Dual action of sulforaphane in the regulation of thioredoxin reductase and thioredoxin in human HepG2 and Caco-2 cells

We have previously demonstrated that sulforaphane is a potent inducer for thioredoxin reductase in HepG2 and MCF-7 cells (Zhang et al. Carcinogenesis 2003, 24, 497-503; Wang et al. J. Agric. Food Chem. 2005, 53, 1417-1421). In this study, we have shown that sulforaphane is not only an inducer for thioredoxin reductase but also an inducer for its substrate, thioredoxin in HepG2, and undifferentiated Caco-2 cells. Sulforaphane acts at two levels in the regulation of thioredoxin reductase/thioredoxin system by the upregulation of the expression of both the enzyme and the substrate. In human hepatoma HepG2 cells, sulforaphane induced thioredoxin reductase mRNA and protein by 4- and 2-fold, respectively, whereas thioredoxin mRNA was induced 2.9-fold and thioredoxin protein was unchanged in whole cell extracts, but an increase in nuclear accumulation (1.8-fold) was observed. Moreover, the induction of thioredoxin reductase was found faster than that of thioredoxin. The effects of PI3K and MAPK kinase inhibitors, LY294002, PD98059, SP600125, and SB202190, have been investigated on the sulforaphane-induced expression of thioredoxin reductase and thioredoxin. PD98059 abrogates the sulforaphane-induced thioredoxin reductase at both mRNA and protein levels in HepG2 cells, although other inhibitors were found less effective. However, both PD98059 and LY294002 significantly decrease thioredoxin mRNA expression in HepG2 cells. None of the inhibitors tested were able to modulate the level of expression of either thioredoxin reductase mRNA or protein in Caco-2 cells suggesting that there are cell-specific responses to sulforaphane. In summary, the dietary isothiocyanate, sulforaphane, is important in the regulation of thioredoxin reductase/thioredoxin redox system in cells.     

Sulforaphane, erucin, and iberin up-regulate thioredoxin reductase 1 expression in human MCF-7 cells

Isothiocyanates (ITCs) found in cruciferous vegetables are potentially important anticarcinogenic phytochemicals for many types of cancers including breast cancer. In this study, we have shown that three isothiocyanates, sulforaphane, erucin, and iberin, are potent inducers of thioredoxin reductase 1 (TrxR1) in human breast cancer MCF-7 cells. Sulforaphane, erucin, and iberin at 1 microM induce TrxR1 mRNA 2-3-fold within 8 h of treatment, and induce mRNA 5-7-fold with 12 microM ITC treatments. Selenium did not affect sulforaphane-induced TrxR1 mRNA levels, but significantly enhanced both TrxR1 protein expression (up to 9-fold in erucin treatment) and corresponding activities. These results suggest that dietary ITCs are important factors in the regulation of redox status through the induction of the selenoprotein thioredoxin reductase.     

Phytochemicals and antioxidant properties in wheat bran

Bran samples of seven wheat varieties from four different countries were examined and compared for their phytochemical compositions and antioxidant activities. Phenolic acid composition, tocopherol content, carotenoid profile, and total phenolic content were examined for the phytochemical composition of wheat bran, whereas the measured antioxidant activities were free radical scavenging properties against 2,2-diphenyl-1-picrylhydrazyl radical, radical cation 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, peroxide radical anion O(2)(.-), and oxygen radical and chelating capacities. The results showed that the tested wheat bran samples differed in their phytochemical compositions and antioxidant properties. Ferulic acid, with a concentration range of 99-231 microg/g, was the predominant phenolic acid in all of the tested bran samples and accounted for about 46-67% of total phenolic acids on a weight basis. The concentrations for alpha-, delta-, and gamma- tocopherols were 1.28-21.29, 0.23-7.0, and 0.92-6.90 microg/g, respectively. In addition, lutein and cryptoxanthin were detected in all of the tested bran samples with levels of 0.50-1.80 and 0.18-0.64 microg/g, respectively. Zeaxanthin was detected in the six bran samples, and the greatest zeaxanthin concentration of 2.19 microg/g was observed in the Australian general purpose wheat bran. Beta-carotene was detected in four of the tested bran samples at a range of 0.09-0.40 microg/g. These data suggest that wheat and wheat bran from different countries may differ in their potentials for serving as dietary sources of natural antioxidants.     

