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Heating decreases epithiospecifier protein activity and increases SF formation in broccoli

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Abstract

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.

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... It is not, however, the only conversion product of this GSL. Its hydrolysis can also result in the formation of nitriles, which may be influenced by plant species [13,27]. According to Singh et al. [12], indole-3-carbinol characterized by low toxicity, may stimulate the production of DNA repair proteins, while preliminary studies suggest its anticancer, antiviral, antifungal, antibacterial, and anti-inflammatory effects. ...
... International Journal of Food Science percentage conversion of GSLs into ITCs in several 7-day brassica sprouts, found that it was highest in two varieties of radish (96.5% and 90%), followed by Tuscan black kale (68.5%), and was only 18.7% in broccoli. According to the findings of Matusheski, Juvik, and Jeffery [27], the primary GRA hydrolysis product in broccoli crushed at room temperature may be nonbioactive SFN nitrile, whereas the SFN yield observed by the authors was low. After hydrolysis was performed by incubating the homogenate at room temperature, a SFN content of 0.08-0.62 ...
... In the case of broccoli, the application of temperatures of 70°C or 100°C led to a significant decrease in the detected hydrolysis products. Interestingly, in the case of sprouts, the effect of even 100°C was much less destructive on the concentration of the obtained hydrolysis products [27]. It appears that heat treatment may primarily degrade the sensory attractiveness and nutrient content of products such as sprouts, while at the same time minimizing the associated microbiological risks. ...
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Sprouts and microgreens which belong to the Brassicaceae family contain significantly more glucosinolates than mature vegetables, and their composition often differs too. These plant growth stages can be a valuable supplement of the aforementioned compounds in the diet. The content and proportion of individual glucosinolates in sprouts and microgreens can be regulated by modifying the length and temperature of cultivation, the type of light, the use of mineral compounds, elicitation, primming, and cold plasma as well as storage conditions. The way in which sprouts are prepared for consumption affects the yield of glucosinolate hydrolysis. Genetic variation leading to different plant responses to the same factors (e.g., type of light) makes it necessary to conduct detailed studies involving species and variety diversity. Heat stress and the use of cold plasma appear to be fairly universal methods for increasing glucosinolate content. Studies on the use of light at different wavelengths do not provide unequivocal results. Despite experiments on the use of seed soaking solutions (e.g., sulfur and selenium compounds), there are no studies in the available literature on the effects of chemical and thermal seed disinfection methods on the glucosinolate content of the obtained sprouts and microgreens.
... Interestingly, it has been found that heating decreases ESP activity and results in increased SRA formation in broccoli florets and sprouts. Matusheski et al. (2004) showed that heating fresh florets to 60 • C for 5 min markedly reduced ESP activity and increased the extraction yield of SRA. The same effect was seen when fresh sprouts were heated to 70 • C for 10 min [34][35][36]. ...
... Matusheski et al. (2004) showed that heating fresh florets to 60 • C for 5 min markedly reduced ESP activity and increased the extraction yield of SRA. The same effect was seen when fresh sprouts were heated to 70 • C for 10 min [34][35][36]. The difference in heat lability of these two enzymes may be exploited in food product development. ...
... and results in increased SRA formation in broccoli florets and sprouts. Matusheski et al. (2004) showed that heating fresh florets to 60 °C for 5 min markedly reduced ESP activity and increased the extraction yield of SRA. The same effect was seen when fresh sprouts were heated to 70 °C for 10 min [34][35][36]. ...
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Our research group previously found that broccoli sprouts possess neuroprotective effects during pregnancy. The active compound has been identified as sulforaphane (SFA), obtained from glucosinolate and glucoraphanin, which are also present in other crucifers, including kale. Sulforaphene (SFE), obtained from glucoraphenin in radish, also has numerous biological benefits, some of which supersede those of sulforaphane. It is likely that other components, such as phenolics, contribute to the biological activity of cruciferous vegetables. Notwithstanding their beneficial phytochemicals, crucifers are known to contain erucic acid, an antinutritional fatty acid. The aim of this research was to phytochemically examine broccoli, kale, and radish sprouts to determine good sources of SFA and SFE to inform future studies of the neuroprotective activity of cruciferous sprouts on the fetal brain, as well as product development. Three broccoli: Johnny’s Sprouting Broccoli (JSB), Gypsy F1 (GYP), and Mumm’s Sprouting Broccoli (MUM), one kale: Johnny’s Toscano Kale (JTK), and three radish cultivars: Black Spanish Round (BSR), Miyashige (MIY), and Nero Tunda (NT), were analyzed. We first quantified the glucosinolate, isothiocyanate, phenolics, and DPPH free radical scavenging activity (AOC) of one-day-old dark- and light-grown sprouts by HPLC. Radish cultivars generally had the highest glucosinolate and isothiocyanate contents, and kale had higher glucoraphanin and significantly higher sulforaphane content than the broccoli cultivars. Lighting conditions did not significantly affect the phytochemistry of the one-day-old sprouts. Based on phytochemistry and economic factors, JSB, JTK, and BSR were chosen for further sprouting for three, five, and seven days and subsequently analyzed. The three-day-old JTK and radish cultivars were identified to be the best sources of SFA and SFE, respectively, both yielding the highest levels of the respective compound while retaining high levels of phenolics and AOC and markedly lower erucic acid levels compared to one-day-old sprouts.
... In raw broccoli, the main product of conversion during such processes is sulforaphane nitrile due to the active ESP present in the broccoli matrix. Several approaches have been assessed to enhance sulforaphane formation in broccoli products such as mild heat treatment to selectively inactivate ESP while maintaining myrosinase activity [9], high pressure thermal processing [10], lactic acid fermentation [11], and a combination of mild heat with lactic acid fermentation [12]. For instance, mild heating to inactivate ESP combined with fermentation enabled a ≈16-time increase in sulforaphane yield in broccoli puree compared to non-treated broccoli puree, which was attributed to the selective inactivation of ESP and fermentation-induced increase in the accessibility of glucoraphanin for myrosinase-catalysed conversion into sulforaphane. ...
... The initial measurable content of GR in the non-processed broccoli floret was 3149.35 µmol/kg DW, which is slightly lower than the GR levels of 4.4-16.4 µmol/g DW reported by Matusheski, Juvik [9]. The measurable GR content dramatically decreased from 3149.35 to 689, 813, and 1241 µmol/kg DW, respectively, in the control, pre-heated, and thermosonicated broccoli puree samples ( Figure 1A), most likely due to myrosinasecatalyzed conversion. ...
... In intact broccoli tissue, myrosinase and its substrate glucoraphanin are in separate compartments, and hence there is no myrosinase-catalyzed conversion in intact broccoli. During pureeing and thermophysical treatments, tissue decompartmentalisation occurs, which leads to myrosinase-glucoraphanin interaction and conversion of glucoraphanin to its metabolites (sulforaphane and sulforaphane nitrile, depending on the presence of active ESP and the pH of the matrix) [6,9,12]. The GR content in thermal or TS pre-treated purees were significantly higher (p < 0.05) than those in control puree ( Figure 1A). ...
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The aim of this study was to compare the effects of thermosonication (18 kHz at 60 °C for 7 min) pre-treatment with thermal treatment alone (60 °C for 7 min) of broccoli florets prior to pureeing and fermentation on selected bioactive components of fermented broccoli puree. Both thermal and thermosoncation pre-treatments significantly increased the rate of acidification of broccoli puree compared to control untreated broccoli puree, with the time to reach pH 4 being 8.25, 9.9, and 24 h, respectively, for thermally treated, thermosonicated, and control samples. The highest sulforaphane yield of 7268 µmol/kg dry weight (DW) was observed in the thermosonicated samples, followed by 6227 µmol/kg DW and 3180 µmol/kg DW in the thermally treated and untreated samples, respectively. The measurable residual glucoraphanin content was 1642 µmol/kg DW, 1187 µmol/kg DW, and 1047 µmol/kg DW, respectively, in the thermonsonicated, thermally pre-treated, and control fermented samples, indicating that pre-treatment specially by thermosonication increases the extractability of glucoraphanin. The higher sulforaphane yield in the thermosonicated and thermally pre-treated samples could be due to increased extractability and accessibility of glucoraphanin and interaction with myrosinase in addition to the inactivation of epthiospecifier protein (ESP), which directs conversion away from sulforaphane into sulforaphane nitrile.
... It requires the presence of Fe 2+ and an epithiospecifier protein, ESP, also present in broccoli seeds. 75 To block this reaction and achieve maximum sulforaphane yield, ESP was thermally deactivated by extracting the glucoraphanin into boiling, demineralised water. 75 As a visual control, the upper branch produces a bright yellow suspension (possibly due to iron sulphide), whereas the products of the lower branch are colourless. ...
... 75 To block this reaction and achieve maximum sulforaphane yield, ESP was thermally deactivated by extracting the glucoraphanin into boiling, demineralised water. 75 As a visual control, the upper branch produces a bright yellow suspension (possibly due to iron sulphide), whereas the products of the lower branch are colourless. Boiling water extraction also denatures the original myrosinase enzyme in the broccoli seeds. ...
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A novel Redox Stress Test has been developed to identify symptoms of diseases associated with oxidative stress by observing symptom changes induced by short-term activation of the transcription factor Nrf2, to restore redox homeostasis. The Nrf2 pathway is triggered by a herbal preparation, Broccoli Seed Tea, developed to deliver a therapeutic dose of highly bioavailable sulforaphane, a potent activator of Nrf2. We discuss the rationale behind the tea and describe the methods used to optimise the bioavailability of sulforaphane to match the pharmacodynamics of Nrf2 activation. When consumed by people with Parkinson’s disease, the Redox Stress Test induced powerful and concurrent attenuation of a diverse group of Parkinson’s non-motor symptoms, including fatigue, constipation and urinary urgency. Motor symptoms were strictly unaffected. This observation indicates that oxidative stress may be a common factor contributing to non-motor symptoms involving sites in the CNS and peripheral organs. We tentatively interpret the results in terms of a hypothetical model for Parkinson’s Syndrome which we describe as a multisystem redox disorder with reservoirs of the disease in peripheral organs as well as in the brain. Eliminating the disease in peripheral organs is therefore a prerequisite to stopping disease progression in the brain. According to this model, the redox disorder in the brain provokes progressive neurological damage, which is not recognised as such in the early years. More specific neurological symptoms only come to light many years later, when damage to dopaminergic neurons creates a dopamine deficiency which eventually exceeds the threshold required for normal motor control, generating a new coherent group of neurological symptoms which define movement disorder. Given the apparent ease with which oxidative stress can be quenched in several locations simultaneously, we briefly discuss possible implications for public health, medical research, patients and patient advocacy groups. We note that the Redox Stress Test may have the potential to explore symptoms of other diseases where oxidative stress is believed to play a major role, although this remains subject to validation by further research.
... SFN contents in alfalfa or broccoli sprouts were analyzed on the day of harvest for every subject cohort. Sprouts samples were heated to 60°C in water for 10 min to inhibit epithiospecifier protein activity, [44] followed by homogenization and 1 h incubation in the presence of 2 mg mL -1 Sinapis alba thioglucosidase (Sigma-Aldrich) at 60°C. Following incubation, samples were centrifuged (16 000 x g, 5 min, 25°C), and supernatants were filtered using 0.22-μm nylon Spin-X filters (VWR, Radnor, PA). ...
... Myrosinase and epithiospecific protein from the plants is still present contributing to SFN and SFN-NIT conversion and pre-existing desulfoglucosinolates within the sprouts could be converted to SFN-NIT further complicating detection of the microbiome-effect. [44,75,76] Human feeding experiments conducted with GLS-extracts in the absence of myrosinase will help to further clarify the relationship between SFN-NIT and SFN generated by the gut microbiome. Additionally, while our linear regression model yielded a relatively high R 2 , this measure simply describes the fit of our model and cannot be extrapolated to other study populations. ...
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Scope The glucosinolate glucoraphanin from broccoli is converted to sulforaphane (SFN) or sulforaphane‐nitrile (SFN‐NIT) by plant enzymes or the gut microbiome. Human feeding studies typically observe high inter‐individual variation in absorption and excretion of SFN, however, the source of this variation is not fully known. To address this, a human feeding trial to comprehensively evaluate inter‐individual variation in the absorption and excretion of all known SFN metabolites in urine, plasma, and stool, and tested the hypothesis that gut microbiome composition influences inter‐individual variation in total SFN excretion has been conducted. Methods and Results Participants ( n = 55) consumed a single serving of broccoli or alfalfa sprouts and plasma, stool, and total urine are collected over 72 h for quantification of SFN metabolites and gut microbiome profiling using 16S gene sequencing. SFN‐NIT excretion is markedly slower than SFN excretion (72 h vs 24 h). Members of genus Bifidobacterium , Dorea , and Ruminococcus torques are positively associated with SFN metabolite excretion while members of genus Alistipes and Blautia has a negative association. Conclusion This is the first report of SFN‐NIT metabolite levels in human plasma, urine, and stool following consumption of broccoli sprouts. The results help explain factors driving inter‐individual variation in SFN metabolism and are relevant for precision nutrition.
