In contrast, all three fractions were more potent than the crude extract in terms of anti-inflammatory effect. This suggests synergism effects among anti-inflammatory agents through partial purification. Inhibition of macrophage NO production by binary mixtures of gallic acid or caffeic acid with the anthocyanin-rich fraction indicated potential synergism. These results warrant further efforts to identify the active agents and substantiate synergistic interactions through isobologram analysis.

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.

S-Allylmercaptocysteine (CySSA) from garlic is known to exhibit anti-cancer effects. Apoptosis induction by CySSA was contrasted with S-1-propenylmercaptocysteine (CySSPe) (the major onion analog) in the presence of Na2SeO3 (Se) in breast cancer cells MCF-7. The dose of CySSA or CySSPe alone required to reduce viable cells by 50% was >400μM, and this was reduced to 62μM and 91μM for CySSA+Se and CySSPe+Se, respectively, at molar ratios of 39:1. Synergism of the mixtures was confirmed by isobologram analysis and the treatments evoked enhanced thiol efflux from MCF-7 cells. Apoptosis was confirmed by Annexin-V and propidium iodide staining. Cell cycle arrest occurred at the G2/M and sub-G1 interphases. Both CySSR+Se mixtures reduced the levels of Akt. CySSPe+Se elevated GSK-3 protein levels, whereas CySSA+Se did not. CySSR+Se mixtures enhanced phospho-c-Jun levels, with CySSA+Se more potent than CySSPe+Se. Corresponding increases in phospho-p53, Bax and Bad levels were observed, indicating apoptosis occurred via the mitochondrial pathway. Lack of caspases 6/7 activation implicated a caspase-independent pathway for apoptosis. Reduction of imported CySSR and export of thiols by MCF-7 cells facilitates the reduction of selenite to yield H2Se, a cytotoxic agent. This appears to be the first report of an anti-cancer effect of CySSPe.

6-Dehydroshogaol (6-DHSG) is a bioactive α,β-unsaturated carbonyl compound isolated from fresh ginger with anti-inflammatory and phase II enzyme inducing activities. Here we describe the glutathione (GSH)-dependent metabolism and the effect of this metabolic transformation on the biological activities of 6-DHSG. Compared with other ginger compounds, such as 6-gingerol and 6-shogaol, 6-DHSG showed the most potent anti-inflammatory effect in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. The biological activities of 6-DHSG were attenuated by sulfhydryl antioxidants such as glutathione (GSH) or N-acetyl cysteine (NAC), but not ascorbic acid (ASC). 6-DHSG was metabolised by GSH to form a GSH conjugate (GS-6-DHSG) in RAW 264.7 cells, via a potential mechanism involving the catalytic activity of glutathione-S-transferase (GST). GS-6-DHSG showed reduced biological activities compared with 6-DHSG in multiple biological assays. Together, these results indicate that GSH conjugation attenuates the biological activities of 6-DHSG and other α,β-unsaturated carbonyl compounds.

S-Alk(en)ylmercaptocysteine (CySSR, R = methyl, ethyl, propyl, 1-propenyl, and allyl), which are the putative metabolites of Allium thiosulfinates, were chemically synthesized. CySSR, but not the corresponding monosulfide species S-alk(en)yl cysteine (CySR), were able to induce quinone reductase (QR, a representative phase II enzyme) in Hepa 1c1c7 cells and inhibit nitric oxide (NO, an inflammatory biomarker) formation in lipopolysaccharide (LPS)-activated RAW 264.7 cells. These results indicate the importance of the disulfide bond for the biological activities of CySSR. Glutathione (GSH) and N-acetylcysteine (NAC), but not other types of cellular antioxidants, suppressed multiple biological activities of CySSR in vitro. The inhibitory effects of GSH and NAC on the biological activities of CySSR were correlated with a glutaredoxin (Grx)-dependent intracellular reduction of CySSR to generate cysteine and RSH, which were secreted into the extracellular medium.

Hexahydrocurcumin, 1-dehydro-[6]-gingerdione, 6-dehydroshogaol and 6-shogaol were evaluated for their antioxidant and anti-inflammatory activities in the present study. The relative antioxidant potencies of ginger compounds decreased in similar order of 1-dehydro-[6]-gingerdione, hexahydrocurcumin>6-shogaol>6-dehydroshogaol in both 1,1-diphenyl-2-picyrlhydrazyl (DPPH) radical-scavenging and trolox equivalent antioxidant capacity (TEAC) assays. All tested compounds could attenuate lipopolysaccharide (LPS)-elicited increase of prostaglandin E2 (PGE(2)) in murine macrophages (RAW 264.7) in a concentration-dependent manner but hexahydrocurcumin of 7μM and 6-shogaol of 7μM. The strongest inhibitory effect was observed for 6-dehydroshogaol and 6-shogaol at 14μM with the inhibition of 53.3% and 48.9%, respectively. Furthermore, both 6-dehydroshogaol and 1-dehydro-[6]-gingerdione significantly suppressed the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins in a concentration-dependent fashion. These results contribute to our theoretical understanding of the potential beneficial effects of consuming ginger as a food and/or dietary supplement.