Evaporative light-scattering analysis of sulforaphane in broccoli samples: Quality of broccoli products regarding sulforaphane contents

Broccoli sulforaphane has received attention as a possible anticarcinogen. Sulforaphane analysis is difficult due to the lack of a chromophore for spectrometric detection. Hence, we developed a method for determining sulforaphane by using high-performance liquid chromatography (HPLC) coupled with an evaporative light-scattering detector (ELSD). Sulforaphane was extracted from acid-hydrolyzed broccoli samples, followed by solid-phase extraction and reversed-phase HPLC. Sulforaphane was detected by ELSD and concurrently identified by electrospray ionization time-of-flight mass spectrometry. The recovery of sulforaphane from broccoli samples was above 95%. The detection limit was 0.5 mug. The present method was sensitive enough to determine sulforaphane in mature broccoli, broccoli sprouts, and commercial broccoli products. Sulforaphane concentration in broccoli sprout (1153 mg/100 g dry weight) was about 10 times higher than that of mature broccoli (44-171 mg/100 g dry weight). Therefore, the broccoli sprout is recommended as a source of sulforaphane-rich products. In contrast, we found that sulforaphane could not be detected in most of broccoli products, suggesting present commercial broccoli products having low quality.     

Comparison of isothiocyanate metabolite levels and histone deacetylase activity in human subjects consuming broccoli sprouts or broccoli supplement

Increased consumption of cruciferous vegetables such as broccoli may reduce the risk of various cancers. Myrosinase is required to convert dietary glucosinolates from broccoli into bioactive isothiocyanates. We evaluated isothiocyanate excretion profiles in healthy subjects who consumed broccoli sprouts or broccoli supplement (no myrosinase) with equivalent glucosinolate content. Urinary metabolites of two major isothiocyanates, sulforaphane and erucin, were measured by liquid chromatography coupled with tandem mass spectrometry. Peak excretion of sulforaphane and erucin was higher and occurred sooner in subjects who consumed broccoli sprouts as compared to subjects who consumed the supplement. A subject-dependent shift in the ratio of urinary sulforaphane to erucin metabolites was observed in both groups, indicating conversion of sulforaphane to erucin. Lower histone deacetylase activity was observed in the peripheral blood mononuclear cells only in subjects consuming sprouts. Fresh broccoli sprouts differ from broccoli supplements in regards to excretion of isothiocyanates and bioactivity in human subjects.     

Germination-enhancing and zinc-sparing roles for selenium in broccoli

Large areas of China have soils low in both available selenium (Se) and zinc (Zn). In order to investigate whether Se supplied as either selenate or selenite can increase germination and growth compared with low-Se controls we used broccoli, an important vegetable with anticancer effects, especially when biofortified with Se. Broccoli was grown under both Zn adequacy and Zn deficiency to determine whether interactions between these minerals affect plant growth. Selenite and selenate at a wide range of doses increased the speed and extent of germination. Both inorganic Se forms increased early root and shoot growth at low concentrations, with selenite having a stronger effect than selenate. A sand culture trial showed a similar growth increase due to low-dose Se under Zn deficiency but not under Zn adequacy. Conversely, at high Se levels, the results provided evidence from biomass, water use, photosynthesis and gas exchange that broccoli growth was inhibited at high Se levels, with selenite being more toxic than selenate. In this broccoli trial, the two Se forms were equally effective in increasing leaf Se concentration, whereas in most plants selenite is largely converted to organic Se forms and stored in the roots. This study suggests that Se, supplied either as selenate or selenite, may improve germination and growth in broccoli, especially on Zn-deficient soils. Field trials conducted on soils which are very low in both plant-available Se and Zn are needed.     

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

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

Phytochemicals and dietary fiber components in rye varieties in the HEALTHGRAIN Diversity Screen

Ten rye varieties grown in one location were analyzed for their contents of dietary fiber (arabinoxylan and beta-glucan) and phytochemicals (folate, tocols, phenolic acids, alkylresorcinols, and sterols). The varieties included old and modern varieties from five European countries. Significant differences were observed in the contents of all phytochemicals in whole grains and in the fiber contents in the flour and bran. The old French varieties Haute Loire and Queyras had high contents of most phytochemicals, whereas the Polish varieties Dankowskie-Zlote and Warko were relatively poor in phytochemicals. The varieties with a high content of folate tended to have low alkylresorcinol contents and vice versa. Furthermore, high contents of arabinoxylans were associated with high contents in tocols and sterols. The 10 selected rye samples comprising old populations and old and modern varieties from different ecological regions of Europe demonstrate high natural variation in their composition and show that landraces and old populations are useful genetic resources for plant breeding. The contents of single phytochemicals can likely be affected by breeding, and they may be adjusted by the right selection of genotype.     