... These conditions guarantee microbiological safety without an increase in temperature during the process (around 35 • C), while maintaining the nutritional value and fresh characteristics of the product [19]. On the other hand, the US can increase the shelf-life of vegetable products through cell disruption without negatively impacting their bioactive compounds [20,21]. The effectiveness of these emerging technologies has been demonstrated in different foods. ...
... Then, carrots were cut into transverse slices, while broccoli stalks were cut into longitudinal portions, taking care to remove browned areas. After that, the cut vegetables were blanched in a microwave oven to inactivate the browning enzymes [2,21,24]. For that, samples of 300 g of each product were exposed twice to 700 W during 2 min, with 1 min between the first exposition and the second. ...
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Vegetable beverages are a convenient strategy to enhance the consumption of horticultural commodities, with the possibility of being fortified with plant by-products to increase functional quality. The main objective was to develop a new veggie beverage from broccoli stalks and carrot by-products seasoned with natural antioxidants and antimicrobial ingredients. Pasteurization, Ultrasound (US), and High Hydrostatic Pressure (HHP) and their combinations were used as processing treatments, while no treatment was used as a control (CTRL). A shelf-life study of 28 days at 4 °C was assayed. Microbial load, antioxidant capacity, and bioactive compounds were periodically measured. Non-thermal treatments have successfully preserved antioxidants (~6 mg/L ΣCarotenoids) and sulfur compounds (~1.25 g/L ΣGlucosinolates and ~5.5 mg/L sulforaphane) throughout the refrigerated storage, with a longer shelf life compared to a pasteurized beverage. Total vial count was reduced by 1.5–2 log CFU/mL at day 0 and by 6 log CFU/mL at the end of the storage in HHP treatments. Thus, the product developed in this study could help increase the daily intake of glucosinolates and carotenoids. These beverages can be a good strategy to revitalize broccoli and carrot by-products with high nutritional potential while maintaining a pleasant sensory perception for the final consumer.
... The Tang [23] method was used for determining the activity of superoxide dismutase. The content of glucosinolates was determined by Wang [24], and the content of sulforaphane was measured using the method developed by Matusheski [25]. Myrosinase activity was measured with reference to the method developed by Guo [26]. ...
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The impact of exogenous sulfate components on the nutritional quality of vegetables has been well documented. In this study, we examined the effects of adding K2SO4 to broccoli on its nutritional quality, active components, and the genes involved in glucosinolate synthesis. Different concentrations of exogenous K2SO4 of 25, 75, and 150 g·m−2 were applied to the “Naihan Youxiu” broccoli cultivar, while the control treatment received no potassium sulfate. Our primary objective was to gain insights into strategies for enhancing broccoli’s nutritional and active components. The results showed that broccoli’s vitamin C content in each treatment was lower than that in the control treatment. The contents of soluble protein, soluble sugar, polyphenols, and total flavonoids in the treatment of 150 g·m−2 K2SO4 were the highest. They increased by 23.51%, 87.49%, 146.00%, and 22.73% more than the control, respectively. MDA was significantly inhibited after the 75 g·m−2 treatment, whereas SOD had the highest activity under the 75 g·m−2 treatment. Therefore, the 150 g·m−2 treatment was beneficial in improving the nutritional quality and antioxidant capacity of broccoli. The contents of PRO, SIN, NAP, GBC, 4ME, NEO, total indole glucosinolates, and total glucosinolates reached the peak at the 150 g·m−2 K2SO4 treatment, RAA and total aliphatic glucosinolates reached the peak at the 75 g·m−2 K2SO4 treatment, and ERU and 4OH reached the highest at the 25 g·m−2 K2SO4 treatment. The sulforaphane content was the highest in the 150 g·m−2 treatment, and myrosinase activity was the highest in the 75 g·m−2 treatment. It can be seen that the 150 g·m−2 treatment significantly increased the content of glucosinolates, total indole glucosinolates, total glucosinolates, and sulforaphane in broccoli. CYP79B2, CYP83B1, CYP83A1, AOP2, UGT74B1, and MYB34 were significantly up-regulated under 150 g·m−2 K2SO4 treatment and reached the peak value. CYP79F1, CYP81F4, and MAM1 showed significant inhibitory effects when treated with 150 g·m−2 of K2SO4. The expression levels of BCAT4, CYP81F1, ST5a, ST5c, and SUR1 were down-regulated under the 150 g·m−2 K2SO4 treatment, but not significantly. In summary, the K2SO4 150 g·m−2 treatment had the best effect on nutritional quality, antioxidant activity, the content of glucosinolates, total glucosinolates, sulforaphane, and expression of CYP79B2, CYP83B1, CYP83A1, FMO2, UGT74B1, AOP2, and MYB34 genes.
... Furthermore, at low pH, or in presence of specifier proteins, nitrile (CN) and epithionitrile (ETN) formation can occur. ETN are only formed from GLS with a terminal unsaturated double bond (Matusheski, Juvik, & Jeffery, 2004). Epithiospecifier proteins (ESP) are Fe 2+ -dependent and affect the aglucon which is formed upon GLS hydrolysis when glucose is cleaved by myrosinase (Mocniak, Elkin, & Bollinger, 2020). ...
... It has been reported that mild heating (∼60°C) of broccoli for a short duration (∼10 min) can selectively inactivate ESP while retaining the activity of myrosinase, thus enhancing the formation of sulforaphane. 15,16 Like most alkyl isothiocyanates, sulforaphane is a lipophilic molecule, 17 and unlike its hydrophilic precursor, it has poor water solubility and stability. 18 Sulforaphane's poor stability is significantly affected by pH, temperature, light, and oxygen. ...
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Sulforaphane, an organosulfur phytochemical, has been demonstrated to have significant anticancer potential in both in vitro and in vivo studies, exhibiting mechanisms of action that include inducing apoptosis, inhibiting cell proliferation, and modulating key signalling pathways involved in cancer development. However, its instability presents a major obstacle to its clinical application due to its limited bioavailability. This study aimed to improve the stability and thus the bioavailability of sulforaphane from broccoli by microencapsulation with whey (BW) and pea protein (BP) by freeze-drying. BW and BP were characterised by particle size measurement, colour, infrared spectroscopy, scanning electron microscopy, thermogravimetry, and differential scanning calorimetry. Dynamic in vitro gastrointestinal digestion was performed to measure sulforaphane bioaccessibility, in BP, BW and dried broccoli. A Caco-2-HT29-MTX-E12 intestinal absorption model was used to measure sulforaphane bioavailability. The in vitro dynamic gastrointestinal digestion revealed that sulforaphane bioaccessibility of BW was significantly higher (67.7 ± 1.2%) than BP (19.0 ± 2.2%) and dried broccoli (19.6 ± 10.4%) (p < 0.01). In addition, sulforaphane bioavailability of BW was also significantly greater (54.4 ± 4.0%) in comparison to BP (9.6 ± 1.2%) and dried broccoli (15.8 ± 2.2%) (p < 0.01). Microencapsulation of broccoli sulforaphane with whey protein significantly improved its in vitro bioaccessibility and bioavailability. This suggests that whey protein isolate could be a promising wall material to protect and stabilise sulforaphane for enhanced bioactivity and applications (such as nutraceutical formulations).
... ESP hydrolyzes glucoraphanin to nitrile but not SFN in the GSL metabolic pathway [10,35]. Decreased ESP activity can lead to increased SFN formation in broccoli [44]. Consequently, ESP negatively regulates SFN production in broccoli. ...
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Sulforaphane (SFN) is one of the hydrolysates of glucosinolates (GSLs), primarily derived from Brassica vegetables like broccoli. In clinical therapy, SFN has been proven to display antimicrobial, anticancer, antioxidant, and anti-inflammatory properties. However, the antimicrobial effects and mechanism of SFN against plant pathogens need to be further elucidated, which limits its application in agriculture. In this study, the genetic factors involved in SFN biosynthesis in 33 B. oleracea varieties were explored. The finding showed that besides the genetic background of different B. oleracea varieties, myrosinase and ESP genes play important roles in affecting SFN content. Subsequently, the molecular identification cards of these 33 B. oleracea varieties were constructed to rapidly assess their SFN biosynthetic ability. Furthermore, an optimized protocol for SFN extraction using low-cost broccoli curds was established, yielding SFN-enriched extracts (SFN-ee) containing up to 628.44 μg/g DW of SFN. The antimicrobial activity assay confirmed that SFN-ee obtained here remarkably inhibit the proliferation of nine tested microorganisms including four plant pathogens by destroying their membrane integrity. Additionally, the data demonstrated that exogenous application of SFN-ee could also induce ROS accumulation in broccoli leaves. These results indicated that SFN-ee should play a dual role in defense against plant pathogens by directly killing pathogenic cells and activating the ROS signaling pathway. These findings provide new evidence for the antimicrobial effect and mechanism of SFN against plant pathogens, and suggest that SFN-ee can be used as a natural plant antimicrobial agent for crop protection and food preservation.
... 9 Additional studies examining the thermal stability of broccoli myrosinase have found that thermal treatment at 70°C or higher drastically reduces sulforaphane formation. 10,11 Furthermore, adding exogenous myrosinase in the form of mustard or moringa powder to cooked broccoli leads to sulforaphane formation, providing additional evidence that myrosinase denaturation is responsible for reducing sulforaphane formation. 9,12 Therefore, identifying strategies to improve the stability of myrosinases to better withstand cooking temperatures could improve the conversion of glucosinolates to bioactive isothiocyanates, enhancing the overall nutritional properties of cooked cruciferous plants. ...
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Glucosinolates are plant-specialized metabolites that can be hydrolyzed by glycosyl hydrolases, called myrosinases, creating a variety of hydrolysis products that benefit human health. While cruciferous vegetables are a rich source of glucosinolates, they are often cooked before consumption, limiting the conversion of glucosinolates to hydrolysis products due to the denaturation of myrosinases. Here we screen a panel of glycosyl hydrolases for high thermostability and engineer the Brassica crop, broccoli (Brassica oleracea L.), for the improved conversion of glucosinolates to chemopreventive hydrolysis products. Our transgenic broccoli lines enabled glucosinolate hydrolysis to occur at higher cooking temperatures, 20 °C higher than in wild-type broccoli. The process of cooking fundamentally transforms the bioavailability of many health-relevant bioactive compounds in our diet. Our findings demonstrate the promise of leveraging genetic engineering to tailor crops with novel traits that cannot be achieved through conventional breeding and improve the nutritional properties of the plants we consume.
... Present in plants as glucoraphanin(GR) (Inactive form) this is known for its chemopreventive activity and is abundant in cruciferous vegetables such as cauliflower, broccoli and brussel sprouts [78]. Myrosinase, a plant thioglucosidase or bacterial thioglucosidase, present in the colon hydrolyze GR to the equivalent isothiocyanate SFN by mincing or masticating [79]. This may protect against mental diseases by decreasing oxidative stress, neuronal inflammation, and neuronal cell death. ...
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Huntington's disease (HD), a multifaceted neurological disorder, presents a complex clinical scenario. An autosomal dominant neurodegenerative ailment called Huntington's disease is brought on by increase in number of CAG (Cytosine-Adenine-Guanine) repeats, which causes the creation of a mutant Huntingtin protein (mHTT) resulting in neuronal death and mental disabilities in human beings. End signs and symptoms can include significant weight loss, difficulty swallowing or breathing, recurrent aspiration pneumonia, declined health and uncontrolled pain. The excessive production of ROS (Reactive Oxygen Species) in nervous tissues is considered a significant risk factor in most of the neurological diseases including HD. Transcriptional regulation, immunological system, and mitochondrial function are all disrupted by mHTT. Although natural products have shown promise in ameliorating symptoms, it is important to note that no singular "phytoconstituent" has been definitively linked to its therapeutic intervention. Nevertheless, certain naturally occurring compounds have exhibited promising outcomes in preclinical investigations. This article focuses on a few phytoconstituents that are known to have a variety of neuroprotective effects through a wide range of biological activities. By stimulating the Nrf2 (Nuclear factor erythroid 2-related factor) pathway and suppressing NF-κB (Nuclear Factor Kappa-light-chain-enhancer of activated B cells), astaxanthin, berberine, and sulfarophane increase the antioxidant and anti-inflammatory activity, resulting in neuroprotection. Curcumin leads to metal chelating effect and decline in reactive oxygen species which are certainly one among the vital processes to impede and manage the disorders causing neurodegeneration including HD. This affects the upregulation of HSPs (Heat Shock Proteins) which helps in HD management. Naringin reduces level of oxidative stress and inflammation by free radical scavenging, NF-κB stimulates cell survival and prevents apoptosis by upregulating anti-apoptotic genes expression and downregulating proapoptotic genes.
