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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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... The composition of glucosinolate degradation products is dependent on various factors including the pH of the incubation medium and the presence of epithiospecifier protein (ESP; a non-catalytic cofactor of myrosinase) and ferrous ions in the incubation medium [4,18]. Of these factors, the pH (of the GIT) is the major factor that affects the composition of glucosinolate degradation products in the GIT of pigs fed practical diets because of the following two reasons: First, the ESP is susceptible to heat (is inactivated by heat at 60 • C) [19], and thus, the ESP present in canola seed is inactivated during oil extraction. Second, ferrous ions are available in the GIT of pigs because feedstuffs used to formulate practical swine diets contain iron [20], and swine diets are supplemented with iron-containing mineral premixes to meet iron requirements. ...
... Eylen et al. (2007) reported myrosinase inactivation due to its heat treatment at 60 • C for 10 min [15]. Similarly, Matusheski et al. (2004) observed complete ESP inactivation due to heat treatment at ≥50 • C for 10 min [19]. The minimum temperature to which SECM and EPCM are exposed to during their production is 103 • C [26], whereas the minimum temperature to which CPCE is exposed during its production is 50 • C [27]. ...
... Eylen et al. (2007) reported myrosinase inactivation due to its heat treatment at 60 • C for 10 min [15]. Similarly, Matusheski et al. (2004) observed complete ESP inactivation due to heat treatment at ≥50 • C for 10 min [19]. The minimum temperature to which SECM and EPCM are exposed to during their production is 103 • C [26], whereas the minimum temperature to which CPCE is exposed during its production is 50 • C [27]. ...
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Canola co-products are widely included in swine diets as sources of proteins. However, inclusion of canola co-products in diets for pigs is limited by toxicity of glucosinolate degradation products. Aliphatic and aromatic glucosinolates are two major classes of glucosinolates. Glucosinolate degradation products derived from aliphatic glucosinolates (progoitrin) include crambene, epithionitriles, and goitrin, whereas indole-3-acetonitrile, thiocyanate, and indole-3-carbinol are the major aromatic glucosinolates (glucobrassicin)-derived degradation products. At acidic pH (<5.7), progoitrin is degraded by myrosinases to crambene and epithionitriles in the presence of iron, regardless of the presence of epithiospecifier protein (ESP), whereas progoitrin is degraded by myrosinases to goitrin in the absence of ESP, regardless of the presence of iron at neutral pH (6.5). Indole-3-acetonitrile is the major degradation product derived from glucobrassicin in the absence of ESP, regardless of the presence of iron at acidic pH (<4.0), whereas thiocyanate and indole-3-carbinol are the major glucobrassicin-derived degradation products in the absence of ESP, regardless of the presence of iron at neutral pH (7.0). In conclusion, the composition of glucosinolate degradation products is affected by parent glucosinolate composition and hindgut pH. Thus, toxicity of canola co-product-derived glucosinolates can be potentially alleviated by modifying the hindgut pH of pigs.
... In this way, most of the studies designed to check the effects of SFN from vegetable having higher amounts of epithiospecifier protein may not observe physiological benefits. It has been reported that epithiospecifier protein may result in nine times higher production of inactive nitrile than isothiocyanates (Matusheski et al., 2004(Matusheski et al., , 2006Williams et al., 2008). The cleavage of glucosinolates to various components is demonstrated in Figure 3. SFN can deliver cancer-preventive effects only when sufficiently absorbed and available in biologically active form in the body after consumption. ...
... The broccoli can be consumed raw or freshly harvested alongside mildly processed. Heating decreases epithiospecifier protein resulting in higher production of SFN in broccoli (Matusheski et al., 2004). ...
... It has also been observed that long-term storage (10 days) reduces glucoraphanin (80%) content in broccoli. Other than broccoli, mustard seed powder, daikon radish, wasabis, arugula, or coleslaw are also known for their myrosinase-rich constituents (Higdon et al., 2007;Matusheski et al., 2004;Nandini et al., 2020). ...
Article
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Sulforaphane belongs to the active class of isothiocyanates capable of delivering various biological benefits for health promotion and disease prevention. This compound is considered vital to curtail numerous metabolic disorders. Various studies have proven its beneficial effects against cancer prevention and its possible utilization as a therapeutic agent in cancer treatment. Understanding the mechanistic pathways and possible interactions at cellular and subcellular levels is key to design and develop cancer therapeutics for humans. In this respect, a number of mechanisms such as modulation of carcinogen metabolism & phase II enzymatic activities, cell cycle arrest, activation of Nrf2, cytotoxic, proapoptotic and apoptotic pathways have been reported to be involved in cancer prevention. This article provides sufficient information by critical analysis to understand the mechanisms involved in cancer prevention attributed to sulforaphane. Furthermore, various clinical studies have also been included for design and development of novel therapies for cancer prevention and cure. Practical applications Diet and dietary components are potential tools to address various lifestyle-related disorders. Due to plenty of environmental and cellular toxicants, the chances of cancer prevalence are quite large which are worsen by adopting unhealthy lifestyles. Cancer can be treated with various therapies but those are acquiring side effects causing the patients to suffer the treatment regime. Nutraceuticals and functional foods provide safer options to prevent or delay the onset of cancer. In this regard, sulforaphane is a pivotal compound to be targeted as a potential agent for cancer treatment both in preventive and therapeutic regimes. This article provides sufficient evidence via discussing the underlying mechanisms of positive effects of sulforaphane to further the research for developing anticancer drugs that will help assuage this lethal morbidity.
... They contain sulfur groups and are present in numerous species belonging to the Brassicaceae family such as broccoli and cabbage. When the tissue of these plants is damaged, glucosinolates can be hydrolyzed by plant myrosinase or non-enzymatically to form primarily isothiocyanates and/or simple nitriles [70,71]. In Arabidopsis, BCAT4 catalyzes the chain elongation pathway of Met-derived glucosinolate biosynthesis [72]. ...
... Thus, glucosinolate might be a signaling molecule that enhances thermotolerance to promote head formation in HT plants. ESP is also responsible for the products of glucosinolate toward epithionitriles [70,77]. Ectopic expression of Arabidopsis ESP in E. coli triggered the hydrolysis of glucosinolate [77], and heating decreased plant ESP activity [70]. ...
... ESP is also responsible for the products of glucosinolate toward epithionitriles [70,77]. Ectopic expression of Arabidopsis ESP in E. coli triggered the hydrolysis of glucosinolate [77], and heating decreased plant ESP activity [70]. In this study, the expression of broccoli ESP was higher at 27°C than 15°C in both HT and HS genotypes. ...
Article
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Background Head formation of broccoli (Brassica oleracea var. italica) is greatly reduced under high temperature (22 °C and 27 °C). Broccoli inbred lines that are capable of producing heads at high temperatures in summer are varieties that are unique to Taiwan. However, knowledge of the early-activated pathways of broccoli head formation under high temperature is limited. Results We compared heat-tolerant (HT) and heat-sensitive (HS) transcriptome of broccoli under different temperatures. Weighted gene correlation network analysis (WGCNA) revealed that genes involved in calcium signaling pathways, mitogen-activated protein kinase (MAPK) cascades, leucine-rich repeat receptor-like kinases (LRR-RLKs), and genes coding for heat-shock proteins and reactive oxygen species homeostasis shared a similar expression pattern to BoFLC1, which was highly expressed at high temperature (27 °C). Of note, these genes were less expressed in HT than HS broccoli at 22 °C. Co-expression analysis identified a model for LRR-RLKs in survival-reproduction tradeoffs by modulating MAPK- versus phytohormones-signaling during head formation. The difference in head-forming ability in response to heat stress between HT and HS broccoli may result from their differential transcriptome profiles of LRR-RLK genes. High temperature induced JA- as well as suppressed auxin- and cytokinin-related pathways may facilitate a balancing act to ensure fitness at 27 °C. BoFLC1 was less expressed in HT than HS at 22 °C, whereas other FLC homologues were not. Promoter analysis of BoFLC1 showed fewer AT dinucleotide repeats in HT broccoli. These results provide insight into the early activation of stress- or development-related pathways during head formation in broccoli. The identification of the BoFLC1 DNA biomarker may facilitate breeding of HT broccoli. Conclusions In this study, HT and HS broccoli genotypes were used to determine the effect of temperature on head formation by transcriptome profiling. On the basis of the expression pattern of high temperature-associated signaling genes, the HS transcriptome may be involved in stress defense instead of transition to the reproductive phase in response to heat stress. Transcriptome profiling of HT and HS broccoli helps in understanding the molecular mechanisms underlying head-forming capacity and in promoting functional marker-assisted breeding. Electronic supplementary material The online version of this article (10.1186/s12870-018-1613-x) contains supplementary material, which is available to authorized users.
... 21 Because of the various health benefits of sulforaphane, there is a great deal of interest in approaches for maximising the yield of sulforaphane and improve its stability. Matusheski et al. 22 studied the possibility of enhancing sulforaphane yield in broccoli through selective inactivation of ESP while maintaining myrosinase activity through mild heat treatment. They observed that heat treatment of broccoli floret for 5 min at 60°C enables selective inactivation of ESP and maximises sulforaphane yield. ...