Kinetics of the stability of broccoli (Brassica oleracea Cv. Italica) myrosinase and isothiocyanates in broccoli juice during pressure/temperature treatments

The Brassicaceae plant family contains high concentrations of glucosinolates, which can be hydrolyzed by myrosinase yielding products having an anticarcinogenic activity. The pressure and temperature stabilities of endogenous broccoli myrosinase, as well as of the synthetic isothiocyanates sulforaphane and phenylethyl isothiocyanate, were studied in broccoli juice on a kinetic basis. At atmospheric pressure, kinetics of thermal (45-60 degrees C) myrosinase inactivation could be described by a consecutive step model. In contrast, only one phase of myrosinase inactivation was observed at elevated pressure (100-600 MPa) combined with temperatures from 10 up to 60 degrees C, indicating inactivation according to first-order kinetics. An antagonistic effect of pressure (up to 200 MPa) on thermal inactivation (50 degrees C and above) of myrosinase was observed indicating that pressure retarded the thermal inactivation. The kinetic parameters of myrosinase inactivation were described as inactivation rate constants (k values), activation energy (Ea values), and activation volume (Va values). On the basis of the kinetic data, a mathematical model describing the pressure and temperature dependence of myrosinase inactivation rate constants was constructed. The stability of isothiocyanates was studied at atmospheric pressure in the temperature range from 60 to 90 degrees C and at elevated pressures in the combined pressure-temperature range from 600 to 800 MPa and from 30 to 60 degrees C. It was found that isothiocyanates were relatively thermolabile and pressure stable. The kinetics of HP/T isothiocyanate degradation could be adequately described by a first-order kinetic model. The obtained kinetic information can be used for process evaluation and optimization to increase the health effect of Brassicaceae.     

Kinetics of chlorophyll degradation and color loss in heated broccoli juice

Degradation of chlorophyll in broccoli juice occurred at temperatures exceeding 60 degrees C. Chemical analysis revealed that degradation of chlorophyll a and b to pheophytin a and b, respectively, followed first-order kinetics and that chlorophyll a was more heat sensitive than chlorophyll b. Temperature dependencies of chlorophyll a and b degradation rate constants could be described by Arrhenius equations with activation energies (E(a)) of 71.04 +/- 4.89 and 67.11 +/- 6.82 kJ/mol, respectively. Objective greenness measurements, using the -a value as the physical property, together with a fractional conversion kinetic analysis, indicated that green color degradation followed a two-step process. Kinetic parameters for the first degradation step were in accordance with the kinetic parameters for pheophytinization of the total chlorophyll content, as determined by chemical analysis (E(a) approximately 69 kJ/mol). The second degradation step, that is, the subsequent decomposition of pheophytins, was characterized by an activation energy of 105.49 +/- 4.74 kJ/mol.     

Soluble Carbohydrates and Secondary Phytochemicals in Betula as Affected by SO2-Pollution

Seedlings of closely related birch species Betula pendula and B. resinifera, which have moderate similarity in their phytochemical composition, were exposed to a SO₂ concentration of 200 μg m^-3 for 24 days to detect their sensitivity and possible induced changes in the amount of primary and secondary phytochemicals. The species deviated from each other in the production of biomass and phytochemicals. The SO₂ pollution induced no changes in growth nor in the soluble sugar concentrations in the species. However, the chromatographic analyses showed the moderate reduction in the amount of antioxidative and defensive phenolics, i.e. myricitrin, (+)-catechin, 3,4'-dihydroxypropiophenone and proanthocyanidins in both birch species. These results indicate more pronounced sensitivity of secondary metabolism to SO₂ pollution compared with primary metabolism, and the reduced tolerance of birches to other oxidative air pollutants as well as to herbivores in polluted environments.     

