Prenylated xanthones from mangosteen (Garcinia mangostana) activate the AhR and Nrf2 pathways and protect intestinal barrier integrity in HT-29 cells.

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.

S-1-propenylmercaptocysteine protects murine hepatocytes against oxidative stress via persulfidation of Keap1 and activation of Nrf2.

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 ß-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.

Data on chromatographic isolation of cysteine mixed-disulfide conjugates of Allium thiosulfinates and their role in cellular thiol redox modulation.

This data article contains experimental data on the preparation and semi-preparative isolation of S-Alk(en)ylmercaptocysteine (CySSRs, R = allyl, "A", 1-propenyl, "Pe" or methyl, "Me") generated through conjugation reactions between allyl and 1-propenyl enriched thiosulfinates (TS) and cysteine. The data presented are related to the research article "S-Alk(en)ylmercaptocysteine suppresses LPS-induced pro-inflammatory responses in murine macrophages through inhibition of NF-κB pathway and modulation of thiol redox status" (Tocmo and Parkin, in press). In this data article, we included a detailed procedure for CySSR preparation, their purification through semi-preparative chromatography and their toxicity profiles in RAW 264.7 macrophages. Data included also highlight, the ability of CySSRs to modulate intracellular thiol redox status.

S-Alk(en)ylmercaptocysteine suppresses LPS-induced pro-inflammatory responses in murine macrophages through inhibition of NF-κB pathway and modulation of thiol redox status.

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-1ß 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 ß-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.

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

BACKGROUND: 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. RESULTS: 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. CONCLUSION: These findings add to our understanding of the nutraceutical potential of buckwheat as a chemoprophylactic dietary component. © 2018 Society of Chemical Industry.

Identification of bioactive metabolites dihydrocanadensolide, Kojic acid, and vanillic acid in soy sauce using GC-MS, NMR spectroscopy, and single-crystal X-ray diffraction.

Microbial transformations of intrinsic substrates offer immense potential for generating new bioactive compounds in fermented food products. The aim of this work was to characterize the secondary metabolites in soy sauce, one of the oldest fermented condiments. Ethyl acetate extract (EAE) of soy sauce was separated using flash column chromatography, crystallized, and analyzed by nuclear magnetic resonance (NMR), single-crystal X-ray diffraction (SC-XRD), and mass spectroscopy. Dihydrocanadensolide (DHC), an antiulcer agent, was identified in a food for the first time. The natural stereostructure of DHC, which remained controversial for several decades, was determined as (3S,3aS,6R,6aR)-6-butyl-3-methyltetrahydrofuro[3,4-b]furan-2,4-dione using SC-XRD analysis. Kojic acid (KA) and vanillic acid (VA) were also identified from EAE as bioactive metabolic products of fungi and yeasts. Moreover, a new polymorphic form of KA was determined by SC-XRD.

Apoptosis in MCF-7 breast cancer cells induced by S-alkenylmercaptocysteine (CySSR) species derived from Allium tissues in combination with sodium selenite.

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.

Glutathione conjugation attenuates biological activities of 6-dehydroshogaol from ginger.

6-Dehydroshogaol (6-DHSG) is a bioactive α,ß-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,ß-unsaturated carbonyl compounds.

A tissue homogenate method to prepare gram-scale Allium thiosulfinates and their disulfide conjugates with cysteine and glutathione.

The health benefits of Allium vegetables are widely attributed to the enzyme-derived organosulfur compounds called thiosulfinates (TS). However, the lack of a suitable method to prepare TS in good yields has hampered the evaluation of their biological activities. This paper describe a simple enzymatic method using Allium tissue homogenates as a reaction system to prepare gram-scale TS, including those enriched in 1-propenyl groups, which are particularly difficult to obtain. This method is simple, easy to scale up, and requires no column purification step, making it suitable for practical large-scale production of Allium TS. The prepared TS were further utilized to prepare the disulfide conjugates with cysteine and glutathione (CySSR and GSSR, R = methyl, ethyl, propyl, 1-propenyl, and allyl), which are the presumptive metabolites of TS. Among all of the Allium CySSR and GSSR conjugates, the newly prepared glutathione conjugate with 1-propenyl TS, GSSPe, showed the most potent effect to induce quinone reductase (QR, a representative phase II enzyme) in murine hepatoma cells (Hepa 1c1c7) and inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated macrophage cells (RAW 264.7).

S-alk(en)ylmercaptocysteine: chemical synthesis, biological activities, and redox-related mechanism.

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.

Organoselenium compounds modulate extracellular redox by induction of extracellular cysteine and cell surface thioredoxin reductase.

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.

In vitro antioxidant and anti-inflammatory activities of 1-dehydro-[6]-gingerdione, 6-shogaol, 6-dehydroshogaol and hexahydrocurcumin.

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.

ß-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 ß-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 ß-carboline-derived alkaloids to induce phase II enzymes.

Phase II-inducing, polyphenols content and antioxidant capacity of corn (Zea mays L.) from phenotypes of white, blue, red and purple colors processed into masa and tortillas.

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.

Cysteine and glutathione mixed-disulfide conjugates of thiosulfinates: chemical synthesis and biological activities.

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 as >98% 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.

Bioactivities of kernel extracts of 18 strains of maize (Zea mays).

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 (•O(2)(-)). 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 •O(2)(-), with efficacies ranging from 8% to 38%, at the level of 1.5 mg/mL, whereas almost none of the ethanolic extracts scavenged •O(2)(-), 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.

Natrinema gari sp. nov., a halophilic archaeon isolated from fish sauce in Thailand.

Two Gram-negative, rod-shaped, halophilic archaea, designated strains HIS40-3(T) and HDS3-1, were isolated from anchovy fish sauce (nam-pla) collected from two different locations in Thailand. The two strains were able to grow at 20-60 degrees C (optimum 37-40 degrees C), at 1.7-5.1 M NaCl (optimum 2.6-3.4 M NaCl) and at pH 5.5-8.5 (optimum pH 6.0-6.5). Hypotonic treatment with less than 1.7 M NaCl caused cell lysis. The major polar lipids of the isolates were C(20)C(20) and C(20)C(25) derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, two glycolipids and one unidentified lipid. The DNA G+C contents were 64.0-65.4 mol%. In addition to phenotypic and chemotaxonomic characteristics, phylogenetic analysis based on 16S rRNA gene sequence similarities showed that strains HIS40-3(T) and HDS3-1 were related most closely to species of the genus Natrinema. Levels of 16S rRNA gene sequence similarity between strains HIS40-3(T) and HDS3-1 and the type strains of recognized Natrinema species were 99.1-96.6 %. The two novel strains could be distinguished from recognized Natrinema species on the basis of low levels of DNA-DNA relatedness and differences in whole-cell protein patterns and phenotypic properties. Levels of 16S rRNA gene sequence similarity and DNA-DNA relatedness between the two strains were 99.7 and 77.7 %, respectively, suggesting that they should be classified as representing a single species. Based on these taxonomic data, strains HIS40-3(T) and HDS3-1 are considered to represent a novel species of the genus Natrinema, for which the name Natrinema gari sp. nov. is proposed. The type strain is HIS40-3(T) (=BCC 24370(T) =JCM 14663(T) =PCU 303(T)).

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.

Isolation and identification of potential cancer chemopreventive agents from methanolic extracts of green onion (Allium cepa).

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.

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).