To investigate the potential activity of ginger rhizome extracts to induce quinone reductase (QR), we performed bioactivity-guided fractionation using a murine hepatoma cell (Hepa 1c1c7) bioassay. Anti-inflammatory effects were then studied utilizing lipopolysaccharide (LPS)-stimulated mouse macrophage (RAW 264.7) cells. An ethyl acetate-partitioned fraction from ethanolic extract, rich in both QR inducing potency and anti-inflammatory activity, was subjected to repeated silica gel column and preparative thin layer chromatography to yield three compounds. The three isolated compounds were [6]-shogaol, 1-dehydro-[6]-gingerdione and hexahydrocurcumin. [6]-Dehydroshogaol, a minor component in ginger rhizome, was chemically synthesized and used for comparison in the subsequent bioassay based on its excellent QR inducing potency. Results showed that [6]-dehydroshogaol had the highest ability to induce QR activity (CD = 9.23 ± 0.22 μM), followed by 1-dehydro-[6]-gingerdione (CD = 13.24 ± 0.45 μM), and then hexahydrocurcumin (CD = 68.81 ± 3.90 μM). Increasing QR activity in induced cells was associated with elevated expression of NQO-1 protein as confirmed by Western blot. [6]-Dehydroshogaol, [6]-shogaol and 1-dehydro-[6]-gingerdione were also potent inhibitors of nitric oxide (NO) synthesis in activated macrophages. Their IC 50 values ranged from 5.80 ± 1.27 to 25.06 ± 4.86 μM. Hexahydrocurcumin exhibited the weakest inhibitory effect (IC 50 = 304.76 ± 54.80 μM). These findings contribute to our theoretical understanding of the potential beneficial effects of consuming ginger as food and/or dietary supplement.

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 of100 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.

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.

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. 1 H 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.

The presence of high level of histamine is detrimental to the quality and safety of fish sauce. Therefore, this study aimed to study the ability of extremely halophilic archaea to reduce histamine under high salt condition and to examine the enzyme activity potentially involved. Of 156 extremely halophilic archaea isolated from various salt-fermented fishery products, HDS3-1 from an anchovy fish sauce sample fermented for 3 months, exhibited the highest histamine degradation activity when cultured in halophilic medium containing 5 mM histamine (free-base), followed by HDS1-1, HPC1-2, and HIS40-3, respectively. HDS3-1 was classified as Natrinema gari based on 16S rRNA gene sequence similarities and did not exhibit decarboxylase activity toward all tested amino acids. Based on in vitro cytotoxicity assay, the treatment with whole cell extract of HDS3-1 to all cell lines tested resulted in dose-dependent inhibitions of the cell growth with the IC50 values higher than 250 μg ml−1. Histamine-degrading activity of HDS3-1 was located in the intracellular fraction and required 1-methoxy-5-methylphenazinium methylsulfate (PMS) as an electron carrier. The optimal pH, salt concentration, and temperature for histamine degradation were pH 6.5–8, 3.5–5 M NaCl, and 40–55 °C, respectively. The activity was fully retained at pH 6.5–9, in the presence of NaCl above 2.5 M, and at temperature lower than 50 °C. The results suggested that histamine-degrading activity of HDS3-1 was most likely associated with salt-tolerant and thermo-neutrophilic histamine dehydrogenase.

The chemical syntheses of cysteine (CYS) and glutathione (GSH) mixed -disulfide conjugates (CySSR, GSSR, respectively) of mercapto residues representing most of the R groups of thiosulfinates (R = methyl, ethyl, propyl, and allyl) are described. Gram-scale conjugates were prepared as98% pure preparations, with 80% reaction yield for each of the two seminal synthesis steps, with structures confirmed by (1)H NMR and high-resolution MS analyses. These conjugates are derivatives of thiosulfinates that may be evolved in processed foods, in the digestive tract, and through in vivo metabolism. The prepared conjugates were found to be able to induce quinone reductase (QR, a representative phase II enzyme) in murine hepatoma cells (Hepa 1c1c7) and to inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated macrophage cells (RAW 264.7), indicating they have potential cancer preventive and anti-inflammatory activities. Among the prepared conjugates, the allyl conjugates of CYS and GSH, S-allylmercaptocysteine (CySSA) and S-allylmercaptoglutathione (GSSA), showed the most potent activity regarding QR induction and NO production inhibition. The conjugates with saturated R groups were also active and conferred biological activity as cystine and oxidized glutathione exhibited no effects in these cellular assays.