Glucosinolates in mixed-packaged mini broccoli and mini cauliflower under modified atmosphere

Contents of total and individual glucosinolates of mini broccoli cv. Milady and mini cauliflower cv. Clarke were assessed to determine the effect of modified atmosphere packaging on postharvest glucosinolate dynamics of mixed mini Brassica vegetables. Therefore, mixed-packaged mini broccoli and mini cauliflower stored in food trays sealed with two different microperforated biaxial-oriented polypropylene films for up to 7 days at 8 degrees C were analyzed. The results indicate that modified atmosphere at 8% O2 + 14% CO2 was a suitable gaseous combination to maintain aliphatic and indole glucosinolates in mini broccoli for 7 days after an initial decrease at 4 days. In contrast, modified atmosphere at 1% O2 + 21% CO2 resulted in the best retention of indole glucosinolates of mini cauliflower for 7 days and also of aliphatic glucosinolates after an initial decrease at 4 days. Thus, to maintain glucosinolates and external appearance and to prevent off-odor, mini broccoli and mini cauliflower should be packed separately in suitable altered gas composition.     

The influence of boron nutrition on nitrogen partitioning in broccoli plants

Abstract

Broccoli (Brassica oleracea var italica cv. Premium Crop) plants were germinated in soil, transferred to vermiculite three weeks later and grown in the greenhouse, supplied continuously with boron concentrations ranging from 0.0 to 12.5 mg L^‐1. At commercial maturity, the partitioning of nitrogen into soluble (nitrate, ammonium, amino acids) and insoluble components of the foliage (young and old leaves) and the florets was investigated. Both boron deficiency and toxicity increased the per cent soluble nitrogen, particularly as nitrate. Boron toxicity, but not deficiency, consistently affected the concentration and relative composition of amino acids. The actual nitrogen partitioning, including the relative amino acid composition, was dependent upon the developmental stage and of the plant organs, and whether boron was present in deficient or toxic levels. Together with information about the composition of transport fluids from a previous study, the data are interpreted as evidence for a reduction in the availability of carbon under conditions of boron stress.     

Effects of microwave cooking conditions on bioactive compounds present in broccoli inflorescences

Cooking as a domestic processing method has a great impact on food nutrients. Most Brassica (Brassicaceae, Cruciferae) vegetables are mainly consumed after being cooked, and cooking considerably affects their health-promoting compounds (specifically, glucosinolates, phenolic compunds, minerals, and vitamin C studied here). The microwave cooking process presents controversial results in the literature due to the different conditions that are employed (time, power, and added water). Therefore, the aim of this work was to study the influence of these conditions during microwave cooking on the human bioactive compounds of broccoli. The results show a general decrease in the levels of all the studied compounds except for mineral nutrients which were stable under all cooking conditions. Vitamin C showed the greatest losses mainly because of degradation and leaching, whereas losses for phenolic compounds and glucosinolates were mainly due to leaching into water. In general, the longest microwave cooking time and the higher volume of cooking water should be avoided to minimize losses of nutrients.     

Health-promoting compounds in broccoli as influenced by refrigerated transport and retail sale period

Total aliphatic and indole glucosinolates, phenolic compounds (flavonoids and hydroxycinnamoyl derivatives), and vitamin C contents were evaluated in freshly harvested broccoli (Brassica oleracea L., var. italica, cv. Marathon) inflorescences. These were film-wrapped and stored for 7 days at 1 degrees C to simulate a maximum period of commercial transport and distribution. After cold storage, inflorescences were kept for 3 days at 15 degrees C to simulate a retail sale period. For wrapping, low-density polyethylene (LDPE) of 11 microm thickness was used. Gas composition was about 17% O(2) and 2% CO(2) during cold storage and about 16% O(2) and 3-4% CO(2) during shelf life within packages. The predominant glucosinolates were 4-methylsulfinylbutyl-glucosinolate (glucoraphanin), 3-indolylmethyl-glucosinolate (glucobrassicin), and 1-methoxy-3-indolylmethyl-glucosinolate (neoglucobrassicin). The predominant hydroxycinnamoyl derivatives were identified as 1,2,2'-trisinapoylgentiobiose, 1,2-diferuloylgentiobiose, 1,2'-disinapoyl-2-feruloylgentiobiose, and 3-O-caffeoyl-quinic (neochlorogenic acid). Results showed major losses at the end of both periods, in comparison with broccoli at harvest. Thus, the respective losses, at the end of cold storage and retail periods, were 71-80% of total glucosinolates, 62-59% of total flavonoids, 51-44% of sinapic acid derivatives, and 73-74% caffeoyl-quinic acid derivatives. Slight differences in all compound concentrations between storage and retail sale periods were detected. Distribution and retail periods had minimal effects on vitamin C. Weight loss was monitored at the end of both periods.     