... SFN is an isothiocyanate that contains sulfur that is naturally occurring and is present in cruciferous vegetables including brussels sprouts, broccoli, cabbage, and cauliflower [109]. In animal models, SFN has been demonstrated to be beneficial in avoiding a variety of chemically generated malignancies and inhibiting the development of preexisting tumors [110À112]. ...
... When broccoli is exposed to temperatures between 60°C and 70°C for a period of 5 to 10 minutes, its effectiveness as a protein may be jeopardized. Under these circumstances, the production of SFN experiences a notable rise of 3 to 7 times [7]. Whenever feasible, opt for raw or recently harvested broccoli. ...
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The isothiocyanate's chemoprevention properties are reported to be present in cruciferous veggies through a variety of mechanisms. Sulforaphane, a phytochemical found in green leafy vegetables, has shown promise in the prevention and treatment of several cancers, including those of the prostate, breast, colon, skin, urinary bladder, and oral cavities. These malignancies include those that affect these organs. This substance is naturally present in broccoli sprouts, kale, cabbage, cauliflower, and garden cress. Broccoli should be a regular part of your diet because it contains a variety of bioactive substances such as vitamins, polyphenols, sulfides, glucosinolates, and antioxidants. Sulforaphane may be used as an inexpensive replacement or dietary supplement for chemo preventive therapy, according to the findings of epidemiological and experimental studies. Phase 2 detoxification enzymes like glutathione transferases, epoxide hydrolase, NAD(P)H: quinone reductase, and glucuronosyltransferases, as well as epoxide hydrolase and epoxide hydrolase, are produced when the body is stimulated. This is a useful tactic for preventing cancer and fending off the harm that electrophiles and reactive oxygen species can cause. Isothiocyanates are widely distributed in the Cruciferae family and Brassica genus of food plants, which include both broccoli and cauliflower. The most typical form of these substances is glucosinolate precursors. Sulforaphane and 4-methylsulfinylbutyl isothiocyanate, two of these isothiocyanates, are particularly powerful inducers of phase 2 enzymes. It is feasible to successfully extract glucosinolates and isothiocyanates from plants by homogenizing them at a temperature of around 50 degrees Celsius in a solution of equal parts dimethyl sulfoxide, dimethylformamide, and acetonitrile. This method avoids the hydrolysis of glucosinolates by myrosinase. It's interesting to note that glucoraphanin, the precursor to sulforaphane, is 10-100 times more abundant in 3-day-old sprouts of various cruciferous vegetables, including broccoli and cauliflower than it is in fully grown veggies. Dimethylbenz(a)anthracene-treated rats displayed notable reductions in mammary tumor occurrence, quantity, and rate of growth when fed extracts from 3-day-old broccoli sprouts. These extracts' primary enzyme-inducing components were either glucoraphanin or sulforaphane. As a result, crucifer sprouts might provide an equivalent amount of cancer prevention to eating far larger quantities of the same mature vegetable species.
... The higher BCN content in the HY isolated using MHG can be explained by the presence of an epithiospecific protein due to its interaction with the enzyme myrosinase, which redirects the reaction toward the formation of epithionitrile or nitrile depending on the glucosinolate structure [25]. The epithiospecific protein is heat sensitive and its activity decreases significantly at high temperatures (>50 • C), which is probably the reason for the lower content of BCN and higher content of BITC in the HY isolated using MAE extraction technique [26]. The obtained results agree with those published so far [2,8,27]. ...
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Tropaeolum majus L. is a traditional medicinal plant with a wide range of biological activities due to the degradation products of the glucosinolate glucotropaeolin. Therefore, the goals of this study were to identify volatiles using gas chromatography–mass spectrometry analysis (GC-MS) of the hydrosols (HYs) isolated using microwave-assisted extraction (MAE) and microwave hydrodiffusion and gravity (MHG). Cytotoxic activity was tested against a cervical cancer cell line (HeLa), human colon cancer cell line (HCT116), human osteosarcoma cell line (U2OS), and healthy cell line (RPE1). The effect on wound healing was investigated using human keratinocyte cells (HaCaT), while the antibacterial activity of the HYs was tested against growth and adhesion to a polystyrene surface of Staphylococcus aureus and Escherichia coli. Antiphytoviral activity against tobacco mosaic virus (TMV) was determined. The GC-MS analysis showed that the two main compounds in the HYs of T. majus are benzyl isothiocyanate (BITC) and benzyl cyanide (BCN) using the MAE (62.29% BITC and 15.02% BCN) and MHG (17.89% BITC and 65.33% BCN) extraction techniques. The HYs obtained using MAE showed better cytotoxic activity against the tested cancer cell lines (IC50 value of 472.61–637.07 µg/mL) compared to the HYs obtained using MHG (IC50 value of 719.01–1307.03 μg/mL). Both concentrations (5 and 20 µg/mL) of T. majus HYs using MAE showed a mild but statistically non-significant effect in promoting gap closure compared with untreated cells, whereas the T. majus HY isolated using MHG at a concentration of 15 µg/mL showed a statistically significant negative effect on wound healing. The test showed that the MIC concentration was above 0.5 mg/mL for the HY isolated using MAE, and 2 mg/mL for the HY isolated using MHG. The HY isolated using MHG reduced the adhesion of E. coli at a concentration of 2 mg/mL, while it also reduced the adhesion of S. aureus at a concentration of 1 mg/mL. Both hydrosols showed excellent antiphytoviral activity against TMV, achieving100% inhibition of local lesions on the leaves of infected plants, which is the first time such a result was obtained with a hydrosol treatment. Due to the antiphytoviral activity results, hydrosols of T. majus have a promising future for use in agricultural production.
... A second sample was similarly processed 6 days postharvest. For SFN measurements, samples were heated to 60 • C in water for 10 min to inhibit epithiospecifier protein activity [25], followed by homogenization and 1 h incubation with 2 mg/mL myrosinase (Sinapis alba thioglucosidase, MilliporeSigma, St. Louis, MO, USA) at 60 • C to fully convert GRN to SFN (SFN equivalence). For GRN measurements, samples were heated to 100 • C in water for 10 min to inactivate all enzyme activities, followed by homogenization, and extracted 3 times in water. ...
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Cruciferous vegetable consumption is associated with numerous health benefits attributed to the phytochemical sulforaphane (SFN) that exerts antioxidant and chemopreventive properties, among other bioactive compounds. Broccoli sprouts, rich in SFN precursor glucoraphanin (GRN), have been investigated in numerous clinical trials. Broccoli microgreens are similarly rich in GRN but have remained largely unexplored. The goal of this study was to examine SFN bioavailability and the microbiome profile in subjects fed a single serving of fresh broccoli microgreens. Eleven subjects participated in a broccoli microgreens feeding study. Broccoli microgreens GRN and SFN contents and stability were measured. Urine and stool SFN metabolite profiles and microbiome composition were examined. Broccoli microgreens had similar GRN content to values previously reported for broccoli sprouts, which was stable over time. Urine SFN metabolite profiles in broccoli microgreens-fed subjects were similar to those reported previously in broccoli sprouts-fed subjects, including the detection of SFN-nitriles. We also reported the detection of SFN metabolites in stool samples for the first time. A single serving of broccoli microgreens did not significantly alter microbiome composition. We showed in this study that broccoli microgreens are a significant source of SFN. Our work provides the foundation for future studies to establish the health benefits of broccoli microgreens consumption.
... The content of glucosinolates and their breakdown products in Brassica spp. is dependent on several environmental and developmental factors (Velasco et al., 2007). The I3C and SRA HPLC data ( Figure 2) indicate that the kale genotypes used in this work are a potential good source of glucoraphanin, representing the native glucosinolate precursor of the sulphoraphane, well noted for its health promoting properties (Matusheski et al., 2004). ...
... The alkenyl GSLs give rise to epithioalkanes in the presence of epithiospecifier protein (Lambrix et al. 2001). Myrosinase and epithiospecifier proteins, in particular, have a lower thermal tolerance (70 and 60 degrees Celsius, respectively) (Matusheski et al. 2004). Thus, the cooking processes have been extensively researched to maximize the higher recovery of ITCs (Bongoni et al. 2014;Dosz and Jeffery 2013;Rungapamestry et al. 2006). ...
... Assessment of Epithiospecifier Protein Activity. To evaluate the effect of leaf age on ESP activity, the protocol described by Matusheski et al. 24 was modified and adapted. Briefly, 50 μL of ESP extract, 350 μL of a 50 mM sodium acetate buffer (pH = 5.5) containing 1 mM dithiothreitol and 0.2 mM of FeSO 4 , 50 μL of 0.5 U/mL myrosinase, 10 μL of 25.5 mM L(+)-ascorbic acid, and 50 μL of allyl GLS (5 mg/mL) were mixed and incubated for 1 h at room temperature. ...
Article
The health-beneficial effects of Brassica vegetables are mainly attributed to their high contents of glucosinolates and the products of their hydrolysis, especially isothiocyanates. Distribution of glucosinolates across plant organs can strongly vary. Here, we investigated the effect of leaf age on glucosinolate accumulation and hydrolysis in two leafy Brassica vegetables, pak choi and giant red mustard. We also evaluated the activity of the hydrolyzing enzyme myrosinase across the leaves. Finally, we assessed whether glucosinolates are transported from older leaves to younger leaves. Young leaves of both species contained more than 3-fold more glucosinolates than older ones. Accordingly, more isothiocyanates were released in the young leaves. Myrosinases fully hydrolyzed all of the amounts of glucosinolates regardless of the leaf age. Moreover, older leaves were observed to supply younger leaves with glucosinolates. Thus, this study suggests that consumers can improve the nutritional value of food by incorporating young leaves of leafy Brassicas in their diet.
... Sulforaphane and sulforaphane nitrile are byproducts of glucosinolate hydrolysis by cytosolic endogenous myrosinase. 13 The myrosinase enzyme activity, the presence of other proteins like epithiospecifier protein (ESP), and conditions like temperature and pH play a role in this hydrolysis process and compounds that are generated. 14 Today, mineral deficiency, especially of those minerals that are essential for the nourishment of both human beings and animals through various agricultural products, is a determining factor in crop production. ...
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Due to the growing world population and increasing environmental stress, improving the production, nutritional quality, and pharmaceutical applications of plants has become an urgent need. Therefore, the current research was designed to investigate the impact of seed priming using plant-growth-promoting bacteria (PGPB) along with selenium nanoparticles (SeNPs) treatment on chemical and biological properties of three Brassica oleracea cultivars [Southern star (VA1), Prominence (VA2), Monotop (VA3)]. With this aim, one out of five morphologically different strains of bacteria, namely, JM18, which was further identified via 16S rRNA gene sequencing as a Nocardiopsis species with strong plant-growth-promoting traits, isolated from soil, was used. To explore the growth-promoting potential of Nocardiopsis species, seeds of three varieties of B. oleracea were primed with JM18 individually or in combination with SeNP treatment. Seed treatments increased sprout growth (fresh and dry weights) and glucosinolate accumulation. The activity of myrosinase was significantly increased through brassica sprouts and consequently enhanced the amino-acid-derived glucosinolate induction. Notably, a reduction in effective sulforaphane nitrile was detected, being positively correlated with a decrease in epithiospecifier protein (EP). Consequently, the antioxidant activities of VA2 and VA3, determined by the ferric reducing antioxidant power (FRAP) assay, were increased by 74 and 79%, respectively. Additionally, the antibacterial activities of JM18-treated cultivars were improved. However, a decrease was observed in SeNP-and JM18 + SeNP-treated VA2 and VA3 against Serratia marcescens and Candida glabrata and VA1 against S. marcescens. In conclusion, seed priming with the JM18 extract is a promising method to enhance the health-promoting activities of B. oleracea sprouts.
... These volatile sulfur compounds are mainly products of sulfur metabolism, including the enzymatic hydrolysis of sulfurcontaining amino acids and glucosinolates [29,30]. The enzyme MGL catalyzes the production of methyl mercaptan from methionine [83]. FA-treated broccoli produced a lower level of volatile sulfur compounds, relative to the control group of broccolis, which may be attributed to the ability of FA to inhibit the expression of APR, APK, and APS, and prevent the excess accumulation of sulfur products. ...
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Introduction: Folic acid (FA) is a critical metabolite in all living organisms and an important nutritional component of broccoli. Few studies have been conducted on the impact of an exogenous application of FA on the postharvest physiology of fruits and vegetables during storage. In this regard, the mechanism by which an exogenous application of FA extends the postharvest quality of broccoli is unclear. Objective: This study utilized a multicomponent analysis to investigate how an exogenous application of FA effects the postharvest quality of broccoli. Methods: Broccoli was soaked in 5 mg L-1 FA for 10 min and the effect of the treatment on the appearance and nutritional quality of broccoli was evaluated. These data were combined with transcriptomic, metabolomic, and DNA methylation data to provide insight into the potential mechanism by which FA delays senescence. Results: The FA treatment inhibited the yellowing of broccoli during storage. CHH methylation was identified as the main type of methylation that occurs in broccoli and the FA treatment was found to inhibit DNA methylation, promote the accumulation of endogenous FA and chlorophyl, and inhibit ethylene biosynthesis in stored broccoli. The FA treatment also prevented the formation of off-odors by inhibiting the degradation of glucosinolate. Conclusions: FA treatment inhibited the loss of nutrients during the storage of broccoli, delayed its yellowing, and inhibited the generation of off-odors. Our study provides deeper insight into the mechanism by which the postharvest application of FA delays postharvest senescence in broccoli and provides the foundation for further studies of postharvest metabolism in broccoli.