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.

An esterase gene, designated estB , was isolated from a genomic library of Lactobacillus casei LILA. Nucleotide sequencing of the estB gene revealed a 954-bp open reading frame encoding a putative peptide of 35.7 kDa. The deduced amino acid sequence of EstB contained the characteristic GXSXG active-site serine motif identified in most lipases and esterases. An EstB fusion protein containing a C-terminal 6-histidine tag was constructed and purified to electrophoretic homogeneity by affinity chromatography. The native molecular weight of EstB was 216.5±2.5 kDa, while the subunit molecular weight was 36.7±1.0 kDa. Optimum pH, temperature, and NaCl concentration for EstB were determined to be pH 7.0, 50 to 55°C, and 15% NaCl, respectively. EstB had significant activity under conditions simulating those of ripening cheese (pH 5.1, 10°C, and 4% NaCl). Kinetic constants (K M and V max ) were determined for EstB action on a variety of ethyl esters and ester compounds consisting of substituted phenyl alcohols and short n -chain fatty acids. For comparison purposes, EstA from Lb. helveticus CNRZ32 was purified to electrophoretic homogeneity and its substrate selectivity determined in a similar fashion. Different substrate selectivities were observed for EstB and EstA.

Free-radical scavenging, reducing, and phase II enzyme-inducing activities of aqueous and 5% aqueous ethanol extracts of freeze-dried root tissue of four beet (Beta vulgaris L.) strains (red, white, orange, and high-pigment (red) phenotypes) were determined. Aqueous and ethanolic tissue extracts of the regular and high-pigment red phenotypes were most capable of inhibiting metmyoglobin/H2O2-mediated oxidation of 2-2‘-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and 2,2‘-azobis-(2-amidinopropane) dihydrochloride (AAPH)-mediated bleaching of β-carotene. These same extracts were also most efficient at reducing ABTS radical cation and inducing quinone reductase in murine hepatoma (Hepa 1c1c7) cells in vitro. Keywords: Quinone reductase; phase II enzyme; antioxidant; chemopreventive; Hepa 1c1c7 cells; beetroot; betalains

Quinone reductase (QR)-inducing activity of aqueous and ethanolic extracts of common vegetables was determined using a murine hepatoma (Hepa 1c1c7) cell bioassay. QR induction observed for aqueous extracts reached 13-fold for corn, 8-fold for kale, and 5-fold for snap beans. QR induction of ethanol extracts reached about 3-fold for both garlic and snap beans, and about 2-fold for both red beetroots and kale. These aqueous extracts were effective inhibitors (kale ∼ red beetroots > corn ∼ green beans > garlic) of the generation of 2-2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical cation (ABTS•+) by metmyoglobin and H2O2, whereas the ethanolic extracts were not inhibitory. Aqueous extracts reduced pre-formed ABTS•+ more effectively (> 95% inhibition) than did ethanolic extracts (∼50 to 90% inhibition). All vegetable extracts inhibited oxyradical-mediated β-carotene bleaching, where kale and red beetroot extracts exhibited the strongest protective effect.

Esterification reaction selectivity in homogeneous organic media was evaluated for Candida antarctica B and Pseudomonas cepacia lipases toward members of an homologous series of even carbon number, saturated n -fatty acids (FAs) of 4–16 carbons (C4–C16), glycerol, and glycerol analogues ( n -propanol, isopropanol, 1,2-, and 1,3-propanediols). Dual FA optima were observed in almost all reaction mixtures, with C. antarctica and P. cepacia lipases generally favoring C10/C6 and C8/C16, respectively. Several explanations are offered to account for multiple FA optima. Reaction selectivity toward FAs was more influenced by steric than electronic features of FA substrates. Relative selectivity and discriminatory power among the series of FAs was modulated by the specific alcohol acceptor used. This may be founded on partial sharing of binding pockets by alcohol and FA co-substrates. Evidence was obtained that implicates the sn -2 alcohol functional group as being a determinant of FA selectivity and lipase discriminatory power.