Bioavailability and kinetics of sulforaphane in humans after consumption of cooked versus raw broccoli

The aim of this study was to determine the bioavailability and kinetics of the supposed anticarcinogen sulforaphane, the hydrolysis product of glucoraphanin, from raw and cooked broccoli. Eight men consumed 200 g of crushed broccoli, raw or cooked, with a warm meal in a randomized, free-living, open cross-over trial. Higher amounts of sulforaphane were found in the blood and urine when broccoli was eaten raw (bioavailability of 37%) versus cooked (3.4%, p ) 0.002). Absorption of sulforaphane was delayed when cooked broccoli was consumed (peak plasma time ) 6 h) versus raw broccoli (1.6 h, p ) 0.001). Excretion half-lives were comparable, 2.6 and 2.4 h on average, for raw and cooked broccoli, respectively (p ) 0.5). This study gives complete kinetic data and shows that consumption of raw broccoli results in faster absorption, higher bioavailability, and higher peak plasma amounts of sulforaphane, compared to cooked broccoli.     

氮营养对青花菜花球酚类物质及抗氧化能力的影响

采用无机基质盆栽,研究不同氮营养水平（5、10、15和20 mmol/L）对两品种青花菜花球生长、总酚、类黄酮含量及抗氧化能力的影响。结果表明,提高氮浓度水平,有利于青花菜株高、花球直径、花球鲜重和干重的增加;两品种花球的总酚含量随着氮浓度的增加呈降低的变化趋势,当供氮浓度高于10 mmol/L,总酚含量显著降低。花球中主要含有槲皮素和山萘黄素两种黄酮醇物质,而且山萘黄素含量是槲皮素的1.48倍;在低氮 （5 mmol/L）处理下,两品种花球的槲皮素含量和绿优2号的山萘黄素含量显著高于其他处理。增加供氮浓度,花球的二苯代苦味酰基（DPPH, 1,1-diphenyl-2-picrylhydrazyl）自由基清除率和铁还原抗氧化能力(FRAP, Ferric reducing /antioxidant power assay) 值呈下降趋势,限制了其抗氧化能力。相关性分析表明,两品种花球的FRAP值与酚类物质的正相关性显著高于DPPH自由基清除率。两品种相比较,中晚熟品种马拉松的花球直径、鲜重和干重显著高于中早熟品种绿优2号,而其总酚含量和抗氧化能力明显低于绿优2号。以上结果说明,通过氮营养水平能够调控青花菜酚类物质合成和抗氧化能力,从而为生产高营养品质的青花菜产品提供了可能的途径。     

Influence of temperature, modified atmosphere packaging, and heat treatment on aroma compounds in broccoli

The aroma compounds in broccoli stored in different modified atmospheres were studied. The packaging materials used were oriented polypropylene (OPP), poly(vinyl chloride) (PVC), and low-density polyethylene (LDPE) containing an ethylene-absorbing sachet. All samples were stored for either 1 week at a constant temperature of 10 degrees C or for 3 days at 4 degrees C, followed by 4 days at 10 degrees C. The atmospheres that developed inside the packaging materials differed significantly. The broccoli samples were analyzed raw and after cooking, with regard to volatile compounds, using gas-phase (headspace) extraction followed by gas chromatography-mass spectrometry (GC-MS) and GC-olfactometry. Dimethyl sulfide (DMS), dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), hexanal, 3-cis-hexen-1-ol, nonanal, ethanol, and a group of thiocyanates were selected for a detailed study because these compounds cause off-odor and can be used as indicators of stress. Significant differences were found in the aroma profiles of the broccoli samples relative to the packaging materials used for storage. Storage in OPP (14% O(2), 10.5% CO(2)) resulted in most of the off-odors, while storage in LDPE (6% O(2), 7% CO(2)) and PVC (17.9% O(2), 4% CO(2)) was found to maintain the concentration of DMS, DMDS, and DMTS during storage. Heat treatment of the broccoli increased the content of aroma compounds as well as the number of compounds containing sulfur.