... Broccoli, an edible plant in Brassica, is rich in a variety of functional, active ingredients beneficial to human health, such as isothiocyanates (ITCs), sulforaphane (SFN), glucosinolates (GLs), phenolic compounds, etc. [1][2][3]. Shapiro et al. [4] showed that high consumption of broccoli could reduce the incidence of cancer, which is mainly related to ITCs [5]. In plants, ITCs are hydrolysates produced by hydrolysis of GLs under the action of myrosinase (MYR, EC3.2.1.147) ...
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Broccoli sprouts have high isothiocyanate and selenium accumulation capacity. This study used a combination of methods, including physiological and biochemical, gene transcription and proteomic, to investigate the isothiocyanate and endogenous selenium accumulation mechanisms in broccoli sprouts under exogenous sodium selenite treatment during germination. Compared with the control, the sprouts length of broccoli sprouts under exogenous selenium treatment was significantly lower, and the contents of total phenol and malondialdehyde in 6-day-old broccoli sprouts were substantially higher. The contents of isothiocyanate and sulforaphane in 4-day-old were increased by up-regulating the relative expression of genes of UGT74B1, OX-1, and ST5b. The relative expression of BoSultr1;1, BoSMT, BoHMT1, and BoCOQ5-2 genes regulating selenium metabolism was significantly up-regulated. In addition, 354 proteins in 4-day-old broccoli sprouts showed different relative abundance compared to the control under selenium treatment. These proteins were classified into 14 functional categories. It was discovered that metabolic pathways and biosynthetic pathways of secondary metabolites were significantly enriched. The above results showed that exogenous selenium was beneficial in inducing the accumulation of isothiocyanate and selenium during the growth of broccoli sprouts.
... Moreover, there was also a significant correlation and effect of EGCG dose on the degree of cataract opacity in diabetic rat models. This illustrates that the higher the administration of EGCG, the milder the lens opacity that occurs [14][15][16] . According to our research hypothesis, EGCG is a compound primarily contained in green tea and has a potent antioxidant effect. ...
Article
Aim: To evaluate the effect of epigallocatechin gallate (EGCG) in preventing lens opacity and the aggregation of lens αB-crystallin in model rats of diabetes mellitus (DM). Methods: This experimental study included Wistar rats for DM as in vivo models and divided into 5 groups. The treatment groups were administered EGCG by orally for 20d and were then assessed for their degree of lens opacity with binocular microscope and lens αB-crystallin expression from Western blot analyze. Results: Pearson correlation test and regression analysis on EGCG exposure and final random blood sugar (RBS) obtained a significance level of P<0.05. EGCG exposure can significantly lower RBS with an R 2 of 0.5634 (56.34%). The same analysis on EGCG exposure and the degree of lens opacity obtained a significance level of P<0.05 and increased exposure to EGCG can significantly lower the degree of lens opacity with an R 2 of 0.8577 (85.77%). Correlation analysis between EGCG and the expression of lens αB-crystallin can be concluded that the higher the EGCG exposure administered, the higher the native lens αB-crystallin expression and the lower the aggregate lens αB-crystallin expression. There was also significant effect in which every 1 mg/kg body weight dose of EGCG can increase the native lens αB-crystallin expression by 0.0063 and decrease the aggregate lens αB-crystallin expression by 0.0076. Conclusion: The administration of EGCG at a dose of 300, 600, and 1200 mg shows a significant effect on preventing lens opacity and aggregation of αB-crystallin in diabetic rat models and this research could be a biomolecular prevention of cataract.
... The SFN content in pure unprocessed broccoli was 2.05 µmol/g dry weight (dw), which was reduced down to 1.06 µmol/g dw after vacuum cooking [90]. As it is known, broccoli processing at low temperatures (less than 50 • C) is more suitable, as it promotes SFN formation [95], as the epithiospecific protein activity is prominent at this temperature [96]. However, the same authors suggested that a cooking temperature of up to 60 • C allows the formation of SFN. ...
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In the last decade, most of the evidence on the clinical benefits of including cruciferous foods in the diet has been focused on the content of glucosinolates (GSL) and their corresponding isothiocyanates (ITC), and mercapturic acid pathway metabolites, based on their capacity to modulate clinical, biochemical, and molecular parameters. The present systematic review summarizes findings of human studies regarding the metabolism and bioavailability of GSL and ITC, providing a comprehensive analysis that will help guide future research studies and facilitate the consultation of the latest advances in this booming and less profusely researched area of GSL for food and health. The literature search was carried out in Scopus, PubMed and the Web of Science, under the criteria of including publications centered on human subjects and the use of Brassicaceae foods in different formulations (including extracts, beverages, and tablets), as significant sources of bioactive compounds, in different types of subjects, and against certain diseases. Twenty-eight human intervention studies met inclusion criteria, which were classified into three groups depending on the dietary source. This review summarizes recent studies that provided interesting contributions, but also uncovered the many potential venues for future research on the benefits of consuming cruciferous foods in our health and well-being. The research will continue to support the inclusion of GSL-rich foods and products for multiple preventive and active programs in nutrition and well-being.
... It is well-known that the formation of these nitriles is affected by the presence of epithiospecifier protein. Our results are in accordance with the findings of Matusheski et al. (2004) who reported that this protein is heat-sensitive. In our previous paper, two approaches were used, autolysis followed by hydrodistillation and exogenous myrosinase hydrolysis followed by solvent extraction (Bla zevi c et al. 2011). ...
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Two wild-growing Brassicaceae plants of Croatian origin, Aurinia leucadea (Guss.) K. Koch and Lepidium draba L., were investigated to uncover glucosinolates via GC/MS analysis of their degradation products. The main constituents of Aurinia leucadea (Guss.) K.Koch distillate were hex-5-enenitrile (28.8%) and but-3-enyl isothiocyanate (18.8%), while 4,5-epithiopentanenitrile (50%) and 5,6-epithiohexanenitrile (18.5%) were the main volatile compounds in autolysate. 4-(Methylsulfanyl)butyl isothiocyanate (96.4%) constituted almost the entire Lepidium draba L. distillate, while the autolysate was characterized by 4-(methylsulfinyl)butyl isothiocyanate (57.3%). So, regarding the glucosinolate degradation products, the main glucosinolates of A. leucadea were glucobrassicanapin and gluconapin, and of L. draba glucoerucin and glucoraphanin.
... The interaction of the ESP with the enzyme myrosinase diverts the reaction towards the production of epithionitriles or nitriles, depending on the glucosinolate structure (33). Given that ESP is thermally sensitive, it is known that its activity decreases significantly at 50 °C or higher (34), which is the main reason for the low presence of BCN in the essential oil of T. majus. On the other hand, soaking for 1 h in water before MHG technique enabled the formation of BCN, which explains its high percentage in the the seed extract. ...
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Research background. Plant Tropaeolum majus L. (garden nasturtium) belongs to the family Tropaeolaceae and contains benzyl glucosinolate. The breakdown product of benzyl glucosinolate, benzyl isothiocyanate (BITC), exhibits various biological activities such as antiproliferative, antibacterial and antiinflammatory. In order to optimize the content of biologically active volatile compounds in plant extract and essential oil, the use of appropriate extraction technique has a crucial role. Experimental approach. The current study investigates the effect of two modern extraction methods, microwave-assisted distillation (MAD) and microwave hydrodiffusion and gravity (MHG), on the chemical composition of volatile components present in the essential oil and extract of garden nasturtium (T. majus L. var. altum) seeds. Investigation of the biological activity of samples (essential oil, extract and pure compounds) was focused on the antiproliferative effect against different cancer cell lines: cervical cancer cell line (HeLa), human colon cancer cell line (HCT116) and human osteosarcoma cell line (U2OS), and the antibacterial activity which was evaluated against the growth and adhesion of Staphylococcus aureus and Escherichia coli to polystyrene surface. Results and conclusions. Essential oil and extract of garden nasturtium (T. majus) seeds were isolated by two extraction techniques: MAD and MHG. BITC and benzyl cyanide (BCN) present in the extract were identified by gas chromatography-mass spectrometry. Essential oil of T. majus showed higher antiproliferative activity (IC50
... Our results are similar to the results of previous studies that have indicated a significant increase in the sulforaphane content of broccoli subjected to heating [34]. e study of Matusheski et al. [35] also reported that preheating broccoli florets and sprouts to 60°C significantly increased the sulforaphane in vegetable tissue extracts following crushing. In addition, we observed that the contents of two types of isothiocyanates (erucin and PEITC) also increased in the hot air-dried samples (Figure 3). ...
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Cabbage (Brassica oleracea var. capitata f. alba), a cruciferous vegetable, is one of the most widely consumed vegetables worldwide. However, research on whether there are differences in its contents of phytochemicals and biological activities according to the drying method for each portion of the vegetable remains insufficient. The present study investigated the contents of representative polyphenols and isothiocyanates, the antioxidant capacity, and the antiproliferative effects among six leaf layers of cabbage subjected to hot air and freeze-drying. High-performance liquid chromatography analysis showed that most phenolic and flavonoid bioactive compounds were significantly accumulated in the outer cabbage leaf layer (P1), whereas isothiocyanates were most abundant in the leaf layer close to the core of the head (P5). The contents of isothiocyanates, gallic acid, epicatechin, p-coumaric acid, sinapic acid, and myricetin were significantly higher in the hot air-dried sample than in the freeze-dried sample, whereas the contents of catechin hydrate, chlorogenic acid, 4-hydroxybenzoic acid, and rutin hydrate were significantly higher in the freeze-dried sample. Compared to other leaf layers, P1 exhibited high antiproliferative efficacy against pancreatic, breast, and gastric cancer cells. P1 also showed excellent DPPH·(EC 50–4.208 ± 0.033 and 4.611 ± 0.053 mg/mL for hot air and freeze-dried samples, respectively) and ABTS· (2.422 ± 0.068 and 2.224 ± 0.070 mg/mL for hot air and freeze-dried samples, respectively) radical-scavenging effects. These results indicate that the contents of polyphenols and isothiocyanates in cabbage may vary depending on the leaf layer and the drying method. Our findings provide insight for applying appropriate food drying methods that can be used to produce cabbage leaf-based products with enhanced bioactivity.
... ESP hinders the synthesis of ITCs but it has been observed that cooking at about 60 o C denatures the ESP and favors the formation of ITCs rather than epithionitriles. 12,13 Nowadays, researchers are keen to study these compounds as they have been linked to prevention and cure (to some extent), some of the most cataclysmic diseases for human health. For instance, isothiocyanates are reported to react with phase I and phase II enzymes to detoxify carcinogens and excrete them out of the body, inhibit cytochrome p450, and suppress cancer cell proliferation thus preventing cancer. ...
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Isothiocyanates are the highly reactive organo-sulphur phytochemicals and are product of hydrolysis of glucosinolates which are present mainly in the cruciferous vegetables. These compounds due to their unique chemical reactivity possess anti-cancer, anti-inflammatory, and neuroprotective properties. Epidemiological and experimental evidences suggest that isothiocyanates reduce oxidative stress and act as indirect antioxidants as well as antimicrobials, therefore, have received attention from the researchers for their possible application in pharmacological and food industry. However, due to high volatility and heat sensitivity of these bioactive compounds, their extraction is very challenging and requires the application of various innovative technologies. In addition to that, their fate during the processing conditions also needs to be considered as these processes tend to affect their bioavailability. Isothiocyanates exhibit wide range of antimicrobial activity due to their ability of reducing oxygen consumption and depolarizing the mitochondrial membrane in bacterial cells. They are generally regarded as safe (GRAS) compounds and hence are allowed to be added to the food as preservatives. Due to their antimicrobial properties, isothiocyanates incorporated food packaging films have become popular in the last decade. They are known to act as substrates to activate lactoperoxidases (LPO) for extension of shelf life of dairy products due to its bactericidal and bacteriostatic properties. This review addresses the detailed evidences supporting the biological activities, bioavailability and stability, methods of extraction, and explanation for their taste perception, as well as utilization of these isothiocyanates in food packaging as natural antimicrobials or natural preservatives to improve shelf life of foodstuffs.