The influence of immediate and delayed pH control, and added S-methyl-L-cysteine sulfoxide (MCSO) on the progress of pink discoloration in relation to the fate of thiosulfinates in extracts of yellow onion (Allium cepa) bulbs was evaluated over an incubation period of about 3 weeks at 21–24°C. Greatest discoloration occurred in extracts prepared with immediate pH control at 6.1, and least discoloration occurred at pH 3.2, over the pH range of 3.2–6.1. Initial thiosulfinate levels, in descending order of abundance, occurred at pH 6.1 > 5.0 > 4.2 > 3.2 for extracts subject to immediate pH control. However, over 21 days incubation, the lowest residual levels (ranging 20–60% of maximum levels) were observed for extracts adjusted to pH 3.2 and 6.1. Extracts subject to delayed (10 min after tissue disruption) pH control (initial extract pH of 5.2–5.4) displayed the greatest extent of discoloration, in descending order, at pH 4.0 > 5.0, 6.0 > 3.0. In these samples, initial thiosulfinate levels were similar and the greatest residual thiosulfinate levels over the 21-day incubation period were observed at pH 3.0 and 4.0 (about 50% of maximum levels), with the lowest residual levels observed for extracts adjusted to pH 6.0 (about 20% of maximum levels). Tissue extracts supplemented with 9- to 18-fold excess MCSO were subject to modest increases in both extent of discoloration and thiosulfinate levels.

A dipeptidase with prolinase activity from Lactobacillus helveticus CNRZ32, which was designated PepR, was purified to gel electrophoretic homogeneity and characterized. The NH2-terminal amino acid sequence of the purified protein had 96% identity to the deduced NH2-terminal amino acid sequence of the pepR gene, which was previously designated pepPN, from L. helveticus CNRZ32. The purified enzyme hydrolyzed Pro-Met, Thr-Leu, and Ser-Phe as well as dipeptides containing neutral, nonpolar amino acid residues at the amino terminus. Purified PepR was determined to have a molecular mass of 125 kDa with subunits of 33 kDa. The isoelectric point of the enzyme was determined to be 4.5. The optimal reaction conditions, as determined with Pro-Leu as substrate, were pH 6.0 to 6.5 and 45 to 50 degrees C. The purified PepR had a Km of 4.9 to 5.2 mM and a Vmax of 260 to 270 mumol of protein per min/mg at pH 6.5 and 37 degrees C. The activity of purified PepR was inhibited by Zn2+ but not by other cations or cysteine, serine, aspartic, or metal-containing protease inhibitors or reducing agents. Results obtained by site-directed mutagenesis indicated that PepR is a serine-dependent protease. Gene replacement was employed to construct a PepR-deficient derivative of CNRZ32. This mutant did not differ from the wild-type strain in its ability to acidify milk. However, the PepR-deficient construct was determined to have reduced dipeptidase activity compared to the wild-type strain with all dipeptide substrates examined.

Procedures were developed for the synthesis, purification and analysis of propanediol (PD) esters of n-fatty acids (FA). Mono- (MAPD) and di-acylated (DAPD) species were synthesized from PD and FA using an immobilized lipase (Candida antarctica B) in tert-butanol. MAPD and DAPD were isolated using silica gel column chromatography as approximately 95% pure preparations. Normal phase gradient LC provided for resolution of MAPD, DAPD and FA. UV220 nm detection provided a detection limit of about 1 microgram, and a linear response range of up to 2000 micrograms. Response factors were determined for MAPD, DAPD and FA components comprised of n-fatty acyl lengths of 4-16.

An endopeptidase gene (pepE) was isolated from a previously constructed genomic library of Lactobacillus helveticus CNRZ32. The pepE gene consisted of a 1,314-bp open reading frame encoding a putative peptide of 52.1 kDa. Significant identity was found between the deduced amino acid sequence of pepE and the sequences for aminopeptidase C from Lactobacillus delbrueckii subsp. lactis DSM7290, L. helveticus CNRZ32, Streptococcus thermophilus CNRZ302, and Lactococcus lactis subsp. cremoris AM2. A recombinant PepE fusion protein containing an N-terminal six-histidine tag was constructed and purified to electrophoretic homogeneity. Characterization of PepE revealed that it was a thiol-dependent protease having a monomeric mass of 50 kDa, with optimum temperature, NaCl concentration, and pH for activity at 32 to 37 degrees C, 0.5%, and 4.5, respectively. PepE had significant activity under conditions which simulate those of ripening cheese (10 degrees C, 4% NaCl, pH 5.1). PepE hydrolyzed internal peptide bonds in Met-enkephalin and bradykinin; however, hydrolysis of alpha-, beta-, and kappa-caseins was not detected.

The effect of selenium compounds on extracellular redox modulating capacity was studied in murine macrophage RAW 264.7 cells and differentiated human THP-1 monocytes. The arylselenium compounds benzeneselenol (PhSeH), dibenzyl diselenide (DBDSe), diphenyl diselenide (DPDSe), and ebselen were capable of inducing extracellular cysteine accumulation via a cystine- and glucose-dependent process. Extracellular cysteine production was dose-dependently inhibited by glutamate, an inhibitor of cystine/glutamate antiporter (Xc(-) transporter), supporting the involvement of Xc(-) transporter for cystine uptake in the above process. These arylselenium compounds also induced cellular thioredoxin reductase (TrxR) expression, particularly at the exofacial surface of cells. TrxR1 knockdown using small interfering RNA attenuated TrxR increases and cysteine efflux induced in cells by DPDSe. Sodium selenite (Na2SeO3), selenomethionine (SeMet), seleno-l-cystine (SeCySS), and Se-methylselenocysteine (MeSeCys) did not have these effects on macrophages under the same treatment conditions. The effects of organoselenium compounds on extracellular redox may contribute to the known, but inadequately understood, biological effects of selenium compounds.

