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Abstract

Broccoli sprouts were directly frozen at -20 °C (DF-20), -40 °C (DF-40) and -80 °C (DF-80) or stored at −20 °C (LN-20), -40 °C (LN-40) and -80 °C (LN-80) after frozen in liquid nitrogen for 5 min or always frozen in liquid nitrogen (LN). The effect of these treatments on glucoraphanin and ascorbic acid content, myrosinase activity, sulforaphane and sulforaphane nitrile formation in broccoli sprouts was investigated. Results showed that glucoraphanin content was not significantly affected by freezing. Treatments of DF-20, DF-40, LN-20 and LN-40 for 24 and 48 h enhanced myrosinase activity and decreased ascorbic acid content. The enhancement of myrosinase activity after freezing was in parallel with more sulforaphane and less sulforaphane nitrile formation. Besides, freezing at -20 °C was more favorable for sulforaphane formation than -40 °C. Directly freezing at -20 °C increased sulforaphane yield by 1.54~2.11 folds in broccoli sprouts of three cultivars investigated in this study.

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Article
Glucosinolates are naturally abundant in many vegetable sources. These compounds have limited health benefit in their original forms, however their derived product, sulforaphane, has been shown to be hugely health beneficial in protecting against certain types of cancer. This work investigated the conversion of glucosinolates (glucoraphanin) to sulforaphane using either an endogenous myrosinase or an exogenous myrosinase under various enzymolysis conditions. It was found that an optimum degradation of glucosinolates to sulforaphane by the endogenous method was achieved under the following conditions: a liquid–solid ratio of 3 ml/g, an enzymolysis time of 8 h, at 25 °C, at pH 4.0, and with the addition of ascorbic acid 0.02 mg/g. This gave 35% conversion rate of glucosinolates to sulforaphane. However, the exogenous approach appeared to be much more efficient in converting glucoraphanin to sulforaphane. At a combined condition of a liquid–solid ratio of 1000 ml/g, 3 h enzymolysis, at 35 °C and pH 5.0, and in the presence of 0.02 mg/g ascorbic acid, as much as 68% of glucoraphanin was found to be degraded to form sulforaphane.
Article
Changes in physiological and biochemical metabolism as well as glucoraphanin and sulforaphane contents of germinating broccoli seeds and sprouts were investigated in this study. Sprout length, root length, and fresh weight increased with germination time. Dry weight varied from 2.5 to 3.0 mg per sprout. A rapid increase in respiratory rate of sprouts occurred between 24 and 36 h of germination and then stayed at a high level. HPLC analysis found that glucoraphanin content increased at the early stage (0-12 h) of germination, decreased to a low value of 3.02 mg/g at 48 h, and then reached the highest value of 6.30 mg/g at 72 h of germination. Sulforaphane content decreased dramatically during the first day of germination, then increased slowly, and reached a high value of 3.38 mg/g at 48 h before declining again.
Article
Tilapia fish (Oreochromis niloticus) were fed with enriched diets containing broccoli and its phytochemical sulforaphane over 30 d. The levels of cytochrome P450, superoxide dismutase, catalase, lipid peroxidation and glutathione-S-transferase activities were measured. Basal value of cytochrome P450 activity was significantly increased as consequence of the broccoli and sulforaphane enriched diets, while no statistically significant changes were found on catalase and lipid peroxidation activities. After benzo(a)pyrene exposure, the cytochrome P450 activity increased to higher levels in the fish feed with broccoli and sulforaphane when compared with the control fish. Activities of antioxidant enzymes also varied but without significant difference with the control fish. Supported by the lower concentrations of BaP metabolites in bile from fish fed with broccoli or with sulforaphane enriched diets (indicating a better xenobiotic elimination) the cytochrome P450 induction could be considered beneficial for the detoxification because this transformation is the first step for PAH elimination by the phase II system. The protection of aquaculture organism against pollution effects by designing special diets able to modulate the enzymes involved in the phase-I and phase-II detoxification mechanism are discussed.