... Freezing at -20°C increased the ITCs formation in broccoli sprouts . Heating at 60°C prior to homogenisation improved the sulforaphane formation but decreased sulforaphane nitrile formation in broccoli florets (Matusheski et al., 2004a). It has been observed that there was higher epithiospecifier protein (ESP) activity resulted in the formation of nitrile during GLs hydrolysis, while there was a decrease in the formation of ITCs . ...
Article
The present work investigated the effects of soaking followed by heating on the metabolism of glucosinolates (GLs) in rocket seeds and sprouts. Soaking for 1 h increased myrosinases (MYR) activity of seeds. Heating at 70°C for 10 min after soaking resulted in the highest isothiocyanates (ITCs) formation in seeds. Both immerse-heating and steam-heating decreased the total GLs content in seeds and 2-day old sprouts. However, steam-heating showed minor effect on total GLs content when compared with immerse-heating. Both methods showed a decreased MYR activity in seeds and sprouts. Nevertheless, heating had a positive impact on ITCs formation. Under immerse-heating, ITCs formation was the highest in seeds and sprouts at 70°C heating. However, under steam-heating, ITCs formation in seeds and sprouts was higher at 70 and 60°C, respectively. The results indicated that steam-heating treatment was effective for improving ITCs formation in rocket seeds and 2-day old sprouts.
... Especially, broccoli seeds contain the highest glucoraphanin; hence, they are a good source of preparation of sulforaphane. However, the conversion rate of glucoraphanin to sulforaphane is very low due to the presence of epithiospecifier protein (ESP) [11]. It has been reported that the heat sensitivity of ESP protein is higher than that of myrosinase. ...
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In this study, microwave pretreatment and grinding treatment were used to enhance sulforaphane formation, then ultrasonic-assisted extraction (UAE) was applied to extract sulforaphane using simultaneous hydrolysis and extraction method. The effects of various parameters, which were ultrasonic time,ultrasonic power, solid-water ratio and solid-ethyl acetate ratio on the extraction rate of sulforaphane were investigated. The results showed that microwave pretreatment enhanced sulforaphane formation. Excessive size reduction did not increase or even reduced extraction rate of sulforaphane. Simultaneous hydrolysis and extraction significantly increased extraction rate of sulforaphane compared to hydrolysis followed by extraction. UAE accelerated mass transfer and the solubilization of the targeted compounds due to the acoustic cavitation effect, thus enhanced enzymatic hydrolysis of glucoraphanin and the extraction rate of sulforaphane. The extraction rate of sulforaphane using UAE with simultaneous hydrolysis and extraction was 4.07-fold of the conventional extraction method. UAE was an effective method to extract sulforaphane from broccoli seeds since it led to higher yield of sulforaphane in a much shorter extraction time.
... The presence of epithiospecifier protein (ESP) disrupts the process of glucoraphanin hydrolysis, reducing the bioavailability of sulforaphane and sulforaphane nitrile, with the nitrile form being less active in its binding to pharmacological targets [102] . Since ESP is temperature insensitive, heating the broccoli at 60 ºC decreases the formation of sulforaphane nitrile [103] . Once absorbed, sulforaphane is conjugated with glutathione and metabolized by the meracaptopurine pathway; it is then excreted as N-acetylcysteine conjugates [104] . ...
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The unending morbidity and mortality that results from cancer, as well as adverse reactions due to chemotherapy and the enormous economic burden of treatment and hospitalization, advocates for the necessity of chemopreventive measures. Cancer chemoprevention refers to the use of agents capable of reversing, reducing, or slowing down the pathology of cancer at various stages. Fortunately, a few therapeutic drugs with relatively low toxicity (e.g., tamoxifen, finasteride), and a sparse number of vaccines (hepatitis B, HPV), are used to prevent specific cancers. In the general population, however, therapeutic options for cancer prevention are not common. Nonetheless, it is generally agreed that diet affects the genesis of cancer, and phytochemicals have the capacity of functioning as cancer chemoprevention agents. This is supported by epidemiological studies and clearly documented with animal models designed to mimic human carcinogenesis. Additionally, some public health strategies, such as recommendations for greater consumption of fruits and vegetables, reflect the merits of cancer chemoprevention. Here, we focus on some well-established natural product cancer chemopreventive agents, including resveratrol (grapes), epigallocatechin-3-gallate (green tea), sulforaphane (cruciferous vegetables), anthocyanins (grapes and berries), curcumin (turmeric), silibinin (milk thistle), and lycopene (tomatoes). As aptly demonstrated by genomic analysis and other methods, the mechanistic underpinning is variable and complex. In addition, responses may be mediated through indirect mechanisms, such as interaction with the microbiome. Furthermore, ancillary applications of chemopreventive agents are worthy of consideration, such as management of sequelae induced by chemotherapy. Recognizing the loss of millions of cancer patients every year, it is obvious that negating malignant metastatic conditions remains of paramount importance. In meeting this objective, cancer chemoprevention offers great promise.
Chapter
The importance of sulphur-containing natural products in primary and secondary metabolism. The role of hydrogen sulphide as a gasotransmitter and its uptake by living systems. The relevance of mustard oil glycosides (glucosinolates) and their enzymic breakdown products. Sulphur compounds of the genus Allium. Different approaches to the production of multiple sulphur compounds by Allium and Brassica species and its implications. The role of sulphur-containing natural products in chronic disease and their impact upon complex biological pathways. Inductive reasoning and natural products. Other potential bioactive compounds in Brassica species. Glucosinolates and their breakdown products and plant pathogenic fungal disease. Dithiocarbamates as potential storage forms of hydrogen sulphide. The importance of extra-oral bitter receptors. Natural products, reductionism, biological activity and statins. The weakness of nutritional epidemiology in the investigation of the effects of natural products.
Chapter
Bladder cancer risk and prevention are motivated by epidemiological studies that elucidated and quantified the potential roles of tobacco, geographic location, fluids, and dietary components on the risk of bladder cancer [1]. Among fruits and vegetables, Brassica vegetables emerged with significant associations with reduced bladder cancer risk. Carcinoma of the urinary bladder presents a considerable health care burden. In the United States, bladder cancer is the sixth most common cancer and the eighth most common cause of cancer death in men [2]. It is the second most prevalent neoplasm in men 60 years or older [3]. Worldwide, almost 400,000 new cases are diagnosed annually, with approximately 150,000 deaths [4]. Bladder cancer recurrence rate is the highest among all malignancies, and it has the highest lifetime treatment costs per patient of all cancers due to its high recurrence rate and ongoing invasive monitoring requirements [5].
Chapter
The importance of this class of vegetables lies not only in their contribution to the vitamin and mineral components of the human diet but also in their beneficial effect on human health, which reflects the action of the glucosinolates, a group of secondary metabolites almost exclusively associated with this plant family. The aliphatic glucosinolates have attracted scientific attention, particularly for the anti-carcinogen isothiocyanate sulforaphane, the major breakdown product of glucoraphanin in broccoli.
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Plants of the Brassicaceae family exhibit a sophisticated defense mechanism against plant pathogens and insect herbivores, leading to the production of diverse hydrolytic derivatives such as thiocyanates, isothiocyanates, nitriles, and epithionitriles. These chemical transformations are catalyzed through the enzymatic action of myrosinase on glucosinolates (GSLs), which are otherwise inactive in the intact plant. The hydrolytic products of GSLs are incredibly effective at protecting plants from pests, weeds, herbivores, bacterial and fungal attack. The impact of GSLs on animal health is multifaceted, with nutritional attributes varying based on chemical structure and concentration. While certain GSLs and their derivatives in Brassica plants are associated with health benefits and cancer prevention, others have been linked to goitrogenic effects in ruminants, with concentrations ranging from 50 to 120 μmol g⁻¹. This variability has implications for the use of rapeseed–mustard meal in food and feed. Despite considerable efforts to modify the chemical profile of GSLs in Brassica species, a comprehensive understanding of their composition and metabolism is still essential for achieving effective and targeted manipulation. This review aims to consolidate both foundational and recent advancements in GSLs research, with a focus on their biological properties, role in plant-pathogen interactions, transport and strategies for managing their accumulation in Brassica seeds. Our goal is to enhance the utilization of this crop by maximizing its potential benefits. This information could be pivotal in developing new varieties with tailored GSLs content and profile, resulting in improved crop yields, enhanced nutritional quality, and greater biofumigation efficacy, thereby benefiting both agriculture and human health.
Article
This study investigated the compositional changes in broccoli (Brassica oleracea L. var. italica) and cauliflower (Brassica oleracea L. var. botrytis) landraces during a four-year breeding program in organic farming, focusing on key bioactive compounds linked to value for cultivation and use (VCU). Our results revealed that many metabolites, notably ascorbic acid, nitriles, sulphides, and total phenols, significantly responded to environmental differences, particularly higher temperatures, and reduced rainfall during plant development. Other traits, like sugar levels, sulforaphane, indole-3-carbinol, and pigments showed stable genetic control and limited variation. This work provides valuable insights for the development of innovative and high VCU material tailored to organic cropping systems and potentially leading to improved final product quality.
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Microgreens contain an abundance of antioxidants (glucosinolates, carotenoids, and phenolic compounds), vitamins (C, E, and K), minerals (iron and magnesium), and other nutrients (fibre and omega‐3 fatty acids). They have been shown to promote cardiovascular health, reduce inflammation, combat cancer, manage diabetes, protect the nervous system, and improve digestion. This review investigates the antioxidant content and health implications of microgreens, providing current insights and prospective outlooks. The types of antioxidants found in microgreens, as well as the factors that influence their content, such as species, growing conditions, and harvesting time, are discussed. The review highlights antioxidants' role in cardiovascular health, inflammation reduction, cancer defence, diabetes management, and nervous system protection. The antioxidant properties of microgreen varieties are discussed. The bioavailability of antioxidants from microgreens, culinary applications, and incorporation into the diet are elaborated. The section of future perspectives covers the commercial potential of microgreens, optimal harvest timing, research directions, and potential functional food and nutraceutical uses. The aim and scope of this review are to provide a comprehensive understanding of the antioxidant content in microgreens and their associated health benefits. It also seeks to identify gaps in current research and suggest avenues for future studies, especially concerning their relevance in human health and nutrition.
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Sulforaphane (SFN) is an isothiocyanate commonly found in cruciferous vegetables. It is formed via the enzymatic hydrolysis of glucoraphanin by myrosinase. SFN exerts various biological effects, including anti-cancer, anti-oxidation, anti-obesity, and anti-inflammatory effects, and is widely used in functional foods and clinical medicine. However, the structure of SFN is unstable and easily degradable, and its production is easily affected by temperature, pH, and enzyme activity, which limit its application. Hence, several studies are investigating its physicochemical properties, stability, and biological activity to identify methods to increase its content. This article provides a comprehensive review of the plant sources, extraction and analysis techniques, in vitro and in vivo biological activities, and bioavailability of SFN. This article highlights the importance and provides a reference for the research and application of SFN in the future.
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Broccoli sprouts have a strong ability to accumulate isothiocyanate and selenium. In this study, the isothiocyanate content increased significantly as a result of ZnSO4 stress. Particularly, based on the isothiocyanate content is not affected, the combined ZnSO4 and Na2SeO3 treatment alleviated the inhibition of ZnSO4 and induced selenium content. Gene transcription and protein expression analyses revealed the changes in isothiocyanate and selenium metabolite levels in broccoli sprouts. ZnSO4 combined with Na2SeO3 was proven to activate a series of isothiocyanate metabolite genes (UGT74B1, OX1, and ST5b) and selenium metabolite genes (BoSultr1;1, BoCOQ5-2, and BoHMT1). The relative abundance of the total 317 and 203 proteins, respectively, in 4-day-old broccoli sprouts varied, and the metabolic and biosynthetic pathways for secondary metabolites were significantly enriched in ZnSO4/control and ZnSO4 combined Na2SeO3/ZnSO4 comparisons. The findings demonstrated how ZnSO4 combined with Na2SeO3 treatment reduced stress inhibition and the accumulation of encouraged selenium and isothiocyanates during the growth of broccoli sprouts.
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The health-beneficial effects of Brassica vegetables are mainly attributed to their high contents of glucosinolates and the products of their enzymatic hydrolysis, especially isothiocyanates. Distribution of glucosinolates across plant organs can strongly vary. Consequently, the effect of leaf age on glucosinolate accumulation as well as hydrolysis was investigated in two leafy Brassica vegetables, pak choi and giant red mustard. Furthermore, activity of the hydrolyzing enzyme, myrosinase, was evaluated across the leaves. Additionally, a possible glucosinolate transport from older to younger leaves was monitored. Young leaves of both species contained of more glucosinolates than old ones. Accordingly, more isothiocyanates were released upon glucosinolate hydrolysis in young leaves. Myrosinases fully hydrolyzed the whole glucosinolates regardless of the leaf age. It was confirmed that older leaves can supply younger leaves with glucosinolates. The results can help to improve the health-beneficial value of Brassicas in the diet by an increased formation of isothiocyanates.