Lipase-mediated esterification and acyl-transfer reactions were evaluated in a series of solvents, ranging in log P (partitioning coefficient of solvent between 1-octanol and water) values from −0.33 to 8.8, and for combinations of substrates of differing polarities. Contrary to the prevailing view, some of the model reaction systems evaluated were most active in solvents having log P values well below the range of 2 to 4 generally believed to be most supportive of biocatalysis in microaqueous organic media. As the polarity of the most polar substrate in the reactive mixtures was progressively increased, there was an attendant decrease in the magnitude and range of solvent log P values yielding maximum activity. When glycerol (log P of −3.0) was used as an acyl-transfer acceptor, log P values of solvents most supportive of activity were −0.33 to 0.80. For acyl-exchange reactions between triacetin (log P of −0.075) and olive oil, solvents having log P of 0.60 to 1.9 were most supportive of activity. For esterification reactions involving benzyl alcohol (log P of 1.1) or dodecanoic acid (log P of 4.8) as the most polar substrate, solvents of log P values > 2.5 and 3.5, respectively, were most supportive of raction. Although there are likely some specific solvent effects, we conclude that the choice of solvent to serve as bulk phase for biocatalytic processes should be made in cognizance of the relative polarity of substrates (and products) of the designed reaction. A secondary feature that should also be considered is the macroscopic phase behavior of reactive mixtures, although this parameter appears to be dependent on the particular source of enzyme.

Eight microbial lipases and one animal tissue lipase were tested for their ability to support glycerolysis reactions with butteroil in the presence and absence of t -butanol to produce monoacylglycerols (MAG) and diacylglycerols (DAG). Two bacterial lipases from Pseudomonas spp. showed the greatest yield of MAG (55–60%) and DAG (24–39%) in the presence or absence of t -butanol. In contrast, lipases from Rhizopus arrhizus, R. javanicus and R. delemar produced yields of MAG and DAG of 23–25% and 36–39%, respectively, but were effective only in the absence of t -butanol. Porcine pancreatic lipase showed limited glycerolysis activity in the presence of t -butanol, yielding 4·6% MAG and 10% DAG, but was not active in the absence of t -butanol. Geotricum candidum, Muco javanicus and Candida cylindracea (C. rugosa) lipases did not support glycerolysis in the presence or absence of t -butanol. It appears possible to selectively use lipases from Pseudonomas and Rhizopus spp. to transform butteroil into blends of MAG and DAG with MAG:DAG ratios of about 0·65 to 2·3.

Aqueous and ethanolic extracts of maize kernels from 18 varieties/strains were prepared for the evaluation of inhibitory activities toward α-glucosidase and scavenging activities toward nitric oxide (NO•) and superoxide (•O2−). All ethanolic extracts of maize strains tested inhibited yeast (Saccharomyces cerevisiae) α-glucosidase with the highest potency (49% to 54%) found for 2 purple and a yellow strains. However, inhibitory effects of maize extracts on rat intestinal α-glucosidase were as a whole about 10% as effective as with the yeast enzyme. Maize extracts were capable of scavenging NO• at the level of 0.25 mg/mL to extents ranging from 24% to 50% and 26% to 57%, respectively, for aqueous and ethanolic extracts. All tested aqueous extracts were also capable of scavenging •O2−, with efficacies ranging from 8% to 38%, at the level of 1.5 mg/mL, whereas almost none of the ethanolic extracts scavenged •O2−, except for one purple strain (approximately 10% effective). The effectiveness in the enzyme inhibition and antioxidant assays did not correlate with total phenolic and/or anthocyanin levels, nor with the nature of pigmentation among the maize strains evaluated. Practical Application: A diversity of pigmented maize strains was evaluated for biological activities related to mitigating oxidative stress and slowing down glucose absorption from the diet. Certain strains tended to be more abundant in these biological activities and have potential to be used in dietary regimes that are designed to promote human health.