... 6 We investigated the feasibility of using lactic acid bacteria (LAB) fermentation for enhancing the yield and stability of sulforaphane in broccoli matrix. Lactic acid bacteria fermentation of broccoli puree using autochthonous lactic acid bacteria doubled sulforaphane yield compared to non-fermented puree and maintained high stability of sulforaphane during storage for about three months at 4°C. 25 Based on our previous work on the impact of lactic acid bacteria fermentation on sulforaphane yield 25 and preceding research that showed that mild heat treatment selectively inactivates ESP and enhance the formation of sulforaphane, 22 we hypothesized that mild heat treatment of broccoli combined with fermentation may further increase the yield of sulforaphane while enabling the production of a microbially safe and stable product. Our earlier study 25 showed that lactic acid bacteria fermentation enables control of undesirable spoilage and pathogenic microorganisms following processing and during storage for up to three months at 4 and 25°C. ...
... The influence of heat treatment conditions on the yield of sulforaphane from glucoraphanin hydrolysis in in-pack heated broccoli florets are shown in Fig. 3. In-pack heat treatment at all the studied conditions increased sulforaphane yield compared to raw broccoli puree in line with previous reports. 22,27 Mild thermal treatments of broccoli florets results in a relatively higher level of inactivation of epithiospecifier protein (ESP) compared to myrosinase, which reduces the formation of sulforaphane nitrile while enhancing sulforaphane production. 22 For treatments at 60°C, increasing treatment time from 0 to 5 min resulted in an increase in sulforaphane yield. ...
Article
This study evaluated for the first time the feasibility of mild preheating treatment of broccoli florets combined with lactic acid bacteria fermentation for enhancing sulforaphane yield in broccoli puree. The optimum preheating condition for in-pack processing of broccoli florets was 3 min treatment at 65 °C increasing sulforaphane yield in broccoli puree from 817 μmol/kg dry weight (DW) in the untreated broccoli to 3983 μmol/kg DW. Preheating of broccoli florets in-pack (65 °C/3 min) combined with lactic acid bacteria fermentation further enhanced the sulforaphane content to 13121 μmol/kg DW, which is ~16 times more compared to raw broccoli. The sulforaphane content of the preheated-fermented puree remained stable (~94% retention) for two weeks at 4 °C. The results indicate that a combination of judicious heat treatment of broccoli florets with lactic acid bacteria fermentation enables production of safe and high sulforaphane content broccoli products with potential health benefits.
... Among high nutritive sprouts, broccoli sprouts (Brassica oleracea) are of great interest due to their antioxidant, anticancer and anti-inflammatory activities, given their high content of antioxidants, vitamins, minerals and glucosinolates (GLs) and their hydroxylated derivatives (Matusheski, Juvik, & Jeffery, 2004). For instance, sprouting induced the parent glucosinolate of sulforaphane to 10 times than its value in seeds and mature plants (Fahey, Zhang, & Talalay, 1997). ...
... GLs are released from vacuoles of myrosin cells when the cells of broccoli are crushed. Sulforaphane (SF) is the most dominant and active GLs in broccoli, SF and SF nitrile are products of GLs hydrolysis through endogenous cytosolic myrosinase (Matusheski et al., 2004). This hydrolysis process and the products that released depend on the activity of myrosinase enzyme, the presence of additional proteins such as epithiospecifier protein (ESP) and hydrolysis conditions such as temperature and pH (Williams, Critchley, Pun, Nottingham, & O'Hare, 2008). ...
... This hydrolysis process and the products that released depend on the activity of myrosinase enzyme, the presence of additional proteins such as epithiospecifier protein (ESP) and hydrolysis conditions such as temperature and pH (Williams, Critchley, Pun, Nottingham, & O'Hare, 2008). The capacity of SF as anti-carcinogen was proved through its effectivity to induce the quinone reductase (QR) in Hepa lclc7 cell culture by saving the mammal DNA against damage through arresting the bioactivated carcinogens (Matusheski et al., 2004). For example, it showed high activity as antiproliferative by inhibiting the growth and inducing https://doi.org/10.1016/j.foodchem.2020.127102 ...
Article
Sprouting process enhances plant bioactive compounds. Broccoli (Brassica oleracea L) sprouts are well known for their high levels of glucosinolates (GLs), amino acids, and antioxidants, which offer outstanding biological activities with positive impact on plant metabolism. elevated CO2 (eCO2, 620ppm) was applied for 9 days to further improve nutritive and health-promoting values of three cultivars of broccoli sprouts i.e., Southern star, Prominence and Monotop. eCO2 improved sprouts growth and induced GLs accumulation e.g., glucoraphanin, possibly through amino acids production e.g., high methionine and tryptophan. There was increases in myrosinase activity, which stimulated GLs hydrolysis to yield health promoting sulforaphane. Interestingly low levels of ineffective sulforaphane nitrile was detected and positively correlated with reduced epithiospecifier protein after eCO2 treatment. High glucoraphanin and sulforaphane levels in eCO2 treated sprouts improved anticarcinogenic and anti-inflammatory properties of their extracts. In conclusion, eCO2 treatment enrich broccoli sprouts with health promoting metabolites and bioactivities.
... Myrosinase initiates the hydrolysis of glucosinolates, resulting in formation of unstable intermediates, which rearrange to form ITCs; while ESP interacts with the unstable intermediates to divert ITC formation into nitriles, which has not shown any anti-cancer potential. Interestingly, the substrate specificity of ESP varies by chemical structure of glucosinolates, showing a high efficiency on the hydrolysis of aliphatic glucosinolates compared with aromatic glucosinolates (Cole, 1978;Kaoulla, MacLeod, & Gil, 1980;Matusheski, Juvik, & Jeffery, 2004;Matusheski et al., 2006;Petroski & Tookey, 1982;Wittstock & Burow, 2007). Also the presence of ESP varies in vegetables with a strong activity in broccoli but not in mustard or horseradish (Cole, 1978;Kaoulla et al., 1980;Matusheski et al., 2004Matusheski et al., , 2006Petroski & Tookey, 1982;Wittstock & Burow, 2007). ...
... Interestingly, the substrate specificity of ESP varies by chemical structure of glucosinolates, showing a high efficiency on the hydrolysis of aliphatic glucosinolates compared with aromatic glucosinolates (Cole, 1978;Kaoulla, MacLeod, & Gil, 1980;Matusheski, Juvik, & Jeffery, 2004;Matusheski et al., 2006;Petroski & Tookey, 1982;Wittstock & Burow, 2007). Also the presence of ESP varies in vegetables with a strong activity in broccoli but not in mustard or horseradish (Cole, 1978;Kaoulla et al., 1980;Matusheski et al., 2004Matusheski et al., , 2006Petroski & Tookey, 1982;Wittstock & Burow, 2007). Therefore, the ITC yield could differ considerably in vegetables even if they contain similar type and/or similar amount of glucosinolates. ...
... Using the ITC yield from the raw cruciferous vegetable as the reference, we found that boiling, stewing, and chip-baking reduced ITC yields; whereas stir-frying, steaming, and microwaving increased ITC showed that heating at 60-70°C for 5-10 min destroys ESP but spares myrosinase, resulting in an increases of ITC yields by approximately three-fold to seven-fold, but further temperature increase caused declines of ITC yields, probably due to inactivation of myrosinase (Matusheski et al., 2004;Sosińska & Obiedziński, 2011 Figure 2). Besides affecting hydrolysis efficiency, cooking also changes the amount of available glucosinolates. ...
Article
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Cruciferous vegetables are primary sources of dietary isothiocyanates (ITCs), a group of phytochemicals showing promising cancer‐chemopreventive activities in multiple cancer models. However, no study has thoroughly examined how cooking affects the yields of ITCs from cruciferous vegetables. In this study, a high‐performance liquid chromatography (HPLC)‐based cyclocondensation assay was performed to examine the ITC yields from four major cruciferous vegetables (broccoli, cabbage, cauliflower, and kale) under six cooking conditions (stir‐frying, steaming, microwaving, boiling, stewing, and chip‐baking for kale only) and measured the level of ITCs under the raw condition for a comprehensive list of cruciferous vegetables and ITC‐containing condiments. A wide range of ITC yields was found across vegetables and condiments. Cooking significantly altered the ITC yields, showing an averagely four‐fold increase by lightly cooking (stir‐frying, steaming, and microwaving) and a 58% decrease by heavily cooking (boiling, stewing, and chip‐baking). These findings will provide the evidence‐based cooking guidance on cruciferous vegetable consumption and help better estimate dietary ITC exposure in epidemiologic studies. Cooking significantly altered the isothiocyanate yields, showing an averagely four‐fold increase by lightly cooking (stir‐frying, steaming, and microwaving) and a 58% decrease by heavily cooking (boiling, stewing, and chip‐baking).
... 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). ...
Article
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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.
... These phenomena are mostly influenced by the duration and method of cooking, the type of vegetable matrix and the extent of cellular disruption, and the chemical structure of the glucosinolates (Conaway et al., 2000;Oliviero et al., 2018). One report suggested that myrosinases from broccoli sprouts are more heat-resistant than those from mature broccoli (Matusheski et al., 2004). In this study, the enzymes in broccoli were thermally inactivated at approximately 70 o C, whereas myrosinases in immature broccoli sprouts remained active at 100 o C (Matusheski et al., 2004). ...