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Broccoli sprouts have been considered as functional foods which have received increasing attention because they have been highly prized for glucosinolates, phenolics, and vitamins in particular glucosinolates. One of hydrolysates-sulforaphane from glucoraphanin is positively associated with the attenuation of inflammatory, which could reduce diabetes, cardiovascular and cancer risk. In recent decades, the great interest in natural bioactive components especially for sulforaphane promotes numerous researchers to investigate the methods to enhance glucoraphanin levels in broccoli sprouts and evaluate the immunomodulatory activities of sulforaphane. Therefore, glucosinolates profiles are different in broccoli sprouts varied with genotypes and inducers. Physicochemical, biological elicitors, and storage conditions were widely studied to promote the accumulation of glucosinolates and sulforaphane in broccoli sprouts. These inducers would stimulate the biosynthesis pathway gene expression and enzyme activities of glucosinolates and sulforaphane to increase the concentration in broccoli sprouts. The immunomodulatory activity of sulforaphane was summarized to be a new therapy for diseases with immune dysregulation. The perspective of this review served as a potential reference for customers and industries by application of broccoli sprouts as a functional food and clinical medicine.
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Sulforaphane is a bioactive metabolite with anti-inflammatory activity and is derived from the glucosinolate glucoraphanin, which is highly abundant in broccoli sprouts. However, due to its inherent instability its use as a therapeutic against inflammatory diseases has been limited. There are few studies to investigate a whole food approach to increase sulforaphane levels with therapeutic effect and reduce inflammation. In the current study, using a mouse model of inflammatory bowel disease, we investigated the ability of steamed broccoli sprouts to ameliorate colitis and the role of the gut microbiota in mediating any effects. We observed that despite inactivation of the plant myrosinase enzyme responsible for the generation of sulforaphane via steaming, measurable levels of sulforaphane were detectable in the colon tissue and feces of mice after ingestion of steamed broccoli sprouts. In addition, this preparation of broccoli sprouts was also capable of reducing chemically-induced colitis. This protective effect was dependent on the presence of an intact microbiota, highlighting an important role for the gut microbiota in the metabolism of cruciferous vegetables to generate bioactive metabolites and promote their anti-inflammatory effects.
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Current clinical management of major mental disorders, such as autism spectrum disorder, depression and schizophrenia, is less than optimal. Recent scientific advances have indicated that deficits in oxidative and inflammation systems are extensively involved in the pathogenesis of these disorders. These findings have led to expanded considerations for treatment. Sulforaphane (SFN) is a dietary phytochemical extracted from cruciferous vegetables. It is an effective activator of the transcription factor nuclear erythroid-2 like factor-2, which can upregulate multiple antioxidants and protect neurons against various oxidative damages. On the other hand, it can also significantly reduce inflammatory response to pathological states and decrease the damage caused by the immune response via the nuclear factor-κB pathway and other pathways. In this review, we introduce the biological mechanisms of SFN and the pilot evidence from its clinical trials of major mental disorders, hoping to promote an increase in psychiatric clinical studies of SFN.
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The myrosinase-glucosinolate system is involved in a range of biological activities affecting herbivorous insects, plants and fungi. The system characteristic of the order Capparales includes sulphur-containing substrates, the degradative enzymes myrosinases, and cofactors. The enzyme-catalyzed hydrolysis of glucosinolates initially involves cleavage of the thioglucoside linkage, yielding D-glucose and an unstable thiohydroximate-Ο-sulphonate that spontaneously rearranges, resulting in the production of sulphate and one of a wide range of possible reaction products. The products are generally a thiocyanate, isothiocyanate or nitrile, depending on factors such as substrate, pH or availability of ferrous ions. Glucosinolates in crucifers exemplify components that are often present in food and feed plants and are a major problem in the utilization of products from the plants. Toxic degradation products restrict the use of cultivated plants, e.g. those belonging to the Brassicaceae. The myrosinase-glucosinolate system may, however, have several functions in the plant. The glucosinolate degradation products are involved in defence against insects and phytopathogens, and potentially in sulphur and nitrogen metabolism and growth regulation. The compartmentalization of the components of the myrosinase-glucosinolate system and the cell-specific expression of the myrosinase represents a unique plant defence system. In this review, we summarize earlier results and discuss the organisation and biochemistry of the myrosinase-glucosinolate system.
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Ten broccoli [Brassica oleracea L. (Botrytis Group)] accessions were grown in several environments to estimate glucosinolate (GS) variability associated with genotype, environment, and genotype × environment interaction and to identify differences in the stability of GSs in broccoli florets. Significant differences in genetic variability were identified for aliphatic GSs but not for indolyl GSs. The percentage of GS variability attributable to genotype for individual aliphatic compounds ranged from 54.2% for glucoraphanin to 71.0% for progoitrin. For total indolyl GSs, the percentage of variability attributable to genotype was only 12%. Both qualitative and quantitative differences in GSs were detected among the genotypes. Ten-fold differences in progoitrin, glucoraphanin, and total aliphatic GS levels were observed between the highest and lowest genotypes. Only two lines, Eu8-1 and VI-158, produced aliphatic GSs other than glucoraphanin in appreciable amounts. Differences in stability of these compounds among the cultivars were also observed between fall and spring plantings. Results suggest that genetic factors necessary for altering the qualitative and quantitative aliphatic GS profiles are present within existing broccoli germplasm, which makes breeding for enhanced cancer chemoprotectant activity feasible.
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The putative anticarcinogenic activity of Brassica vegetables has been associated with the presence of certain glucosinolates. 4-Methylsulphinylbutyl isothiocyanate (sulphoraphane), derived from the corresponding glucosinolate found in broccoli, has previously been identified as a potent inducer of the anticarcinogenic marker enzyme quinone reductase [NADP(H):quinone-acceptor oxidoreductase] in murine hepatoma Hepa 1c1c7 cells. We have therefore produced a broccoli hybrid with increased levels of this anticarcinogenic glucosinolate and tested the ability of extracts to induce quinone reductase. A 10-fold increase in the level of 4-methylsulphinylbutyl glucosinolate was obtained by crossing broccoli cultivars with selected wild taxa of the Brassica oleracea (chromosome number, n = 9) complex. Tissue from these hybrids exhibited a >100-fold increase in the ability to induce quinone reductase in Hepa 1c1c7 cells over broccoli cultivars, due to both an increase in 4-methylsulphinylbutyl glucosinolate content and increased percentage conversion to sulphoraphane.
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Sulforaphane is an isothiocyanate that is present naturally in widely consumed vegetables and has a particularly high concentration in broccoli. This compound has been shown to block the formation of tumors initiated by chemicals in the rat. Although sulforaphane has been proposed to modulate the metabolism of carcinogens, its mechanism of action remains poorly understood. We have previously demonstrated that sulforaphane inhibits the reinitiation of growth and decreases the cellular viability of quiescent human colon carcinoma cells (HT29). Moreover, the weak effect observed on differentiated CaCo2 cells suggests a specific anticancer activity for this compound. Here we investigated the effect of sulforaphane on the growth and viability of HT29 cells during their exponentially growing phase. We observed that sulforaphane induced a cell cycle arrest in a dose-dependent manner, followed by cell death. This sulforaphane-induced cell cycle arrest was correlated with an increased expression of cyclins A and B1. Moreover, we clearly demonstrated that sulforaphane induced cell death via an apoptotic process. Indeed, a large proportion of treated cells display the following: (a) translocation of phosphatidylserine from the inner layer to the outer layer of the plasma membrane; (b) typical chromatin condensation; and (c) ultrastructural modifications related to apoptotic cell death. We also showed that the expression of p53 was not changed in sulforaphane-treated cells. In contrast, whereas bcl-2 was not detected, we observed increased expression of the proapoptotic protein bax, the release of cytochrome c from the mitochondria to the cytosol, and the proteolytic cleavage of poly(ADP-ribose) polymerase. In conclusion, our results strongly suggest that in addition to the activation of detoxifying enzymes, induction of apoptosis is also involved in the sulforaphane-associated chemoprevention of cancer.
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Glucosinolates are anionic thioglucosides that have become one of the most frequently studied groups of defensive metabolites in plants. When tissue damage occurs, the thioglucoside linkage is hydrolyzed by enzymes known as myrosinases, resulting in the formation of a variety of products that are active against herbivores and pathogens. In an effort to learn more about the molecular genetic and biochemical regulation of glucosinolate hydrolysis product formation, we analyzed leaf samples of 122 Arabidopsis ecotypes. A distinct polymorphism was observed with all ecotypes producing primarily isothiocyanates or primarily nitriles. The ecotypes Columbia (Col) and Landsberg erecta (Ler) differed in their hydrolysis products; therefore, the Col x Ler recombinant inbred lines were used for mapping the genes controlling this polymorphism. The major quantitative trait locus (QTL) affecting nitrile versus isothiocyanate formation was found very close to a gene encoding a homolog of a Brassica napus epithiospecifier protein (ESP), which causes the formation of epithionitriles instead of isothiocyanates during glucosinolate hydrolysis in the seeds of certain Brassicaceae. The heterologously expressed Arabidopsis ESP was able to convert glucosinolates both to epithionitriles and to simple nitriles in the presence of myrosinase, and thus it was more versatile than previously described ESPs. The role of ESP in plant defense is uncertain, because the generalist herbivore Trichoplusia ni (the cabbage looper) was found to feed more readily on nitrile-producing than on isothiocyanate-producing Arabidopsis. However, isothiocyanates are frequently used as recognition cues by specialist herbivores, and so the formation of nitriles instead of isothiocyanates may allow Arabidopsis to be less apparent to specialists.
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Glucoraphanin in Brassica vegetables breaks down to either sulforaphane or sulforaphane nitrile depending on the conditions, and sulforaphane can be further conjugated with glutathione. Using a high-throughput microtitre plate assay and TaqMan real time quantitative RT-PCR to measure mRNA, we show that sulforaphane and its glutathione conjugate, but not the nitrile, increased significantly (P < 0.05) both UGT1A1 and GSTA1 mRNA levels in HepG2 and HT29 cells. These changes were accompanied by an increase in UGT1A1 protein, as assessed by immunoblotting, and a 2-8-fold increase in bilirubin glucuronidation. When treated together, the nitrile derivative did not affect sulforaphane induction. The induction of UGT1A1 and GSTA1 mRNA by sulforaphane was time and concentration dependent. The results show a functional induction of glucuronidation by sulforaphane but not sulforaphane nitrile, and show that the pathway of metabolism of glucosinolates in Brassica vegetables is important in determining the resulting biological and anticarcinogenic activities.
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For the first time the human intestinal effective permeability, estimated from the luminal disappearance and intestinal metabolism of phytochemicals, sulforaphane and quercetin-3,4'-glucoside, as well as the simultaneous changes in gene expression in vivo in enterocytes, has been studied in the human jejunum in vivo (Loc-I-Gut). Both compounds as components of an onion and broccoli extract could readily permeate the enterocytes in the perfused jejunal segment. At the physiologically relevant, dietary concentration tested, the average effective jejunal permeability (Peff) and percentage absorbed (+/- S.D.) were 18.7 +/- 12.6 x 10-4 cm/s and 74 +/- 29% for sulforaphane and 8.9 +/- 7.1 x 10-4 cm/s and 60 +/- 31% for quercetin-3,4'-diglucoside, respectively. Furthermore, a proportion of each compound was conjugated and excreted back into the lumen as sulforaphane-glutathione and quercetin-3'-glucuronide. The capacity of the isolated segment to deconjugate quercetin from quercetin-3,4'-diglucoside during the perfusion was much higher than the beta-glucosidase activity of the preperfusion jejunal contents, indicating that the majority (79-100%) of the beta-glucosidase capacity derives from the enterocytes in situ. Simultaneously, we determined short-term changes in gene expression in exfoliated enterocytes, which showed 2.0 +/- 0.4-fold induction of glutathione transferase A1 (GSTA1) mRNA (p < 0.002) and 2.4 +/- 1.2-fold induction of UDP-glucuronosyl transferase 1A1 (UGT1A1) mRNA (p < 0.02). The changes in gene expression were also seen in differentiated Caco-2 cells, where sulforaphane was responsible for induction of GSTA1 and quercetin for induction of UGT1A1. These results show that food components have the potential to modify drug metabolism in the human enterocyte in vivo very rapidly.