White, blue, red and purple corns (Zea mays L.) were lime-cooked to obtain masa for tortillas. The total phenolics and anthocyanins content, antioxidant activity expressed as total reducing power (TRP), peroxyl radical bleaching (PRAC), total antioxidant activity (TAA) and quinone reductase (QR) induction in the murine hepatoma (Hepa 1 c1c7 cell line) as a biological marker for phase II detoxification enzymes were investigated. Among the extracts prepared from raw corn varieties the highest concentration of total phenolics, anthocyanins, antioxidant index and induction of QR-inducing activity were found in the Veracruz 42 (Ver 42) genotype. The nixtamalization process (masa) reduced total phenolics, anthocyanins and antioxidant activities and the ability for QR induction when was compared to raw grain. Processing masa into tortillas also negatively affected total phenolics, anthocyanin concentration, antioxidant activities, and QR induction in the colored corn varieties. The blue variety and its corresponding masa and tortillas did not induce QR. Ver 42 genotype and their products (masa and tortilla) showed the greatest antioxidant activity and capacity to induce QR.

Long-day yellow onions differing in pungency were used to compare methods for appraisal of pungency. Pyruvate, an established indicator of pungency for Allium species, was determined by the lactate dehydrogenase and 2,4-dinitrophenyl hydrazine methods. Thiosulfinate content, another indicator of pungency, was determined spectrophotometrically by monitoring the absorbance at 515 nm after reaction with N-ethylmaleimide. Significant correlation (R2 = 0.871) existed between thiosulfinate content and pyruvate concentration. In addition, a modified N-ethylmaleimide reaction was used to develop a simple pungency indicator test which adequately detected differences between bulbs. The color produced by this method was analyzed using a HunterLab reflectance colorimeter. The Hunter a value and saturation index (C) correlated with both thiosulfinate content and pyruvate concentration (R2 = 0.735–0.860). The procedure described provides the potential for screening of pungency of large numbers of Allium samples.

Oxidative stress was evoked in the peel tissue of cucumber fruits during chilling at 4°C in the dark. Within 2 days of chilling, tissue reduced glutathione (GSH) and thylakoid α-tocopherol (αToc) levels decreased by 28% and 50%, respectively. Tissue ascorbic acid (AA) and thylakoid β-carotene (CAR) levels decreased by 35% and 60%, respectively, between 5 and 7 days of chilling. Rewarming generally had no effect on, or led to further declines in, the levels of these antioxidants. Lipophilic fluorescent products of lipid peroxidation began to accumulate in polar lipid fractions isolated from peel tissue thylakoids after 5–7 days of chilling or chilling/rewarming. This was accompanied by a 2-fold increase in the phospholipid:glycolipid ratio and a slight decrease in the degree of unsaturation of the glycolipids. Measurements of electrolyte leakage of peel tissue indicated that irreversible injury in cucumber fruits required at least 7 days of continuous chilling. None of these changes occurred for fruit held at 13°C in the dark. These results indicate that oxidative stress is an early response of cucumber fruits to chilling and that lipid peroxidation may contribute to the development of chilling injury.

Biological membranes are rarely considered by food scientists when the deteriorative reactions that take place during the processing or storage of food tissues are studied. Yet, membranes and their deterioration play a major but underestimated role in food losses, and recent biochemical information indicates that at least some of these reactions can be controlled by procedures suited to food materials. Much of the present information available on membrane degradation in food systems is incomplete and speculative. It is known, however, that in order to accomplish their many indispensable functions in cells, membranes are constituted mainly of phospholipids, protein, and some carbohydrates arranged in thin, bimolecular sheet-like structures that serve to compartmentalize cells and their organelles. Membranes have embedded in their asymmetric surfaces complements of catalytic and cytoskeletal proteins that serve permeability and structural functions. Membrane surfaces exhibit fluidity, due partially to the continuous lateral diffusion of lipids and some proteins. Two important consequences of fluidity are the ability of membrane phospholipids to exist in different interconvertible conformational phase structures and the formation of heterogenous lipid domains on the membrane surface. Cellular death leads unavoidably to the initiation of membrane deterioration. While the time course of this series of reactions differs in animal and plant tissue, they are damaged by generally similar mechanisms. These include an initial peroxidative attack on polyunsaturated fatty acids, with the concomitant production of free radicals. Many biological agents can act as accelerating agents in these reactions, including transition metal ions, heme compounds, radiation, illuminated chlorophyll, calcium, and ethylene. Once formed, free radicals catalyze further reactions that can affect all aspects of membrane function and cellular metabolism, and lead ultimately to significant losses in food quality through defects such as chilling injury and cold shortening. These are aggravated by many food-processing steps, especially those that involve tissue disruption. Control of membrane breakdown by exogenous chemical intervention has been practiced, but, at best, this only slows the rate of these reactions. Newer approaches to this problem include dietary treatment of meat animals, modified storage and packaging conditions, and genetic interventions. This review advances the proposition that membrane deterioration can be considered a "universal mechanism" that leads to significant quality losses in food. Perhaps because the study of biological membranes and the biochemical and physiological properties has only begun recently, not much progress has been made in finding practical control mechanisms for these reactions in food systems.(ABSTRACT TRUNCATED AT 400 WORDS)