... One report suggested that myrosinases from broccoli sprouts are more heat-resistant than those from mature broccoli (Matusheski et al., 2004). In this study, the enzymes in broccoli were thermally inactivated at approximately 70 o C, whereas myrosinases in immature broccoli sprouts remained active at 100 o C (Matusheski et al., 2004). Glucosinolates may act as an antioxidant by increasing enzymatic activities that inhibit oxidation, such as those of glutathione-S-transferase and uridine diphosphate-glucuronosyltransferase (Williamson et al., 1998;Vig et al., 2009). ...
Article
In this study, we determined the contents of glucosinolate, polyphenol, and flavonoid, and the antioxidant activities of uncooked, steamed, and boiled cauliflower. Eight glucosinolate peaks were detected, representing glucoiberin, progoitrin, glucoraphanin, sinigrin, gluconapin, glucoiberverin, glucobrassicin, and gluconasturtiin. Boiled cauliflower contained significantly lowered concentrations of glucosinolate, total polyphenol, and total flavonoid compared to uncooked or steamed cauliflower. These results clearly indicate that health-promoting compounds in cauliflower are significantly impacted by different cooking methods: uncooked> steamed> boiled. The amounts of total polyphenols and total flavonoids in uncooked cauliflower extracted with 80% ethanol were higher than extracts of steamed and boiled cauliflower. The highest antioxidant activity was observed in uncooked cauliflower extracted using 80% ethanol compared to those extracted with water at the same concentration. Steamed and boiled cauliflower extracts also showed lower antioxidant activity than uncooked extracts. Based on these results, fresh uncooked cauliflower contains higher contents of health-promoting compounds and elevated antioxidant activity. Moreover, steaming may be more desirable than boiling in order to minimize loss of glucosinolates when storing, pretreating, processing, and cooking cruciferous vegetables.
... Innovative Food Science and Emerging Technologies 58 (2019) 102213 epithiospecifier protein (ESP), nitrile specifier protein (NSP), and thiocyanate-forming protein (TFP) (Bell et al., 2018;Ghawi et al., 2013). In the study by Matusheski, Juvik, and Jeffery (2004), heating broccoli florets and sprouts to 60°C for 5-10 min caused a significant decrease in ESP activity, as a consequence, the formation of sulforaphane nitrile was reduced; however, an increase in sulforaphane, an isothiocyanate, was observed. The temperature sensitivity of ESP might explain the higher formation of thiocyanates upon mild heating in the current study. ...
... The temperature sensitivity of ESP might explain the higher formation of thiocyanates upon mild heating in the current study. On the other hand, heating to 100°C resulted in a significant decrease in total hydrolytic products, sulforaphane nitrile and sulforaphane (Matusheski et al., 2004). Moreover, sulfur containing compounds can be formed through thermal degradation of sulforaphane (Jin, Wang, Rosen, & Ho, 1999). ...
Article
This study focused on investigating quality changes of thermally processed kale purée using an integrated targeted and untargeted approach. Low, medium, and high processing intensities (carried out at 70, 90, and 128 °C) were selected based on predetermined shelf-life targets: frozen-thawed, refrigerated, and ambient storage, respectively. The results show that that physicochemical properties determining consumer acceptability were largely dependent on the treatment intensity. The high intensity treatment resulted in the least favorable quality characteristics (distinct brown color, chlorophyll and vitamin C destruction as well as a phase separation after storage). Enzymes were inactivated with increasing thermal load. Regarding taste related compounds, there was no clear effect of processing and storage on acidity and sugar profiles. The untargeted GC–MS approach showed that increasing the processing intensity resulted in an increase of the formation of furans and sulfides. Storage clearly affected the formation of volatiles that could be, depending on the thermal treatment, attributed to lipid and/or carotenoid oxidation, continuation of Maillard reactions, and enzyme catalyzed reactions. Industrial relevance Compared to other Brassicaceae vegetables such as as broccoli, cauliflower and different types of cabbages, kale so far has received little attention from an industrial processing point of view. However, kale has been reported to contain high nutritional value due to its important content of minerals, bioactive compounds and fibre. With the growing health awareness of consumers and increasingly busy lifestyles, the demand for more convenient fruit and vegetable products with high nutritional content has increased. Therefore, research to obtain more insight on the effect of processing and storage on kale purée is important.
... Occurrence and activity of the epithiospecifier protein (ESP) leads to the generation of ETNs from alkenyl-GLS aglucons as well as nitriles from non-alkenyl-GLS-aglucons (Burow et al., 2006;Matusheski et al., 2006). Many Brassica species release nitriles and ETNs upon GLS hydrolysis, among them B. oleracea and also B. campestris, B. carinata, and B. rapa (Macleod and Rossiter, 1985;Matusheski et al., 2004;Hanschen and Schreiner, 2017;Klopsch et al., 2017;Klopsch et al., 2018;Hanschen et al., 2019). So far, ESPs were characterized in A. thaliana (Lambrix et al., 2001;De Torres Zabala et al., 2005;Hanschen et al., 2018b) and in Brassica species, such as broccoli (B. ...
... To compare the substrate specificities of the three recombinant BoESPs, the protocol of (Matusheski et al., 2004) was modified and adapted. In order to maximize GLS hydrolysis, vitamin C was added and Fe 2+ concentration in the assay was reduced to 0.2 mM to optimize ITC formation while maintaining ETN formation from alkenyl GLS in the assay: Briefly, 50 µl of purified ESP (containing in total 6.5 µg ESP as determined by Bradford assay (Bradford, 1976), 350 µl of a 50 mM sodium acetate buffer (pH 5.5) containing 1 mM dithiothreitol and 0.2 mM of FeSO 4 , 10 µl of a 25.5 mM vitamin C solution, 50 µl of 0.5 U ml -1 myrosinase and 50 µl of a 10 mM solution of the GLS to be tested were mixed in an extraction tube and incubated for 1 h at room temperature. ...
Article
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Glucosinolates present in Brassicaceae play a major role in herbivory defense. Upon tissue disruption, glucosinolates come into contact with myrosinase, which initiates their breakdown to biologically active compounds. Among these, the formation of epithionitriles is triggered by the presence of epithiospecifier protein (ESP) and a terminal double bond in the glucosinolate side chain. One ESP gene is characterized in the model plant Arabidopsis thaliana (AtESP; At1g54040.2). However, Brassica species underwent genome triplication since their divergence from the Arabidopsis lineage. This indicates the presence of multiple ESP isoforms in Brassica crops that are currently poorly characterized. We identified three B. oleracea ESPs, specifically BoESP1 (LOC106296341), BoESP2 (LOC106306810), and BoESP3 (LOC106325105) based on in silico genome analysis. Transcript and protein abundance were assessed in shoots and roots of four B. oleracea vegetables, namely broccoli, kohlrabi, white, and red cabbage, because these genotypes showed a differential pattern for the formation of glucosinolate hydrolysis products as well for their ESP activity. BoESP1 and BoESP2 were expressed mainly in shoots, while BoESP3 was abundant in roots. Biochemical characterization of heterologous expressed BoESP isoforms revealed different substrate specificities towards seven glucosinolates: all isoforms showed epithiospecifier activity on alkenyl glucosinolates, but not on non-alkenyl glucosinolates. The pH-value differently affected BoESP activity: while BoESP1 and BoESP2 activities were optimal at pH 6-7, BoESP3 activity remained relatively stable from pH 4 to 7. In order test their potential for the in vivo modification of glucosinolate breakdown, the three isoforms were expressed in A. thaliana Hi-0, which lacks AtESP expression, and analyzed for the effect on their respective hydrolysis products. The BoESPs altered the hydrolysis of allyl glucosinolate in the A. thaliana transformants to release 1-cyano-2,3-epithiopropane and reduced formation of the corresponding 3-butenenitrile and allyl isothiocyanate. Plants expressing BoESP2 showed the highest percentage of released epithionitriles. Given these results, we propose a model for isoform-specific roles of B. oleracea ESPs in glucosinolate breakdown.
... This thermolabile enzyme shows 90% of degradation after treatments at 80 C for 10e60 min. When the blanching protocol was modified to heat samples only to 76 C, lipoxygenase but not peroxidase and myrosinase was inactivated and 82% of the sulforaphane present in unheated broccoli remained (Matusheski et al., 2004). At 66 C, there was significantly more sulforaphane formation than in the unblanched control (Matusheski et al., 2004). ...
... When the blanching protocol was modified to heat samples only to 76 C, lipoxygenase but not peroxidase and myrosinase was inactivated and 82% of the sulforaphane present in unheated broccoli remained (Matusheski et al., 2004). At 66 C, there was significantly more sulforaphane formation than in the unblanched control (Matusheski et al., 2004). This is attributable to the inactivation of the thermally unstable ESP, which are also present in the vegetable tissues, and can hydrolyze the GLS to the nonbioactive compound nitriles. ...
... In broccoli (Brassica oleracea var. italica), the main glucosinolate is glucoraphanin (GFN), and its hydrolysis at neutral pH yields preferably sulforaphane (SFN) which is one of the most potent foodderived anticancer compounds and also exhibits antimicrobial activity [30]. These facts encouraged the study of the myrosinase-glucosinolate system in broccoli in recent years. ...
... The molecular docking simulations were carried out at different pH (1.0, 3.0, 5.0, 7.0), based on the pH range reported for epithiospecifier (ESP) and nitrile-specifier protein (NSP) [16,[21][22][23][24]28,30,31]. The five aglycones considered in this study (3MSOP, 2PROP, 4MSOB, 4OHI3M, and I3M, see Table 2) were chosen based on the precursor glucosinolates detected in broccoli inflorescences. ...