Article
Broccoli (Brassica oleracea L., Italica Group) has been recognized as a source of glucosinolates and their isothiocyanate metabolites that may be chemoprotective against human cancer. A predominant glucosinolate of broccoli is glucoraphanin and its cognate isothiocyanate is sulforaphane. Sulforaphane has been shown to be a potent inducer of mammalian detoxication (Phase 2) enzyme activity and to inhibit chemical-induced tumorigenesis in animal models. Little is known about phenotypic variation in broccoli germplasm for Phase 2 enzyme (e.g., quinone reductase) induction potential. Thus, this study was undertaken to evaluate: 1) quinone reductase induction potential (QRIP) diversity among a population of broccoli inbreds; 2) QRIP levels in selected lines; 3) correlation of QRIP with other horticultural characteristics; and 4) QRIP expression in a sample of synthesized hybrids. In 1996, 71 inbreds and five hybrid checks (all field-grown), ranged from a QRIP of nearly zero to 150,000 units/g fresh weight (FW) (mean of 34,020 units/g FW). These values were highly correlated with methylsulphinylalkyl glucosinolate (MSAG; primarily glucoraphanin) concentrations that ranged from 0.04 to 2.94 μmol·g-1 FW. A select subset of lines evaluated in 1996 were reevaluated in 1997. QRIP and MSAG values in this second year were similar to and correlated with those observed in 1996 (r = 0.73, P < 0.0001 and r = 0.79, P < 0.0001, respectively). In addition, both QRIP and MSAG concentration were highly correlated with days from transplant to harvest. Average F1 hybrid values for QRIP and MSAG in 1997 fell typically between their parental means, but were often closer to the mean of the low parent. Results of this study indicate that divergent QRIP expression can effectively be used to select enhanced inbred lines to use in development of value-added hybrids. Evidence is also provided that there is a significant genetic component to both QRIP and MSAG concentration, and that selection for either one may provide an effective means for developing broccoli hybrids with enhanced chemoprotective attributes. Chemical names used: 4-methylsulphinylbutyl glucosinolate (glucoraphanin) and 4-methylsulphinylbutyl isothiocyanate (sulforaphane).
Article
The putative anticarcinogenic activity of Brassica vegetables has been associated with the presence of certain glucosinolates. 4-Methylsulphinylbutyl isothiocyanate (sulphoraphane), derived from the corresponding glucosinolate found in broccoli, has previously been identified as a potent inducer of the anticarcinogenic marker enzyme quinone reductase [NADP(H):quinone-acceptor oxidoreductase] in murine hepatoma Hepa lclc7 cells. We have therefore produced a broccoli hybrid with increased levels of this anticarcinogenic glucosinolate and tested the ability of extracts to induce quinone reductase. A 10-fold increase in the level of 4-methylsulphinylbutyl glucosinolate was obtained by crossing broccoli cultivars with selected wild taxa of the Brassica oleracea (chromosome number, n = 9) complex. Tissue from these hybrids exhibited a >100-fold increase in the ability to induce quinone reductase in Hepa lclc7 cells over broccoli cultivars, due to both an increase in 4-methylsulphinylbutyl glucosinolate content and increased percentage conversion to sulphoraphane.
Article
A new hydrolysis product derived from 3-butenylglucosinolate in seeds of certain strains of Brassica campestris Yellow Sarson is described. The structure, 1-cyano-3,4-epithiobutane is proposed. If the seeds are heated at 115° for 30 min before hydrolysis, 3-butenyl isothiocyanate is the main product.
Article
Nasturtium officinale contains four glucosinolates, the major representative being 2-phenethylglucosinolate. On autolysis of seeds or leaves, isothiocyanates were the main products of glucosinolate degradation but no thiocyanate was detected. The application of heat during extraction caused an increase in nitrile formation to dominance over isothiocyanates. A (benzyl) thiocyanate-forming extract of Lepidium sativum seeds did not provoke generation of any thiocyanate from glucosinolates of N. officinale (or Barbarea praecox), but it did impose accentuated nitrile-forming properties on the systems. The conclusion is reached that some glucosinolate-containing Cruciferae are predominantly nitrile-producing and some predominantly isothiocyanate-producing, all other factors being constant.
Article
Lepidium sativum seeds were dry heated at 125° for varying periods, and also for 30 min at various temperatures. Autolysates were then analysed for benzylglucosinolate degradation products. Whilst heating for 4 hr 20 min at 125° was sufficient to prevent formation of benzyl thiocyanate, just over 7.5 hr at 125° was required before benzyl isothiocyanate also ceased to be produced. This indicates the presence of a discrete, thiocyanate-forming factor in L. sativum seeds, separate from thioglucosidase. After 7.5 hr at 125°, benzyl cyanide continued to be formed, proving that it can be obtained (in relatively small amounts) directly from the glucosinolate even without the influence of any thioglucosidase. In general, isothiocyanate was the more favoured product of glucosinolate degradation following heat treatment of seeds, until the point of thioglucosidase inactivation was approached when nitrile formation took over. It is suggested that the thiocyanate-forming factor is an isomerase causing Z-E isomerization of the glucosinolate aglucone, but that only those glucosinolates capable of forming particularly stable cations are then able to undergo E-aglucone rearrangement to thiocyanate.
Article
Air-dried leaves of Farsetia aegyptia and F. ramosissima have been analysed for their glucosinolates; the former was shown to contain at least six but chiefly allylglucosinolate, whilst the latter contains at least five but mainly but-3-enylglucosinolate with some 4-(methylthio)butylglucosinolate. Without the addition of extraneous thioglucosidase enzyme, both species gave predominantly nitrile degradation products of glucosinolates; but if extra enzyme were added, corresponding isothiocyanates became the major products instead. Varying the pH from the natural level for the plant also considerably affected the ratios of glucosinolate products.
Article
Glucosinolates were evaluated in 5 groups and 65 accessions of Brassica oleracea (50 broccoli, 4 Brussels sprouts, 6 cabbage, 3 cauliflower, and 2 kale) grown under uniform cultural conditions. Glucosinolates and their concentrations varied among the different groups and within each group. The predominant glucosinolates in broccoli were 4-methylsulfinylbutyl glucosinolate (glucoraphanin), 3-butenyl glucosinolate (gluconapin), and 3-indolylmethyl glucosinoate (glucobrassicin). Glucoraphanin concentration in broccoli ranged from 0.8 mu mol g(-1) DW in EV6-1 to 21.7 mu mol g(-1) DW in Brigadier. Concentrations of the other glucosinolates in broccoli varied similarly over a wide range. In Brussels sprouts, cabbage, cauliflower, and kale, the predominant glucosinolates were sinigrin (8.9, 7.8, 9.3, and 10.4 mu mol g(-1) DW, respectively) and glucobrassicin (3.2, 0.9, 1.3, and 1.2 mu mol g(-1) DW, respectively). Brussels sprouts also had significant amounts of gluconapin (6.9 mu mol g(-1) DW). Wide variations in glucosinolate content among genotypes suggest differences in their health-promoting properties and the opportunity for enhancement of their levels through genetic manipulation.
Article
Epithiospecifier protein (ESP) activity was determined in the seeds of two cultivars of Brassica napus, in B. campestris and in Lepidium sativum. All four types of seeds contained susceptible substrates for ESP (that is, glucosinolates with terminal unsaturation in their side-chain), although L. sativum contained only a very small amount of one. Results suggest that Fe2+ is essential for ESP activity, but its presence certainly promoted the effects of ESP to a considerable extent, and even at a very low level (e.g. 6 × 10−11 mol Fe2+. Further evidence was gained for the intramolecular nature of the reaction which results in cyanoepithioalkane formation.
Article
An active thioglucoside glucohydrolase extract was prepared from commercial mustard powder and its effect on the degradation of two pure glucosinolates was investigated. During reaction in a distilled water medium the pH of the solution decreased markedly and the ratio of products (isothiocyanate and nitrile) varied considerably. After 20–30 min, when the pH had fallen to ca 5.6, isothiocyanate production ceased whilst nitrile continued to be produced and in amounts which increased linearly with time for at least 40 min. This behaviour can be correlated with the changing pH of the medium. In controlled pH experiments it was confirmed that nitrile formation is favoured at lower pH levels and that the ratio of nitrile to isothiocyanate is directly related to the hydrogen ion concentration of the medium. No reason could therefore be found for the observed formation of nitrile in some natural systems at pHs greater than 7.
Article
Cabbage seeds contain 5 glucosinolates and on autolysis produce, in particular, 1-cyano-2-3-epithiopropane. Watercress seeds contain 4 glucosinolates, but none capable of forming a cyanoepithioalkane. Cabbage exhibited behaviour commensurate with possession of an epithiospecifier protein (ESP) whilst watercress did not show any such activity.
Article
An epithiospecifier protein present in turnip tissue gives rise to 1-cyano-epithioalkanes during autolysis. Volatile hydrolysis products are produced from glucosinolates during autolysis of seeds, seedlings and plant tissue more than 6 weeks after sowing.
Article
The effects of pH on the enzymic degradation of benzylglucosinolate in Lepidium sativum seed autolysates were investigated both with and without addition of the enzyme co-factor ascorbic acid. Benzyl cyanide, isothiocyanate, thiocyanate and alcohol were identified in autolysates, although only traces of the alcohol were obtained. The nitrile was always the major product (80% of total glucosinolate products) even at pH 8 and 9 when the usually accepted, proton-dependent mechanism of nitrile production cannot be operative. Thiocyanate was always the second most abundant product. In the absence of added ascorbate, isothiocyanate production decreased with increasing pH, again contrary to accepted theory. L. sativum seeds thus constitute an inherently nitrile-producing system which exhibits ‘anomalous’ glucosinolate degradation. In the absence of added ascorbate, thiocyanate was the only product which was formed in approximately constant amounts, whatever the pH, so its mechanism of production is not necessarily pH-dependent. The presence of added ascorbate in general promoted enzyme activity and showed a maximum effect at ca pH 5, although minimum isothiocyanate formation was observed at that pH. At pH 4 and below, there was less glucosinolate degradation in the presence of added ascorbate than in its absence, and the conclusion is reached that at relatively high acidities the enzyme co-factor behaves as an inhibitor.
Article
Epi-progoitrin (I), the principal thioglucoside of crambe seed, has at least two patterns of degradation in wetted seed meal. Under some conditions, the product is a "cyano" fraction (IV) consisting of (S)-1-cyano-2-hydroxy-3-butene (III) and unknown substances containing sulfur, instead of the expected (R)-goitrin (II). Formation of II is increased by diluting the meal with water, by increasing the temperature, by raising the pH, by dry heating of the seed meal, or by storage of the seed under ambient conditions (compared with cold storage). Under conditions of test, enzyme hydrolysis to form IV only is faster than when II is also formed. Tests with Brassica napus (rapeseed) show a similar phenomenon in seed from this related plant.
Article
An extraction and preparative HPLC method has been devised to simultaneously purify sulforaphane and sulforaphane nitrile from the seed of Brassica oleracea var. italica cv. Brigadier. The seed was defatted with hexane, dried, and hydrolyzed in deionized water (1:9) for 8 h. The hydrolyzed seed meal was salted and extracted with methylene chloride. The dried residue was redissolved in a 5% acetonitrile solution and washed with excess hexane to remove nonpolar contaminants. The aqueous phase was filtered through a 0.22-μm cellulose filter and separated by HPLC using a Waters Prep Nova-Pak HR C-18 reverse-phase column. Refractive index was used to detect sulforaphane nitrile, and absorbance at 254 nm was used to detect sulforaphane. Peak identification was confirmed using gas chromatography and electron-impact mass spectrometry. Each kilogram of extracted seed yielded approximately 4.8 g of sulforaphane and 3.8 g of sulforaphane nitrile. Standard curves were developed using the purified compounds to allow quantification of sulforaphane and sulforaphane nitrile in broccoli tissue using a rapid GC method. The methodology was used to compare sulforaphane and sulforaphane nitrile content of autolyzed samples of several broccoli varieties. Keywords: Brassica oleracea; glucosinolates; broccoli; sulforaphane; sulforaphane nitrile; isothiocyanate; HPLC
Article
Three major glucosinolates in broccoli, i.e., glucoiberin, glucoraphanin, and glucobrassicin were greatly reduced by both water and steam blanching. Water blanching produced the most significant glucosinolate loss. Blanched brussels sprouts did not exhibit this significant reduction of glucosinolates. This inhibition of glucosinolate loss probably is due to the physical configuration of brussels sprouts, i.e., a tight, compact vegetable that is more resistant to the leaching effects of blanching compared to the loose structure of broccoli. Thus, flavor and the numerous physiological changes and attributes associated with glucosinolates are retained to a much greater degree in blanched brussels sprouts than in blanched broccoli.
Article
Changes in the volatile chemicals produced on autolysis were monitored during the development of cauliflower, Chinese cabbage, fodder rape and radish. Compounds were identified from gas-liquid chromatography retention times, and mass spectrometry, seven of the mass spectrographs not having previously been recorded. Of the 23 compounds identified overall, 19 were extracted from seeds which, whether aged or freshly harvested, contained the maximum concentration of volatiles encountered. A rapid decrease in the concentrations of volatile hydrolysis products occurred during the first 4 weeks of development. Thereafter a steady increase and change in compounds was recorded, usually related to biologically significant events. The enzymic production of these volatile chemicals is discussed with respect to the action of thioglucosidase and epithiospecifier protein.