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

Mise en evidence, grâce a des chelatants, des formes actives de l'oxygene liberees du cours de la photo-inactivation. Mise en evidence du mecanisme structurelle d'inactivation de l'enzyme

Phase II xenobiotic metabolizing enzymes confer amelioration of risk arising from potentially carcinogenic chemicals derived both endogenously, and exogenously, from food and the environment. In this study, efforts were made to isolate and identify potentially cancer preventive constituents from methanolic extracts of green onion (Allium cepa) directed by the quinone reductase (QR) induction bioassay using murine hepatoma (Hepa 1c1c7) cells. Crude methanolic extracts of green onion tissue were solvent-partitioned, and subsequently fractionated by flash chromatography, thin layer chromatography and high pressure preparative liquid chromatography to afford pure QR-inducing isolates. Multiple isolates were found active at inducing QR. One newly identified compound, 5-hydroxy-3-methyl-4-propylsulfanyl-5H-furan-2-one (3), and four known compounds: 5-(hydroxymethyl) furfural (1), acetovanillone (2), methyl 4-hydroxyl cinnamate (4) and ferulic acid methyl ester (5), were isolated and identified as active agents.

An esterase gene (estC) was isolated from a genomic library of Lactobacillus casei LILA. The estC gene consisted of a 777 bp open reading frame encoding a putative peptide of 28.9 kDa. A recombinant EstC fusion protein containing a C-terminal six-histidine tag was constructed and purified to electrophoretic homogeneity. Characterization of EstC revealed that it was a serine-dependent dimeric enzyme. Optimum temperature, NaCl concentration, and pH for EstC were determined to be 30 degrees C, 0% NaCl, and pH 5.5, respectively. EstC had significant activity under conditions simulating those of ripening cheese (10 degrees C, 4% NaCl, and pH 5.1). Kinetic constants (KM and Vmax) were determined for EstC action on a variety of ethyl esters and ester compounds consisting of substituted phenyl alcohols and short n-chain fatty acids. For comparison purposes, the previously studied EstA from Lactococcus lactis MG1363 was purified to electrophoretic homogeneity and its substrate selectivity determined in a similar fashion. Different substrate selectivities were observed for EstC and EstA.

Thiosulfinates (TSs) have been implicated as a principle source of the antiplatelet property of raw onion and garlic juice. The in vitro responses of human platelets to dosages of four TSs were measured using whole blood aggregometry and compared by regression analysis. Of the compounds evaluated, methyl methane-TS (MMTS), propyl propane-TS (PPTS), and 2-propenyl 2-propene-TS (allicin) are present in freshly cut Allium vegetables, whereas ethyl ethane-TS (EETS) has not been detected. All TSs were synthesized using a model reaction system. PPTS and allicin had the strongest antiplatelet activity at 0.4 mM, inhibiting aggregation by 90 and 89%, respectively. At the same concentration, EETS and MMTS were significantly weaker, inhibiting 74 and 26%, respectively. Combinations of TSs were not additive in their inhibition of aggregation, indicating that the antiplatelet potential of Allium extracts cannot be easily predicted by quantifying organosulfur components. EETS, PPTS, and allicin were significantly more potent platelet inhibitors than aspirin at nearly equivalent concentrations.

Chilling at 4 degrees C in the dark induced lipid degradation in cucumber (Cucumis sativa L.) fruit upon rewarming at 14 degrees C. Rates of ethane evolution by fruits rewarmed after 3 days of chilling were up to four-fold higher than those evolved by unchilled (14 degrees C) fruits (0.02-0.05 picomoles gram fresh weight(-1) hour(-1)). This potentiation of lipid peroxidation occurred prior to irreversible injury (requiring 3 to 7 days of chilling) as indicated by increases in ethylene evolution and visual observations. Decreases in unsaturation of peel tissue glycolipids were observed in fruits rewarmed after 3 days of chilling, indicating the plastids to be the site of the early phases of chilling-induced peroxidation. Losses in unsaturation of tissue phospholipids were first observed only after chilling for 7 days. Phospholipase D activity appeared to be potentiated in fruits rewarmed after 7 days of chilling as indicated by a decrease in phosphatidylcholine (and secondarily phosphatidylethanolamine) with a corresponding increase in phosphatidic acid. These results indicate that lipid peroxidation may have a role in conferring chilling injury.