Article
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Glucosinolates are secondary plant metabolites of Brassicaceae. They exert their effect after enzymatic hydrolysis to yield aglycones, which become nitriles and epithionitriles through the action of epithiospecifier (ESP) and nitrile-specifier proteins (NSP). The mechanism of action of broccoli ESP and NSP is poorly understood mainly because ESP and NSP structures have not been completely characterized and because aglycones are unstable, thus hindering experimental measurements. The aim of this work was to investigate the interaction of broccoli ESP and NSP with the aglycones derived from broccoli glucosinolates using molecular simulations. The three-dimensional structure of broccoli ESP was built based on its amino-acid sequence, and the NSP structure was constructed based on a consensus amino-acid sequence. The models obtained using Iterative Threading ASSEmbly Refinement (I-TASSER) were refined with the OPLS-AA/L all atom force field of GROMACS 5.0.7 and were validated by Veryfy3D and ERRAT. The structures were selected based on molecular dynamics simulations. Interactions between the proteins and aglycones were simulated with Autodock Vina at different pH. It was concluded that pH determines the stability of the complexes and that the aglycone derived from glucoraphanin has the highest affinity to both ESP and NSP. This agrees with the fact that glucoraphanin is the most abundant glucosinolate in broccoli florets.
... The reported values of glucoraphanin, which is the precursor of sulforaphane, have included ranges of 0.8-21.7 μMol/g dry weight (DW) (Kushad et al., 1999) and 4.4-16.4 μMol/g DW (Matusheski, Juvik, & Jeffery, 2004). Varietal differences and postharvest storage conditions affect glucoraphanin content and myrosinase activity, and further a high glucoraphanin content does not necessarily result in a high production of isothiocyanates (Rangkadilok et al., 2002; https://doi.org/10.1016/j.lwt.2020.109443 ...
... The more heat labile epithiospecifier protein must be inactivated (e.g. by heat treatment of 5-10 min at 60°C for broccoli juice) whilst maintaining sufficient activity of myrosinase (Van Eylen, Oey, Hendrickx, & Van Loey, 2007). It is known that varietal differences and slight changes in heat processing conditions have a significant effect on sulforaphane content (Dosz and Jefferey, 2013;Kushad et al., 1999;Matusheski et al., 2004;Tabart, Pincemail, Kevers, Defraigne, & Dommes, 2018). ...
Article
There is significant interest in developing high sulforaphane-containing broccoli-based ingredients as sulforaphane has potential for reducing the risk of a number of diseases. Glucoraphanin in broccoli is converted to sulforaphane by myrosinase when the plant tissue is disrupted. The instability of sulforaphane at high temperature and in aqueous environments presents challenges for developing sulforaphane-containing ingredients. Variations in heat treatment conditions of broccoli suspensions, pH, oil addition and storage conditions influenced the level of sulforaphane. The sulforaphane content of aqueous broccoli suspensions as a function of pH (pH 4.0, 5.0 and 6.0) after a heat treatment (60 °C/5 min) and holding for 4 h at 25 °C were examined. A pH of 5 was optimum for production of sulforaphane. The addition of oil to broccoli suspensions prior to freeze drying retarded the rate of degradation of sulforaphane during storage of powders. There was little difference between degradation rate of sulforaphane in (broccoli-oil) powders made from pH 5 or pH 6 (broccoli-oil) emulsions. There are significant hurdles for the development of high sulforaphane broccoli powders for use as functional food ingredients or supplement.
... The rate of hydrolysis and the concentration of various products resulting from hydrolysis of GR depends on the reaction conditions (e.g., temperature, pH, ferrous ion concentrations) and presence of epithiospecifier protein (ESP) [13]. The production of sulforaphane (1-isothiocyanato-4-methylsulfinylbutane; SF) is favored at elevated temperature (above 50 °C) and natural pH, while sulforaphane nitrile synthesis is favored at lower temperature (below 50 °C), acidic pH, and the presence of ESP [7,[14][15][16]. ...
... Increase in SF content in broccoli subjected to mild heat treatment is commonly attributed to thermal inactivation of the thermolabile epithiospecifier protein (ESP), which acts as non-catalytic cofactor for myrosinase that promotes the formation of SF-nitrile from GR instead of SF [11]. Matusheski et al. [15] observed a significant inactivation of ESP in broccoli after 5 and 10 min heating at 40 °C and 50 °C, respectively, and complete inactivation after 5 min heating at 60 °C, which was accompanied by higher SF formation. Nevertheless, the heatinduced increase in extractability and accessibility of GR [7] may also contribute to the observed higher yield of SF in mild-heat-treated broccoli. ...
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A large proportion of broccoli biomass is lost during primary production, distribution, processing, and consumption. This biomass is rich in polyphenols and glucosinolates and can be used for the production of bioactive rich ingredients for food and nutraceutical applications. This study evaluated thermosonication (TS) (18 kHz, 0.6 W/g, 40–60 °C, 3–7 min) for the pre-treatment of broccoli florets to enhance enzymatic conversion of glucoraphanin into the bioactive sulforaphane. TS significantly increased sulforaphane yield, despite a decrease in myrosinase activity with increasing treatment intensity. The highest sulforaphane yield of ~2.9 times that of untreated broccoli was observed for broccoli thermosonicated for 7 min at 60 °C, which was 15.8% higher than the corresponding yield for thermal processing without sonication (TP) at the same condition. This was accompanied by increase in the residual level of glucoraphanin (~1.8 and 2.3 time respectively after TP and TS at 60 °C for 7 min compared to control samples) indicating that treatment-induced release of bound glucoraphanin from the cell wall matrix and improved accessibility could be at least partially responsible for the enhanced sulforaphane yield. The result indicates the potential of TS for the conversion of broccoli biomass into high sulforaphane broccoli-based ingredients.
... ESP is a myrosinase accessory protein that does not catalyze GRP hydrolysis by itself, but instead directs the decomposition of the thiohydroximate O-sulfinate intermediate to SFN, thereby decreasing SF production (Matusheski & Jeffery, 2001;Matusheski, Juvik, & Jeffery, 2004). ESP has been identified in many nitrile-forming cruciferous plants, the plant Arabidopsis lacking this protein do not form nitriles, whereas addition of purified ESP increases nitrile formation from GLS in vitro (Lambrix, Reichelt, Mitchell-Olds, Kliebenstein, & Gershenzon, 2001). ...
... Therefore, inhibiting ESP activity in broccoli and increasing the partial conversion of GRP to SF can enhance the health benefits of broccoli (Matusheski & Jeffery, 2001). SF production increased significantly in fresh broccoli florets and sprouts heated for 5 and 10 min at 60 • C, since myrosinase is stable at this temperature whereas ESP activity decreases significantly above 50 • C (Matusheski et al., 2004). Rungapamestry, Duncan, Fuller, and Ratcliffe (2006) also reported that heating cabbage to 50-60 • C maximized SF yield, and Alvarez-Jubete et al. (2014) found that microwaving broccoli at a high temperature below 70 • C increased SF production compared to that following room temperature hydrolysis. ...
Article
Salmonella is a genus responsible for a majority of foodborne disease outbreaks affecting public health worldwide, and Salmonella enterica subsp. enterica serotype Enteritidis (S. Enteritidis) is the most common pathogen that presents alarming rates of antimicrobial resistance. The aim of this study was to analyse the antimicrobial resistance of 124 S. Enteritidis strains collected from retail foods in 39 cities across China and to conduct a comprehensive molecular characterisation of their antimicrobial resistance genes (ARGs) and class 1 integrons through PCR amplification, antibiotic susceptibility testing, and conjugation assays. The strains exhibited a multidrug resistance rate as high as 68.5% and were frequently resistant to nalidixic acid (97.6%) and ampicillin (74.2%). Correspondingly, the analysis of quinolone genes revealed that 97.5% of the strains harboured single mutations in gyrA; some possessed the plasmid-mediated quinolone resistance genes qnrS (0.8%), oqxAB (2.4%), as well as the blaTEM-1 (67.7%); and the extended spectrum beta-lactamase (ESBL) genes blaCTX-M-55 were detected in 2.4% of the strains. Class 1 integrons were observed in 11.3% (14/124) of the strains and integrated three different gene cassettes, namely, aadA22 (1/14), dfrA1-aadA1 (5/14), and dfrA17-aadA5 (8/14). Conjugation experiments and antimicrobial susceptibility testing showed that the integrons including antimicrobial resistance genes could be transferred to the recipient strain via mobile plasmids, rendering the recipient strain resistant. Both multilocus variable-number tandem-repeat analysis (MLVA) and core genome multilocus sequence typing (cgMLST) show a better discrimination in this study, hence a combination of MLVA and cgMLST analyses would improve the accuracy and cost-effectiveness. This study provides a systematic and comprehensive analysis of the antimicrobial resistance profiles of S. Enteritidis strains isolated from retail food in China. The present data will help to gain insight into the public health impact of S. Enteritidis and guide the development of control strategies.
... broccoli, brussel sprouts, and cabbages. These vegetables typically contain ~1 uM/g of sulphoraphane [90]. It should be noted that cooking may decrease their sulphoraphane content [90]. ...