Article
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Article
Consumption of vegetables, especially crucifers, reduces the risk of developing cancer. Although the mechanisms of this protection are unclear, feeding of vegetables induces enzymes of xenobiotic metabolism and thereby accelerates the metabolic disposal of xenobiotics. Induction of phase II detoxication enzymes, such as quinone reductase [NAD(P)H:(quinone-acceptor) oxidoreductase, EC 1.6.99.2] and glutathione S-transferases (EC 2.5.1.18) in rodent tissues affords protection against carcinogens and other toxic electrophiles. To determine whether enzyme induction is responsible for the protective properties of vegetables in humans requires isolation of enzyme inducers from these sources. By monitoring quinone reductase induction in cultured murine hepatoma cells as the biological assay, we have isolated and identified (-)-1-isothiocyanato-(4R)-(methylsulfinyl)butane [CH3-SO-(CH2)4-NCS, sulforaphane] as a major and very potent phase II enzyme inducer in SAGA broccoli (Brassica oleracea italica). Sulforaphane is a monofunctional inducer, like other anticarcinogenic isothiocyanates, and induces phase II enzymes selectively without the induction of aryl hydrocarbon receptor-dependent cytochromes P-450 (phase I enzymes). To elucidate the structural features responsible for the high inducer potency of sulforaphane, we synthesized racemic sulforaphane and analogues differing in the oxidation state of sulfur and the number of methylene groups: CH3-SOm-(CH2)n-NCS, where m = 0, 1, or 2 and n = 3, 4, or 5, and measured their inducer potencies in murine hepatoma cells. Sulforaphane is the most potent inducer, and the presence of oxygen on sulfur enhances potency. Sulforaphane and its sulfide and sulfone analogues induced both quinone reductase and glutathione transferase activities in several mouse tissues. The induction of detoxication enzymes by sulforaphane may be a significant component of the anticarcinogenic action of broccoli.
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The potential of some intrinsic (MgCl2, ascorbic acid, pH) and extrinsic (temperature, pressure) factors for controlling/altering activity of myrosinase from broccoli was investigated in this paper. A combination of MgCl2 and ascorbic acid was found to enhance enzyme activity. Concentrations resulting in optimal activity were determined as 0.1 g/liter and 2 g/liter, respectively. Both in the absence and presence of this enzyme activator, the optimal pH was situated between 6.5 and 7, corresponding to the natural pH of fresh broccoli juice. At atmospheric pressure, the enzyme was optimally active at a temperature about 30 degrees C. Application of low pressure (50 to 100 MPa) slightly enhanced the activity while at higher pressure (300 MPa), the activity was largely reduced. Future work should focus on the extension of this work to real food products in order to take cellular disruption into account. In intact vegetable tissues, the enzyme myrosinase is present in compartments separated from its substrate, the glucosinolates. Hence, enzymatic hydrolysis can merely occur after cellular disruption. In this respect, processes such as cutting, cooking, freezing, or pressurizing of the vegetables will have a large effect on the glucosinolate hydrolysis by myrosinase. This work could then be the basis for controlling glucosinolate hydrolysis in food preparation and processing.
Article
Epidemiological and laboratory studies suggest that dietary broccoli may prevent or delay a variety of cancers. Broccoli and other crucifers contain a relatively unique family of secondary metabolites called glucosinolates. Glucoraphanin, the major glucosinolate in broccoli, is hydrolyzed by an endogenous plant myrosinase to form either the potent anticarcinogen sulforaphane (SF) or sulforaphane nitrile (SF nitrile). The bioactivities of SF and SF nitrile were compared in rats and in mouse hepatoma cells. Male, 4-week-old, Fischer 344 rats were administered SF or SF nitrile (200, 500, or 1000 micromol/kg) by gavage daily for 5 days. Hepatic, colonic mucosal, and pancreatic quinone reductase and glutathione S-transferase activities were induced by high doses of SF, but not by SF nitrile. When Hepa 1c1c7 cells were exposed to increasing levels of each compound for 24 h, quinone reductase showed a 3-fold maximal induction over control at 2.5 microM SF and a 3.5-fold maximal induction over control at 2000 microM SF nitrile, the highest concentration tested. These results demonstrate that SF nitrile is substantially less potent than SF as an inducing agent of phase II detoxification enzymes. Therefore, glucoraphanin hydrolysis directed toward the production of SF rather than SF nitrile could increase the potential chemoprotective effects of broccoli.
Article
Gastric infection with Helicobacter pylori is a cosmopolitan problem, and is especially common in developing regions where there is also a high prevalence of gastric cancer. These infections are known to cause gastritis and peptic ulcers, and dramatically enhance the risk of gastric cancer. Eradication of this organism is an important medical goal that is complicated by the development of resistance to conventional antimicrobial agents and by the persistence of a low level reservoir of H. pylori within gastric epithelial cells. Moreover, economic and practical problems preclude widespread and intensive use of antibiotics in most developing regions. We have found that sulforaphane [(-)-1-isothiocyanato-(4R)-(methylsulfinyl)butane], an isothiocyanate abundant as its glucosinolate precursor in certain varieties of broccoli and broccoli sprouts, is a potent bacteriostatic agent against 3 reference strains and 45 clinical isolates of H. pylori [minimal inhibitory concentration (MIC) for 90% of the strains is <or=4 microg/ml], irrespective of their resistance to conventional antibiotics. Further, brief exposure to sulforaphane was bactericidal, and eliminated intracellular H. pylori from a human epithelial cell line (HEp-2). In complementary experiments, sulforaphane blocked benzo[a]pyrene-evoked forestomach tumors in ICR mice. This protection resulted from induction of phase 2 detoxication and antioxidant enzymes, and was abrogated in mice lacking the nrf2 gene, which regulates phase 2 enzymes. Thus, the dual actions of sulforaphane in inhibiting Helicobacter infections and blocking gastric tumor formation offer hope that these mechanisms might function synergistically to provide diet-based protection against gastric cancer in humans.