Article
Our objective was to determine whether steam blanching, storage and preparation affected concentrations of sulforaphane (SF), sulforaphane nitrile (SFN), cyanohydroxybutene (CHB), iberin (I) or iberin nitrile (IN) in fresh and frozen broccoli. Broccoli (var. “Arcadia”) was grown in St. Charles, IL over three seasons. Samples were steam blanched (2 min at 93 ± 5°C) within 24h of harvest, frozen and stored at −20°C up to 90 days, and fresh broccoli was stored at 4°C up to 21 days. Samples were analyzed uncooked or microwave cooked. SF, SFN, I, IN and CHB were determined by GC in dichloromethane extracts from lyophilized samples. Rates of loss for CHB and SF were similar during storage of fresh broccoli. Blanching, storage, and microwave cooking decreased (p < 0.01) concentrations of each compound in fresh and frozen broccoli.
Article
Glucosinolates from seventy-nine 8-week-old plant species were hydrolysed and the volatile products identified by GC-MS and related to previous published findings. Known compounds, identified in new plant sources, were 4-methylthiobutyl thiocyanate in Alyssum, 4-methylthiobutyl isothiocyanate in Diplotaxis and Eruca and isopropyl isothiocyanate and 5-vinyl-2-oxazolidinethione in Plantago.
Article
Dichloromethane extracts of juices from fresh cabbages, including four known (Brutus, Galaxy, Bentley, Structon) and two unknown cultivars, were analyzed by X-MS for the presence of sinigrin degradation products. Allyl isothiocyanate (AITC), which has been reported to be the important aroma compound of freshly disrupted cabbage was not detected in any of the dichloromethane extracts of cabbage juice. Instead, 1-cyano-2,3-epithiopropane, which is one of the isomers of AITC, was the primary volatile compound in all cabbage extracts. AITC was detected in relative trace concentration only when cabbage juice was injected into the GC, using a wide bore, packed column. Thus, the relative importance of AITC and 1-cyano-2,3-epithiopropane to the aroma of freshly disrupted cabbage should be considered.
Article
Allylglucosinolate is converted to 1-cyano-2,3-epithiopropane by interaction of the thioglucoside glucohydrolase and epithiospecifier protein from several genera: Brassica, Crambe, Armoracia and Sinapis. The interactions occur across genetic lines, indicating non-specific requirements for epithiospecifier protein and thioglucoside glucohydrolase.
Article
The purification of the omega-(methylsulfinyl)alkyl glucosinolate hydrolysis products 1-isothiocyanato-3-(methylsulfinyl)propane (IMSP), 1-isothiocyanato-4-(methylsulfinyl) butane (IMSB), 4-(methylsulfinyl)-butanenitrile (MSBN), and 5-(methylsulfinyl) pentanenitrile (MSPN) from the seeds of broccoli and Lesquerella fendieri (Gray) S. Watson is described. The procedure uses solvent extraction of autolyzed defatted seed meals, followed by purification of the hydrolysis products using gel filtration chromatography and reversed-phase high-performance liquid chromatography (HPLC). Purity and confirmation of the compounds were monitored and verified using gas chromatography with flame ionization detection (GC-FID), thin-layer chromatography (TLC), gas chromatography-mass spectroscopy (GC-MS), and nuclear magnetic resonance (NMR). The techniques are useful for the preparative-scale isolation of structurally related glucosinolate hydrolysis products arising from omega-(methylsulfinyl)alkyl glucosinolates and should facilitate more extensive studies into the biological effects of these naturally occurring compounds.
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.
Article
We describe a rapid and direct assay of NAD(P)H:(quinone-acceptor) oxidoreductase (EC 1.6.99.2) activity in cultured cells suitable for identifying and purifying inducers of this detoxication enzyme. Hepa 1c1c7 murine hepatoma cells are plated in 96-well microtiter plates, grown for 24 h, and exposed to inducing agents for another 24 h. The cells are then lysed and quinone reductase activity is assayed by the addition of a reaction mixture containing an NADPH-generating system, menadione (2-methyl-1,4-naphthoquinone), and MTT [3-(4,-5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide]. Quinone reductase catalyzes the reduction of menadione to menadiol by NADPH, and MTT is reduced nonenzymatically by menadiol resulting in the formation of a blue color which can be quantitated on a microtiter plate absorbance reader. The reaction is more than 90% dicoumarol inhibitable and menadione dependent. The results are comparable to those obtained by harvesting cells from larger plates, preparing cytosols, and carrying out spectrophotometric measurements.
Article
Sonicated extract of crambe seed meal prepared in the presence of ferrous ion and dithiothreitol enzymatically converts epi-progoitrin to glucose, HSO4−, and a mixture of 1-cyano-2-hydroxy-3,4-epithiobutanes (50–70%) and 1-cyano-2-hydroxy-3-butene (30–50%). A fraction of the extract precipitating between 60 and 70% saturated ammonium sulfate contains thioglucosidase that converts epi-progoitrin essentially to 1-cyano-2-hydroxy-3-butene. Chromatography (on cross-linked dextran) of a 40–60% ammonium sulfate fraction leads to separation of a proteinaceous material (s20 = 2.6 S) that does not hydrolyze epi-progoitrin but, in the presence of thioglucosidase, promotes the formation of 1-cyano-2-hydroxy-3,4-epithiobutanes in amounts proportional to those from crude seed meal extract.
Article
Sulforaphane [1-isothiocyanato-4-(methyl-sulfinyl)butane] was recently isolated from one variety of broccoli as the major and very potent inducer of phase 2 detoxication enzymes in murine hepatoma cells in culture. Since phase 2 enzyme induction is often associated with reduced susceptibility of animals and their cells to the toxic and neoplastic effects of carcinogens and other electrophiles, it was important to establish whether sulforaphane could block chemical carcinogenesis. In this paper we report that sulforaphane and three synthetic analogues, designed as potent phase 2 enzyme inducers, block the formation of mammary tumors in Sprague-Dawley rats treated with single doses of 9,10-dimethyl-1,2-benzanthracene. The analogues are exo-2-acetyl-exo-6-isothiocyanatonorbornane, endo-2-acetyl-exo-6-isothiocyanatonorbornane, and exo-2-acetyl-exo-5-isothiocyanatonorbornane. When sulforaphane and exo-2-acetyl-exo-6-isothiocyanatonorbornane were administered by gavage (75 or 150 mumol per day for 5 days) around the time of exposure to the carcinogen, the incidence, multiplicity, and weight of mammary tumors were significantly reduced, and their development was delayed. The analogues endo-2-acetyl-exo-6-isothiocyanatonorbornane and exo-2-acetyl-exo-5-isothiocyanatonorbornane were less potent protectors. Thus, a class of functionalized isothiocyanates with anticarcinogenic properties has been identified. These results validate the thesis that inducers of phase 2 enzymes in cultured cells are likely to protect against carcinogenesis.
Article
Mammalian cells have evolved elaborate mechanisms for protection against the toxic and neoplastic effects of electrophilic metabolites of carcinogens and reactive oxygen species. Phase 2 enzymes (e.g. glutathione transferase, NAD(P)H:quinone reductase, UDP-glucuronosyltransferases) and high intracellular levels of glutathione play a prominent role in providing such protection. Phase 2 enzymes are transcriptionally induced by low concentrations of a wide variety of chemical agents and such induction blocks chemical carcinogenesis. The inducers belong to many chemical classes including phenolic antioxidants. Michael reaction acceptors, isothiocyanates, 1,2-dithiole-3-thiones, trivalent arsenicals, HgCl2 and organomercurials, hydroperoxides, and vicinal dimercaptans. Induction by all classes of inducers involves the antioxidant/electrophile response element (ARE/EpRE). Inducers are widely, but unequally, distributed among edible plants. Search for such inducer activity in broccoli led to the isolation of sulforaphane, an isothiocyanate that is a very potent Phase 2 enzyme inducer and blocks mammary tumor formation in rats.
Article
Induction of phase 2 detoxication enzymes [e.g., glutathione transferases, epoxide hydrolase, NAD(P)H: quinone reductase, and glucuronosyltransferases] is a powerful strategy for achieving protection against carcinogenesis, mutagenesis, and other forms of toxicity of electrophiles and reactive forms of oxygen. Since consumption of large quantities of fruit and vegetables is associated with a striking reduction in the risk of developing a variety of malignancies, it is of interest that a number of edible plants contain substantial quantities of compounds that regulate mammalian enzymes of xenobiotic metabolism. Thus, edible plants belonging to the family Cruciferae and genus Brassica (e.g., broccoli and cauliflower) contain substantial quantities of isothiocyanates (mostly in the form of their glucosinolate precursors) some of which (e.g., sulforaphane or 4-methylsulfinylbutyl isothiocyanate) are very potent inducers of phase 2 enzymes. Unexpectedly, 3-day-old sprouts of cultivars of certain crucifers including broccoli and cauliflower contain 10-100 times higher levels of glucoraphanin (the glucosinolate of sulforaphane) than do the corresponding mature plants. Glucosinolates and isothiocyanates can be efficiently extracted from plants, without hydrolysis of glucosinolates by myrosinase, by homogenization in a mixture of equal volumes of dimethyl sulfoxide, dimethylformamide, and acetonitrile at -50 degrees C. Extracts of 3-day-old broccoli sprouts (containing either glucoraphanin or sulforaphane as the principal enzyme inducer) were highly effective in reducing the incidence, multiplicity, and rate of development of mammary tumors in dimethylbenz(a)anthracene-treated rats. Notably, sprouts of many broccoli cultivars contain negligible quantities of indole glucosinolates, which predominate in the mature vegetable and may give rise to degradation products (e.g., indole-3-carbinol) that can enhance tumorigenesis. Hence, small quantities of crucifer sprouts may protect against the risk of cancer as effectively as much larger quantities of mature vegetables of the same variety.
Article
Isothiocyanates and their naturally occurring glucosinolate precursors are widely consumed as part of a diet rich in cruciferous vegetables. When plant cells are damaged, glucosinolates are released and converted to isothiocyanates by the enzyme myrosinase. Many isothiocyanates inhibit the neoplastic effects of various carcinogens at a number of organ sites. Consequently, these agents are attracting attention as potential chemoprotectors against cancer. As a prerequisite to understanding the mechanism of the protective effects of these compounds, which is thought to involve the modulation of carcinogen metabolism by the induction of phase 2 detoxication enzymes and the inhibition of phase 1 carcinogen-activating enzymes, we examined the fate of ingested isothiocyanates and glucosinolates in humans. Recently developed novel methods for quantifying isothiocyanates (and glucosinolates after their quantitative conversion to isothiocyanates by purified myrosinase) and their urinary metabolites (largely dithiocarbamates) have made possible a detailed examination of the fates of isothiocyanates and glucosinolates of dietary crucifers. In a series of studies in normal volunteers, we made these findings. First, in nonsmokers, urinary dithiocarbamates were detected only after the consumption of cruciferous vegetables and condiments rich in isothiocyanates and/or glucosinolates. In sharp contrast, the consumption of noncrucifers (corn, tomatoes, green beans, and carrots) did not lead to the excretion of dithiocarbamates. Moreover, the quantities of dithiocarbamates excreted were related to the glucosinolate/isothiocyanate profiles of the cruciferous vegetables administered (kale, broccoli, green cabbage, and turnip roots). Second, eating prepared horseradish containing graded doses of isothiocyanates (12.3-74 micromol; mostly allyl isothiocyanate) led to a rapid excretion of proportionate amounts (42-44%) of urinary dithiocarbamates with first-order kinetics. The ingestion of broccoli in which myrosinase had been heat-inactivated also led to proportionate but low (10-20%) recoveries of urinary dithiocarbamates. Broccoli samples subsequently treated with myrosinase to produce the cognate isothiocyanates were much more completely (47%) converted to dithiocarbamates. Finally, when bowel microflora were reduced by mechanical cleansing and antibiotics, the conversion of glucosinolates became negligible. These results establish that humans convert substantial amounts of isothiocyanates and glucosinolates to urinary dithiocarbamates that can be easily quantified, thus paving the way for meaningful studies of phase 2 enzyme induction in humans.
Article
Thermal and pressure inactivation of myrosinase from broccoli was kinetically investigated. Thermal inactivation proceeded in the temperature range 30-60 degrees C. These results indicate that myrosinase is rather thermolabile, as compared to other food quality related enzymes such as polyphenol oxidase, lipoxygenase, pectinmethylesterase, and peroxidase. In addition, a consecutive step model was shown to be efficient in modeling the inactivation curves. Two possible inactivation mechanisms corresponding to the consecutive step model were postulated. Pressure inactivation at 20 degrees C occurred at pressures between 200 and 450 MPa. In addition to its thermal sensitivity, the enzyme likewise is rather pressure sensitive as compared to the above-mentioned food quality related enzymes. By analogy with thermal inactivation, a consecutive step model could adequately describe pressure inactivation curves. At 35 degrees C, pressure inactivation was studied in the range between 0. 1 and 450 MPa. Application of low pressure (<350 MPa) resulted in retardation of thermal inactivation, indicating an antagonistic or protective effect of low pressure.