... These vegetables typically contain ~1 uM/g of sulphoraphane [90]. It should be noted that cooking may decrease their sulphoraphane content [90]. Multiple in vitro studies have shown that sulforaphane can act as a histone deacetylase inhibitor (HDACi) and thereby cause decreased proliferation of PC cells [91]. ...
... Key The effect of domestic cooking processes on myrosinase stability varied among cabbage morphotypes and accessions and will be discussed in more detail later. The stability of myrosinase in different Brassica vegetables under different processing conditions has been discussed by several authors [24][25][26][27]39,55]. Differences in myrosinase stability as a result of cooking can be attributed to the maximum core temperature of the vegetable during heating. ...
... The results obtained in this study are similar to those observed by several authors during the thermal processing of Brassica vegetables [25,29,32,35,55,89]. This study adds to the findings of previous researchers; however, the study is particularly conclusive as it demonstrates similar findings across cabbage morphotypes and accessions. ...
Article
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Glucosinolate hydrolysis products are responsible for the health-promoting properties of Brassica vegetables. The impact of domestic cooking on the myrosinase stability, glucosinolates and hydrolysis products in 18 cabbage accession was investigated. Cabbages were steamed, microwaved, and stir-fried before analysis. Cooking significantly affected myrosinase stability and glucosinolate concentrations within and between cabbage morphotypes. Myrosinase was most stable after stir-frying, with up to 65% residual activity. Steaming and microwaving resulted in over 90% loss of myrosinase activity in some accessions. Stir-frying resulted in the greatest decrease in glucosinolate concentration, resulting in up to 70% loss. Steamed cabbages retained the highest glucosinolates after cooking (up to 97%). The profile and abundance of glucosinolate hydrolysis products detected varied across all cooking methods studied. Cooking reduced the amounts of nitriles and epithionitriles formed compared to raw samples. Steaming led to a significant increase in the concentration of beneficial isothiocyanates present in the cabbage and a significantly lower level of nitriles compared to other samples. Microwaving led to a reduction in the concentrations of both nitriles and isothiocyanates when compared to other cooking methods and raw cabbage. The results obtained help provide information on the optimal cooking methods for cabbage, suggesting that steaming may be the best approach to maximising beneficial isothiocyanate production.
... ESPs are responsible for the conversion of GLs to nitriles [1] and are less heat stable than MYR. Their inactivation allows for the preferential formation of ITCs [21,22]. Any type of prolonged high temperature heating, however, may cause MYR denaturation [5,19,21] and GL thermal degradation [21]. ...
Article
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There are no known physiological-based digestion models that depict glucoraphanin (GR) to sulforaphane (SR) conversion and subsequent absorption. The aim of this research was to make a physiological-based digestion model that includes SR formation, both by endogenous myrosinase and gut bacterial enzymes, and to simulate the SR bioavailability. An 18-compartment model (mouth, two stomach, seven small intestine, seven large intestine, and blood compartments) describing transit, reactions and absorption was made. The model, consisting of differential equations, was fit to data from a human intervention study using Mathwork’s Simulink and Matlab software. SR urine metabolite data from participants who consumed different broccoli products were used to estimate several model parameters and validate the model. The products had high, medium, low, and zero myrosinase content. The model’s predicted values fit the experimental values very well. Parity plots showed that the predicted values closely matched experimental values for the high (r2 = 0.95), and low (r2 = 0.93) products, but less so for the medium (r2 = 0.85) and zero (r2 = 0.78) myrosinase products. This is the first physiological-based model to depict the unique bioconversion processes of bioactive SR from broccoli. This model represents a preliminary step in creating a predictive model for the biological effect of SR, which can be used in the growing field of personalized nutrition.
... Van Eylen et al. (2009) reported that high-pressure treatment of broccoli (Brassica oleracea var. italica) heads at 300 MPa for 35 minutes caused glucoraphanin to be converted into sulforaphane, while Matusheski et al. (2004) found that mild heating of fresh broccoli sprouts or florets to 60 prior to homogenization accelerated this conversion process. Pérez et al. (2014) proposed an optimized process consisting of blanching at 57 for 13 minutes. ...
Article
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Concentration of sulforaphane in broccoli is known to be enhanced during storage under an atmosphere of low O2 (0.002–1.3%) and high CO2 (20.6–25.4%). However, maintaining this concentration at a maximum is problematic. Therefore, storage of broccoli florets at low temperatures was investigated. Results showed that storage for 2 days under hypoxia significantly increased (3.3-fold against initial value) sulforaphane concentration. Subsequently, 4 days storage at three temperature levels was conducted. Sulforaphane levels were not maintained at 20℃ (normal temperature) or 1℃ (cold temperature). However, after storage at -20℃ (frozen) for 4 days, the sulforaphane concentration was not significantly reduced compared with the maximum level on day 2. Meanwhile, this concentration was not significantly higher than those at 20℃ or 1℃ on the same day. Storage under hypoxic conditions is known to affect the flavor and taste of vegetables. Therefore, eight taste factors were objectively measured using an electronic tongue. Astringency and bitterness were stable and independent of storage period and temperature, while other tastes were significantly affected by storage period. The rich umami value at -20℃ was significantly higher than that at 20℃, and in general it was challenging to maintain taste values even for cold or frozen storage.
... In an in vivo study in rats, sulforaphane had substantially more potent anticarcinogenic activity than sulforaphane nitrile, a similarly formed product of glucoraphanin hydrolysis (Matusheski and Jeffery, 2001). Myrosinase activity as a driver of sulforaphane production in broccoli also increased if fresh broccoli florets and sprouts were heated to 60 • C before homogenization instead of 70 • C; this also decreased formation of sulforaphane nitrile (Matusheski et al., 2004). As an alternative to lower temperature heat treatment to retain sulforaphane, and as a possibility for enhancing the bioactive compound content of boiled (and thus inactivated myrosinase) broccoli, research by Ghawi et al. has shown that the addition of powdered mustard seeds to heat processed (e.g. ...
Article
Although extensive resources are dedicated to the development and study of cancer drugs, the cancer burden is expected to rise by about 70% over the next 2 decade. This highlights a critical need to develop effective, evidence-based strategies for countering the global rise in cancer incidence. Except in high-risk populations, cancer drugs are not generally suitable for use in cancer prevention owing to potential side effects and substantial monetary costs (Sporn, 2011). There is overwhelming epidemiological and experimental evidence that the dietary bioactive compounds found in whole plant-based foods have significant anticancer and chemopreventative properties. These bioactive compounds often exert pleiotropic effects and act synergistically to simultaneously target multiple pathways of cancer. Common bioactive compounds in fruits and vegetables include carotenoids, glucosinolates, and polyphenols. These compounds have been shown to target multiple hallmarks of cancer in vitro and in vivo and potentially to address the diversity and heterogeneity of certain cancers. Although many studies have been conducted over the past 30 y, the scientific community has still not reached a consensus on exactly how the benefit of bioactive compounds in fruits and vegetables can be best harnessed to help reduce the risk for cancer. Different stages of the food processing system, from "farm-to-fork," can affect the retention of bioactive compounds and thus the chemopreventative properties of whole foods, and there are opportunities to improve handling of foods throughout the stages in order to best retain their chemopreventative properties. Potential target stages include, but are not limited to, pre- and postharvest management, storage, processing, and consumer practices. Therefore, there is a need for a comprehensive food-system-based approach that not only taking into account the effects of the food system on anticancer activity of whole foods, but also exploring solutions for consumers, policymakers, processors, and producers. Improved knowledge about this area of the food system can help us adjust farm-to-fork operations in order to consistently and predictably deliver desired bioactive compounds, thus better utilizing them as invaluable chemopreventative tools in the fight to reduce the growing burden of cancer worldwide.
... Numerous epidemiological studies have also indicated that sulforaphane (4-methylsulfinylbutyl isothiocyanate) is effective in preventing cancers (Cartea and Velasco 2008;Huang et al. 2016;Matusheski et al. 2004). Previous studies showed that Se-containing organic compounds also play important roles in animal physiological processes as anticarcinogenic agents (Gandin et al. 2018;Chen et al. 2013). ...
Article
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The effects of S and Se treatment on cabbage, especially the interactions of S and Se metabolism with the biosynthesis of glucosinolate (GSL), including glucoraphanin, which is a major aliphatic GSL in cruciferous vegetables and the precursor of the anticancer compound sulforaphane, were examined. Cabbage plants were treated with sulfate and selenite (SeO3²⁻), and the total S, Se, and GSL contents of cabbage head and outer foliage leaves were measured. Results showed that selenite treatment was beneficial to GSL biosynthesis and Se accumulation in cabbage head and outer foliage leaves. GSL synthesis was induced by exogenous selenite-elevated sulfate treatment at certain concentration ratios, i.e., 50-μΜ selenite + 1-mΜ sulfate or 100-μΜ selenite + 4-mΜ sulfate. A high exogenous sulfate concentration was more favorable to GSL accumulation than a low sulfate concentration. According to the relative expression of genes on GSL synthesis, an increase in the GSL content was attributed to the upregulation of gene expression and possible transportation from the outer foliage leaf to the head of cabbage. These results might be helpful for increasing the health benefits of cabbage by supplying exogenous S and Se. Further research should explore the effects of sulfate and selenite on GSL precursor substances to reveal the reason why total GSL contents increased.