Article
Broccoli florets contain low levels of 3-methylsuphinylpropyl and 4-methylsulphinylbutyl glucosinolates. Following tissue disruption, these glucosinolates are hydrolysed to the corresponding isothiocyanates (ITCs), which have been associated with anticarcinogenic activity through a number of physiological mechanisms including the induction of phase II detoxification enzymes and apoptosis. In this paper, we describe the development of ITC-enriched broccoli through the introgression of three small segments of the genome of Brassica villosa, a wild relative of broccoli, each containing a quantitative trait locus (QTL), into a broccoli genetic background, via marker-assisted selection and analysis of glucosinolates in the florets of backcross populations. Epistatic and heterotic effects of these QTLs are described. The ITC-enriched broccoli had 80-times the ability to induce quinone reductase (a standard assay of phase II induction potential) when compared to standard commercial broccoli, due both to an increase in the precursor glucosinolates and a greater conversion of these into ITCs.
Article
Sulforaphane, an isothiocyanate from broccoli, is one of the most potent food-derived anticarcinogens. This compound is not present in the intact vegetable, rather it is formed from its glucosinolate precursor, glucoraphanin, by the action of myrosinase, a thioglucosidase enzyme, when broccoli tissue is crushed or chewed. However, a number of studies have demonstrated that sulforaphane yield from glucoraphanin is low, and that a non-bioactive nitrile analog, sulforaphane nitrile, is the primary hydrolysis product when plant tissue is crushed at room temperature. Recent evidence suggests that in Arabidopsis, nitrile formation from glucosinolates is controlled by a heat-sensitive protein, epithiospecifier protein (ESP), a non-catalytic cofactor of myrosinase. Our objectives were to examine the effects of heating broccoli florets and sprouts on sulforaphane and sulforaphane nitrile formation, to determine if broccoli contains ESP activity, then to correlate heat-dependent changes in ESP activity, sulforaphane content and bioactivity, as measured by induction of the phase II detoxification enzyme quinone reductase (QR) in cell culture. Heating fresh broccoli florets or broccoli sprouts to 60 degrees C prior to homogenization simultaneously increased sulforaphane formation and decreased sulforaphane nitrile formation. A significant loss of ESP activity paralleled the decrease in sulforaphane nitrile formation. Heating to 70 degrees C and above decreased the formation of both products in broccoli florets, but not in broccoli sprouts. The induction of QR in cultured mouse hepatoma Hepa lclc7 cells paralleled increases in sulforaphane formation.
Article
One successful strategy for cancer chemoprevention is modulation of drug metabolizing enzymes, leading to a facilitated elimination of endogenous and environmental carcinogens. Inducers of phase 2 enzymes such as dithiolethiones inhibit tumorigenesis of environmental carcinogens in various animal models and modulate the metabolism of the carcinogen aflatoxin B1 in human clinical trials. Increasing lines of evidence show that the Keap1-Nrf2 complex is a key molecular target of chemopreventive phase 2 enzyme inducers. The transcription factor Nrf2 is a member of the basic leucine-zipper NF-E2 family and interacts with the antioxidant response element (ARE) in the promoter region of phase 2 detoxifying enzymes. A cytoplasmic actin-binding protein, Keap1, is an inhibitor of Nrf2 that sequesters it in the cytoplasm. Inducers dissociate this complex, allowing Nrf2 to translocate to the nucleus. Disruption of the nrf2 gene in mice leads to the loss of chemopreventive efficacy by inducers. This review focuses on (1) the role of Nrf2 in the regulation of phase 2 and antioxidative genes, (2) the molecular actions of dithiolethiones on the Keap1-Nrf2 pathway, and (3) the contribution of Nrf2-regulated gene families to the cytoprotective actions of dithiolethiones and other inducers. Rapidly accumulating data on this pathway is providing insight into the coordinated mammalian defense systems against electrophiles and oxidative stresses and the means by which it may be targeted by small molecules.
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