A crude microsomal fraction isolated from red hake (Urophycis chuss) muscle demethylated trimethylamine-N-oxide (TMAO). Two cofactor systems were capable of stimulating activity; the system of NADH and FMN required anaerobic conditions while the other system, composed of iron and cysteine and/or ascorbate functioned in the presence or absence of oxygen. The components of each cofactor system functioned synergistically and kinetic parameters were established for each. Of several amine compounds common to fish muscle, TMAO was the only substrate demethylated by the microsomes. Activity was inhibited by iodoacetamide, potassium cyanide, and sodium azide under certain conditions, but not by carbon monoxide. An enzymic nature of the reaction was demonstrated by the properties of heat lability, sensitivity to protease treatment, the requirement of microsomes for TMAO demethylation and by the exhibition of typical hyperbolic kinetics with respect to substrate (TMAO). Moreover, TMAO demethylation by the microsomes was 3 to 4 orders of magnitude faster than the non-enzymic reaction and the reaction was specific for dimethylamine (DMA) as product. It appears the two cofactor systems may share a common catalytic unit in the process of TMAO demethylation.

Chilling injury (CI) is a physiological defect of plants and their products that results in reduced quality and loss of product utilization following exposure to low but nonfreezing temperatures. To design more effective control strategies and maximize shelf-life, it is necessary to develop an understanding of the biochemical mechanism(s) responsible for the initiation of CI. Despite considerable efforts in this field of study, there is no general agreement on the cause or nature of CI, or even the primary event(s) triggering low temperature damage. The first unified theory to explain CI was founded on low temperature induced membrane lipid phase transitions leading to a loss of membrane integrity and physiological dysfunction. This was modified to account for the observation that the level of certain high melting phospholipids appears to be related to the chill sensitivity of many plant tissues. Membranes and changes in their physical characteristics are further implicated as having a role in CI by the discovery that chilling stress evokes an elaborate membrane retailoring response that leads to increased fluidity at reduced temperatures. Others have postulated that CI results from the direct effect of reduced temperatures on enzymes or the indirect effect of membrane perturbations on intrinsic enzymes. The redistribution of cellular calcium has most recently been advanced as the primary transducer of CI. The weight of this theory rests on the role of calcium as a secondary messenger for many cellular functions. In this review it is also speculated that lipid peroxidation may have a role in the development of irreversible injury during low temperature stress. Its effect would be similar to the senescent processes of free radical damage to tissue and progressive membrane rigidification.

Detergent treatments were examined for their efficacy in purifying trimethylamine-N-oxide (TMAO) demethylase activity from fish muscle microsomes. Tritons X-100 and X-45, deoxycholate, Brijs, Tweens 20, 65, and 80, and SDS were generally ineffective in solubilizing demethylase activity from this membrane fraction, at concentrations up to 10 mg detergent per mg protein. In all of these cases, specific activity became enriched in the particulate fraction obtained post-treatment. Highest fold-purification was achieved by using 10 mg SDS per mg protein in 5 mM histidine, pH 7.0 at 10-14 degrees C. Activity was relatively stable to the presence of SDS at this level, and with this treatment, TMAO demethylase activity became purified in the resultant particulate fraction 28- and 58-fold for activity stimulated by ascorbate-iron-cysteine and FMN-NADH, respectively. The presence of urea or 2-mercaptoethanol, or sonication of the SDS-microsome suspension during purification resulted in significant losses of recovered activity. This partially purified fraction represented about 1% of the original microsomal protein and SDS-PAGE revealed the presence of several protein components. The partially purified demethylase could utilize the same two cofactor systems as the native microsomes. It displayed a curvilinear dependence on iron for activity and a sigmoidal response for cysteine. Utilization of NADH, FMN, and ascorbate differed for the purified fraction as compared to the microsomes. Substrate inhibition by TMAO was observed for the partially purified preparation, whereas saturation kinetics were previously noted for microsomal activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Mincing stimulated the breakdown of trimethylamine oxide (TMAO) to dimethylamine (DMA) and formaldehyde (HCHO) in red hake muscle at both above and below freezing temperatures. Aging of red hake muscle on ice as fillets (skin on or off) prior to frozen storage resulted in a greater extent of DMA production as compared to fillets derived from fish aged in the whole or gutted form. Frozen storage temperature influenced the extent of DMA production, yet the freezing nadir had no effect on the rate of its formation during storage at -5°C. Additive studies indicated that reducing conditions in the tissue exert considerable influence on the reaction rate in frozen minced tissue. Trimethylamine (TMA), DMA, dimethylaniline and citrate were found to inhibit the reaction in frozen minced tissue.

The development of chilling injury in cucumbers stored at 4°C and 95% RH was evaluated. Irreversible damage due to chilling required 7 to 10 days as indicated by increases in tissue electrolyte leakage (from 30 to 70%), stress ethylene production (to 30-60 pMol g_1 hr−1) and visual manifestations of injury. Low levels of ethane evolution (