... Various Brassica vegetables, especially broccoli, contain glucoraphanin (sulforophane or SFN). Following cutting or chewing, it is hydrolyzed into the corresponding isothiocyanate SFN either by the plant thioglucosidase myrosinase or by bacterial thioglucosidases in the colon [118]. Because of its lipophilicity [119] and molecular size, SFN is likely to passively diffuse into the enterocytes [120]. ...
... As previously mentioned, when the plant tissue is cut, chopped, mixed, or chewed, a β-thioglucosidase called myrosinase is released. This enzyme can improve the bioaccessibility and bioavailability of cruciferous glucosinolates through the formation of anti-cancer metabolites such as isothiocyanates and indole-3-carbinol [113]. When this enzyme is inactivated by the high temperature of cooked vegetables or some extraction methods to formulate dietary supplements, the hydrophilic nature of glucosinolates allows it to be hydrolyzed into active metabolites by the colon intestinal microbiota [114]. ...
Article
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The gastrointestinal (GI) tract is composed of rapidly renewing cells, which increase the likelihood of cancer. Colorectal cancer is one of the most frequently diagnosed GI cancers and currently stands in second place regarding cancer-related mortality. Unfortunately, the treatment of GI is limited, and few developments have occurred in the field over the years. With this in mind, new therapeutic strategies involving biologically active phytocompounds are being evaluated as anti-cancer agents. Vegetables such as broccoli, brussels sprouts, cabbage, cauliflower, and radish, all belonging to the Brassicaceae family, are high in dietary fibre, minerals, vitamins, carotenoids, polyphenols, and glucosinolates. The latter compound is a secondary metabolite characteristic of this family and, when biologically active, has demonstrated anti-cancer properties. This article reviews the literature regarding the potential of Cruciferous vegetables in the prevention and/or treatment of GI cancers and the relevance of appropriate compound formulations for improving the stability and bioaccessibility of the major Cruciferous compounds, with a particular focus on glucosinolates.
... In our study, ITCs were the default rearrangement products of the aglucones (Figure 1) in the GSL extracts treated with the commercial myrosinase, supporting previous studies. 45,46 Figure 2B shows the presence of 14 GSLs before hydrolysis with myrosinase. Figure 2C indicates that after incubation with myrosinase and NAC at 50°C for 4 h, all 14 GSLs were hydrolyzed, peaks corresponding to 12 NAC−ITCs and 3 unknown peaks appeared. ...
Article
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A new method to simultaneously analyze various glucosinolates (GSLs) and isothiocyanates (ITCs) by RP-UHPLC-ESI-MSn has been developed and validated for 14 GSLs and 15 ITCs. It involved derivatization of ITCs with N-acetyl-L-cysteine (NAC). The limits of detection were 0.4-1.6 µM for GSLs and 0.9-2.6 µM for NAC-ITCs. The analysis of Sinapis alba, Brassica napus, and B. juncea extracts spiked with 14 GSLs and 15 ITCs indicated that the method generally had good intra- (≤ 10% RSD) and inter-day precisions (≤ 16% RSD). Recovery of the method was unaffected by the extracts and within 71-110% for GSLs and 66-122% for NAC-ITCs. The method was able to monitor the enzymatic hydrolysis of standard GSLs to ITCs in mixtures. Furthermore, GSLs and ITCs were simultaneously determined in Brassicaceae plant extracts before and after myrosinase treatment. This method can be applied to further investigate the enzymatic conversion of GSLs to ITCs in complex mixtures.
... The highest percentage of this epithionitrile in the essential oil was unexpected and even opposite to the findings of Matusheski et al. who reported that epithiospecifier protein is heat-sensitive. [27] The main difference among volatile compounds in these isolates was the presence of ethyl isothiocyanate which was quantitatively important constituent of autolysate and the main constituent of hydrolysate, but was absent in the essential oil. ...
... During the isolation of EOs by hydrodistillation from B. nigra no epithionitriles were found. The ESP activity is known to significantly decrease at temperature of 50 °C or higher [34]. On the other hand, soaking in water before microwave isolation enabled formation of epithionitriles. ...
Article
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Mustard seeds are used as a food, and spice due to their nutritive value, the presence of biologically active compounds, and specific taste. Its morphology was investigated by optical microscopy and scanning electron microscopy before and after isolation by hydrodistillation and microwave assisted distillation. The chemical composition of volatile compounds in different mustard seeds (Brassica juncea L. and Brassica nigra L.) was determined using GC–MS, after applying various techniques (Clevenger hydrodistillation, microwave assisted distillation—MAD, and microwave hydrodiffusion and gravity—MHG). Allyl isothiocyanate, degradation product of allyl glucosinolate, was the main volatile compound in B. juncea essential oils and extract after all applied extraction techniques (91.07–99.01%). Gastrointestinal stability of allyl isothiocyanate was determined employing two-phase digestion model (gastric and duodenal) by two methods (in vitro digestion method using commercial and ex vivo digestion method using human digestive enzymes). The stability rate of allyl isothiocyanate was higher after the gastric digestion phase by both methods. In B. nigra seeds but-3-enyl and allyl isothiocyanate were the main compounds after Clevenger hydrodistillation (80.58% and 15.39%, respectively). After MAD, and MHG the main compounds were 4,5-epithiopentanenitrile (50.70% and 59.93%, respectively), and 3,4-epithiobutanenitrile (7.61% and 25.97%, respectively), originating from the same glucosinolates, i.e. but-3-enyl and allyl glucosinolate, respectively. Antiproliferative activity of mustard seed EOs and extracts was evaluated against human cancer cell lines (MDA-MB-231 and TCCSUP). The best antiproliferative activity was shown for B. nigra MHG extract against MDA-MB-231 cell line with IC50 value of 9.1 µg/mL.
... It has been reported that sulforaphane yield from glucoraphanin in fresh broccoli and its sprouts is very low, and a non-bioactive sulforaphane nitrile forms along due to the existence of ESP, a modifier protein of myrosinase (Matusheski, Juvik, & Jeffery, 2004;Mithen et al., 2003). On the other hand, the modifier protein, Epithiospecifier modifier protein (ESM1) is conductive to sulforaphane formation from glucoraphanin by interfering with ESP (Angelino & Jeffery, 2014;Ku et al., 2013). ...
Article
CaCl2, Ca²⁺ chelator (EGTA) and Ca²⁺ channel blocker (verapamil) were used to investigate mechanism of glucoraphanin metabolism in broccoli sprouts under ZnSO4 stress. CaCl2 treatment promoted sprout growth, reduced MDA (malonaldehyde) content and electrolyte leakage in sprouts under ZnSO4 stress. The highest MDA content and electrolyte leakage were obtained in ZnSO4 plus verapamil-treated sprouts. In addition, ZnSO4 plus CaCl2 treatment significantly enhanced glucoraphanin content and sulforaphane formation, while an opposite result was observed after ZnSO4 plus EGTA treatment; which were further supported by expression of glucoraphanin biosynthetic and hydrolytic genes as well as myrosinase (MYR) and epithiospecifier protein (ESP) activities. These results indicated that exogenous and endogenous calcium promoted glucoraphanin biosynthesis and the conversion rate of glucoraphanin into sulforaphane. Verapamil treatment also stimulated glucoraphanin biosynthesis, but exerted an adverse influence on sulforaphane formation from the hydrolysis of glucoraphanin because of much higher ESP expression and ESP activity than ZnSO4 treatment.
... Consequently, the cooking process likely denatured broccoli's endogenous thermo-labile myrosinase preventing hydrolysis of the glucosinolates. Sulforaphane was low in raw broccoli but higher levels of sulforaphane nitrile were present, which agrees with previous literature [46][47][48]. The formation of sulforaphane nitrile rather than sulforaphane in raw broccoli is due to the action of epithiospecifier protein (ESP), a non-catalytic cofactor of myrosinase that promotes the formation of epithionitriles from alkenyl glucosinolates. ...
Article
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PurposeStudies on broccoli (Brassica oleracea var. italica) indicate beneficial effects against a range of chronic diseases, commonly attributed to their bioactive phytochemicals. Sulforaphane, the bioactive form of glucoraphanin, is formed by the action of the indigenous enzyme myrosinase. This study explored the role that digestion and cooking practices play in bioactivity and bioavailability, especially the rarely considered dose delivered to the colon.Methods The antimicrobial activity of sulforaphane extracts from raw, cooked broccoli and cooked broccoli plus mustard seeds (as a source myrosinase) was assessed. The persistence of broccoli phytochemicals in the upper gastrointestinal tract was analysed in the ileal fluid of 11 ileostomates fed, in a cross-over design, broccoli soup prepared with and without mustard seeds.ResultsThe raw broccoli had no antimicrobial activity, except against Bacillus cereus, but cooked broccoli (with and without mustard seeds) showed considerable antimicrobial activity against various tested pathogens. The recovery of sulforaphane in ileal fluids post soup consumption was < 1% but the addition of mustard seeds increased colon-available sulforaphane sixfold. However, when sulforaphane was extracted from the ileal fluid with the highest sulforaphane content and tested against Escherichia coli K12, no inhibitory effects were observed. Analysis of glucosinolates composition in ileal fluids revealed noticeable inter-individual differences, with six “responding” participants showing increases in glucosinolates after broccoli soup consumption.Conclusions Sulforaphane-rich broccoli extracts caused potent antimicrobial effects in vitro, and the consumption of sulforaphane-enriched broccoli soup may inhibit bacterial growth in the stomach and upper small intestine, but not in the terminal ileum or the colon.
... 7 ESP is considered to be the myrosinase cofactor and has the ability to catalyze the conversion of glucoraphanin into sulforaphane nitrile, a component that has little biological activity. 8 It has been reported that a higher pH (5−7) enhanced sulforaphane formation whereas a lower pH (<3) would promote the production of sulforaphane nitrile. 9 As we know, the human stomach is a highly acidic environment (pH 1−4), 10 which may be adverse to the formation of sulforaphane. ...
... In such a condition, the formation of SFN increases by 3-7 times. [7] Raw or freshly harvested broccoli should be used whenever possible. A delay of 10 days might result in loss of GPN by nearly 80%. ...
Article
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Isothicyanates present in cruciferous vegetables are known to exhibit chemoprevention by various mechanisms. Presently, there is growing evidence that a phytochemical compound known as sulforaphane in these green leafy vegetables is found to be effective in preventing and treating various cancers such as prostate cancer, breast cancer, colon cancer, skin, urinary bladder and oral cancers. This component is naturally present in the broccoli sprouts, kale, cabbage, cauliflower and garden cress and is available as a commercial supplementary pill called Broccoli extract. Availability of many bioactive substances such as vitamins, polyphenols, sulfides, glucosinolates and antioxidants makes broccoli consumption important in daily diet regularly. Researchers have named it as “Green chemoprevention.” It is easily affordable and more cost‑effective than the traditional chemopreventive drugs. Results from the epidemiological and experimental studies have emphasized the role of sulforophane as a complementary or alternative chemopreventive agent.
... Glucosinolates are the vital groups of health-promoting secondary metabolites in Brassicaceae vegetables. The hydrolysis of glucosinolates through myrosinase activity yields sulforaphane, which plays a beneficial role in the prevention and treatment of several diseases [2]. Many epidemiological studies indicated that diets rich in Brassica vegetables helped with reducing the risk of cancers and regulated immune and inflammatory responses [3,4]. ...
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To investigate the effects of supplementary UV-A intensity on growth and antioxidant compounds in Chinese kale (Brassica alboglabra Bailey) baby-leaves, three different UV-A intensity treatments (5, 10, 15 W·m−2, respectively) were applied 10 days before harvest in artificial light plant factory. In Chinese kale baby-leaves, supplemental 5 and 10 W·m−2 UV-A (UVA-5 and UVA-10) were beneficial for inter-node length, stem diameter, canopy diameter, fresh weight and dry weight, particularly in UVA-10 treatment, while these above-mentioned growth parameters all significantly decreased in UVA-15 treatment. The soluble sugar content decreased under UVA-5, but there was no significant difference under UVA-10 and UVA-15. Soluble protein contents decreased under UVA-5 and UVA-10, but significantly increased under UVA-15. UVA-10 played a predominant role in increasing FRAP and contents of total phenolics and total flavonoids compared to other treatments. Contents of total glucosinolates (GLs), aliphatic GLs and indolic GLs in Chinese kale baby-leaves significantly increased with UV-A intensity increasing, and the highest contents were found under UVA-15. The percentage of total aliphatic GLs (about 80%) was significantly higher than those of total indolic GLs. Glucobrassicanapin and sinigrin were two major individual GLs in Chinese kale baby-leaves, variation trends of which were consistent with the contents of total GLs and aliphatic GLs. From the heatmap analysis, and taking economic benefits into account, UVA-10 might be optimal for the production of high-quality Chinese kale baby-leaves in an artificial light plant factory.
... Matusheski et al. [15] studied the effect of temperature on the formation of SR and sulforaphane nitrile in fresh broccoli florets and broccoli sprouts from several commercial cultivars. They found that heating fresh broccoli florets or broccoli sprouts to 60 °C prior to homogenization increased the content of SR and decreased the content of sulforaphane nitrile, while heating to 70 °C and above decreased the formation of both compounds in broccoli florets, but not in broccoli sprouts. ...
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Broccoli sprouts contain 10–100 times higher levels of sulforaphane than mature plants, something that has been well known since 1997. Sulforaphane has a whole range of unique biological properties, and it is especially an inducer of phase 2 detoxication enzymes. Therefore, its use has been intensively studied in the field of health and nutrition. The formation of sulforaphane is controlled by the epithiospecifier protein, a myrosinase co-factor, which is temperature-specific. This paper studies the influence of temperature, heating time, the addition of myrosinase in the form of Raphanus sativus sprouts in constant ratio to broccoli sprouts, and other technological steps on the final sulforaphane content in broccoli sprout homogenates. These technological steps are very important for preserving sulforaphane in broccoli sprouts, but there are some limitations concerning the amount of sulforaphane. We focused, therefore, on the extraction process, using suitable β-cyclodextrin, hexane and ethanol, with the goal of increasing the amount of sulforaphane in the final extract, thus stabilizing it and reducing the required amount sulforaphane needed, e.g., as a dietary supplement.
... italica) 9 . On the basis of earlier work and preliminary results, the author, a retired research scientist diagnosed with Parkinson's disease in 2018, established a method to make a standardized broccoli-seed tea containing sulforaphane [9][10][11][12] , based upon dose calculations reviewed in 12 . ...
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Over the last two decades, cutting-edge research has demonstrated how the transcription factor Nrf2 can be activated to attenuate oxidative stress and inflammation in neurons but this hypothesis has not been officially tested on Parkinson’s disease patients. Here we report on a series of eight independent (n-of-1) experiments carried out at their own initiative by a group of eight people with Parkinson’s disease using the potent Nrf2 activator sulforaphane, prepared from broccoli seeds. The results show a marked reduction in the mean scores for non-motor symptoms compared to motor symptoms over the six-week duration of the experiment. These results suggest that quite different processes are involved in the development of these two symptoms groups and that it may be possible to slow or interrupt the process driving non-motor symptoms. The results are provisionally interpreted in terms of a cascade of events in the pathogenesis of Parkinson’s disease. In this model, age-related chronic oxidative stress triggers mitochondrial dysfunction in dopaminergic neurons and is the cause of non-motor symptoms. Upregulating Nrf2 can potentially reverse this step. The second step of the cascade is the loss of the neurons as a consequence of energy shortages due to the mitochondrial dysfunction. This step is believed to be the cause of motor symptoms and is most likely irreversible.
... Myrosinase activity has been shown to be positively correlated with the sulforaphene content in cold-stored radish roots (Lee et al. 2017). Sulforaphene is a by-product of GRH hydrolysis, which acts as a GSL precursor (Matusheski et al. 2004). The decrease in the GRH content and increase in the GRE content after the drying treatment could be related to their common metabolic pathway (Nugroho et al. 2020). ...
Article
The aboveground parts of radish (Raphanus sativus L.) are typically discarded after harvesting the roots. However, numerous bioactive compounds have been identified in radish leaves, and dried leaf products have garnered attention as a health food owing to their high fiber content. Here, we assessed the morphological and physiological characteristics of the leaves of 14 commercial radish cultivars and the effects of postharvest drying treatment on their glucosinolate (GSL) contents. The morphological and physiological characteristics of radish leaves exhibited considerable variation, depending on the cultivar. ‘Baekbong’ and ‘Baekja’ presented higher values for the assessed morphological and physiological characteristics than other cultivars. ‘Baekja’ had the best growth parameters, including leaf length and fresh and dry weights, among the tested cultivars. The total and individual GSL content was the highest in the cultivars ‘Sincheongilpum’ and ‘Taebaek’ at harvest. However, after 10 d of postharvest drying in a greenhouse at ambient temperature, the GSL content was the highest in the cultivars ‘Cheongbok plus’ and ‘Taebaek’. The results of the multivariate data analyses indicated that ‘Taebaek’ is a highly distinctive cultivar in terms of shoot physiology, morphology, and GSL content. The GSL content and profiles were differentially affected by both radish cultivar and postharvest drying treatment.
... Similar results have been observed for white and red cabbage (Yen and Wei, 1993) where a treatment at 70°C for 30 min was necessary to reach 90% loss in myrosinase activity while myrosinase in crude extract was sensitive from 40-50°C. In the same way, Matusheski et al. (2004) have noticed that broccoli myrosinase was remained fully active after treating broccoli tissue at 60°C for 10 min. Peréz et al. (2014) have reported that myrosinase was stable up to 70°C. ...
Thesis
Ce travail de thèse est dédié à la mise en place d’une stratégie de valorisation optimale et raisonnée des cultures intermédiaires piège à nitrates de B. juncea (moutarde brune) dans un concept de bioraffinerie et de développement durable. Les graines de moutarde sont riches en huile (>35%) mais également en glucosinolates (≈ 120 µmol/g), facteurs antinutritionnels produisant de ce fait des tourteaux impropres à l’alimentation du bétail alors qu’ils sont riches en protéines de qualité. Ce travail de thèse a pour ambition de lever ce verrou en extrayant ces facteurs antinutritionnels de la graine pour exploiter leur activité biologique comme agent de contrôle des ennemis de cultures. Le schéma de bioraffinage proposé intègre les acteurs en amont et en aval de la chaîne de transformation. La première partie de travail a porté sur l’inactivation de la myrosinase, enzyme endogène responsable de l’hydrolyse des glucosinolates (sinigrine and gluconapine). Trois procédés d’inactivation ont été testés : (1) le chauffage conventionnel (2) le chauffage micro-ondes (MO) et (3) l’inactivation par CO2 supercritique (SC-CO2). Les trois procédés se sont montrés efficaces. Un chauffage à 80°C pendant 70 min a été retenu pour inactiver l’enzyme (98%) sans dégrader les composés d’intérêt (glucosinolates, protéines) tout en améliorant l’extractibilité de l’huile. Les graines ont ensuite subi un double pressage à 80 bars pendant 60 min à l’aide d’une presse hydraulique du laboratoire pour en extraire 84% d’huile et obtenir un tourteau enrichi en protéines (35.8-40.8%) et en glucosinolates intacts (144-158 µmol/g). La récupération des GSL a été entreprise en couplant une extraction hydro-alcoolique avec différents prétraitements (broyage, décharges électriques de haute tension, ultrasons) pour intensifier l’extraction des GSL tout en limitant la co-extraction des protéines. Une optimisation préliminaire d’extraction chimique a montré que 90% des GSL pouvaient être récupérés via une solution hydro-alcoolique d’éthanol (40% v:v) à 40°C sans altérer la qualité protéique du tourteau résiduel (36-40% protéines). Ces conditions viennent ainsi contrebalancer le procédé classique d’extraction/élimination des GSL par méthanol à haute température (75°C), contestable sur le plan environnemental, énergétique et sanitaire. L’extraction assistée par DEHT (U = 40 kV, tDEHT = 3.5 ms) a permis d’extraire dans des conditions douces (T = 30°C, eau et à faible coût énergétique (233 kJ/kg)) la quasi-totalité des GSL (98 %) dans leur état intact tout en minimisant la co-extraction des protéines qui sont préservées dans le résidu solide. Enfin, la purification du jus riche en GSL a été étudiée selon deux voies : chromatographie d’échange d’ions (IEC) et ultrafiltration (UF). La récupération des glucosinolates par IEC a été optimisée en batch via l’utilisation d’une résine fortement basique (PA312LOH) qui a permis de séparer les GSL des protéines. 72.9% de la sinigrine a été récupérée après élution avec une solution de NaCl (1.0 M, 30°C, 300 rpm, 40 mL/g résine) avec une pureté 79.6%. Les expériences réalisées en dynamique (purification sur colonne) ont permis la récupération de 28% de la gluconapine (2.6 BV/h, pH 4.0). Le procédé membranaire (UF) a présenté de meilleures performances avec 98% et 60% de sinigrine et gluconapine récupérés respectivement dans un perméat à 90% de pureté (membrane PES 10 kDa, 5 bars, 500 rpm). L’extrait purifié obtenu par UF (90 %) riche en sinigrine (3.36 mg/ml) et en gluconapine (0.16 mg/ml) sera utilisée pour développer une formule phytobiotique qui sera utilisée en biofumigation. Le résidu solide détoxifié (<20 µmol GSL/g MS) et riche en protéines (~40%) pourra être valorisé en alimentation animale.
... 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.
... Among glucosinolates products, isothiocyanates are preferred due to their significant health-promoting properties like chemopreventive, antimicrobial, antiinflammatory and antithrombotic effects (Traka and Mithen, 2009). Isothiocyanate formation is favored by several factors like acidification (below pH 5) and thermal degradation of specific proteins (ESP) (70 C/5 min was enough to inactivate ESP) (Hanschen et al., 2017;Matusheski et al., 2004). Nevertheless, heat treatments may also inactivate endogenous plant myrosinase, as it was observed after blanching at 86 C (Dosz and Jeffery, 2013). ...
Article
The effect of revalorized Bimi leaves (B) and/or mustard (M) addition, as supplementary ingredients, to develop an innovative kale (K) pesto sauce was studied. Microbial, physicochemical (color, total soluble solids content -SSC-, pH and titratable acidity –TA-) and sensory quality were studied during 20 days at 5 °C. Bioactive compounds changes (total phenolics, total antioxidant capacity and glucoraphanin contents) were also monitored throughout storage. The high TA and pH changes in the last 6 days of storage were avoided in the K+B pesto when adding mustard, due to the antimicrobial properties of this brassica seed. SSC was increased when B + M were added to the K pesto, which positively masked the kale-typical bitterness. Mustard addition hardly change yellowness of the K pesto, being not detected in the sensory analyses, showing K+B+M pesto the lowest color differences after 20 days of shelf life. The addition of Bimi leaves to the K pesto enhanced its phenolic content while mustard addition did not negatively affect such total antioxidant compounds content. Finally, mustard addition effectively aimed to glucoraphanin conversion to its bioactive products. Conclusively, an innovative kale pesto supplemented with Bimi by-products was hereby developed, being its overall quality well preserved up to 20 days at 5 °C due to the mustard addition.
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Red cabbage contains glucosinolates, precursors to health-promoting isothiocyanates. However, raw cabbage often releases mainly epithionitriles and nitriles from glucosinolates. To increase isothiocyanate formation, the effect of acid usage in the preparation of red cabbage was evaluated. Moreover, the effects of the chosen boiling method (acidic boiled red cabbage versus neutral boiled blue cabbage) on glucosinolate degradation were investigated using UHPLC-DAD-ToF-MS and GC-MS. The addition of vinegar significantly increased isothiocyanate formation of cabbage salad from 0.09 to 0.21 µmol/ g fresh weight, while lemon juice only slightly increased isothiocyanate formation. Acidic boiled red cabbage degraded glucosinolates and increased nitrile formation, while in neutral boiled blue cabbage, glucosinolates were stable. However, shortly boiled blue cabbage (5 min) had the highest isothiocyanate levels (0.08 µmol/ g fresh weight). Thus, for a diet rich in isothiocyanates it is recommended to acidify raw cabbage salads and prepare shortly boiled blue cabbage instead of red cabbage.
Chapter
The sedentary lifestyle coupled with continuously changing food habits and search for nutrient-dense enriched protective foods has resulted in increased demand and consumption of natural foods. Among natural dietary ingredients, vegetables are the daily consumed dietary ingredients packed with vitamins, minerals, antioxidants, and an array of bioactive phytochemicals. Amongst vegetable crops, cruciferous vegetables like broccoli, cabbage, cauliflower, arugula, horseradish, mustard green, bok choy, brussels sprouts, etc. are the crops which are perceived far important than the mere table items for daily consumption owing to their rich functional bioactive profile. Presence of the sulfur-rich compounds (methyl cysteine sulfoxide and glucosinolates), coloring pigments (carotenoids, anthocyanins), minerals (Se, Fe, K, Ca), vitamins (B complex and C), dietary fiber, and other bioactive compounds (phytoalexins, terpenes, tocopherols, hydroxycinnamic acid, chlorogenic acid, ferulic acid, synapic acid and flavonols) give them the distinctive nutraceutical status with well documented therapeutic benefits. Most of the research effort in the last decade has been directed to effectively find out the exact mode of action of these bioactive compounds on health with their minimum effective concentration, means to ensure their effective delivery to the target organs, and increased bioavailability of these compounds. Though, there is substantial evidence based on in vivo and in vitro findings that scientifically demonstrate their benefits more research needs to be conducted with an exploration of the unknown beneficial activities as well as the unwanted effects. Future research should be directed towards the functional enrichment either through genetic modifications or through regulation of pathways for ensuring the national and health security to the general population and the health-conscious people.
Article
An in vitro study was conducted to determine effects of reducing pH of porcine in vitro fermentation medium on compositions of glucosinolate degradation products and porcine in vitro fermentation characteristics for solvent-extracted canola meal (SECM) and cold-pressed canola cake (CPCC). Two canola co-products were subjected to porcine in vitro fermentation for 72 h. Accumulated gas production during microbial fermentation was recorded and modeled to estimate kinetics of gas production. Glucosinolate degradation products after microbial fermentation were determined and fermentation medium pH after incubation was recorded. Total and individual volatile fatty acids (VFA) concentrations per unit weight of dry matter (DM) of feedstuffs were determined. On DM basis, SECM and CPCC contained 6.15 and 11.1 µmol/g of glucosinolates, respectively. Goitrin concentration for CPCC was lower (P < 0.05) than that for SECM. Isothiocyanate and indole-3-acetonitrile concentrations for CPCC were lower (P < 0.05) than those for SECM, whereas thiocyanate production for CPCC was greater (P < 0.05) than that for SECM. A reduction in fermentation medium pH from 6.2 to 5.2 increased (P < 0.05) goitrin and indole-3-acetonitrile concentrations and decreased (P < 0.05) isothiocyanates concentration for SECM, but did not affect concentration of these 2 glucosinolate degradation products for CPCC. Fermentation medium pH after in vitro fermentation for SECM tended to be greater (P = 0.081) than that for CPCC. Canola co-product type and fermentation medium pH did not interact on gas production parameters. However, a reduction in fermentation medium pH from 6.2 to 5.2 resulted in increased (P < 0.05) lag time and reduced (P < 0.05) fractional rate of degradation and total gas production. Canola co-product type and fermentation medium pH did not interact on total or individual VFA production per gram of DM of feedstuff fermented. However, reducing fermentation medium pH from 6.2 to 5.2 increased (P < 0.05) total VFA and acetate productions, and tended to reduce (P = 0.083) branched-chain VFA production SECM and CPCC. In conclusion, a reduction in fermentation medium pH from 6.2 to 5.2 resulted in increased production of goitrin and indole-3-acetonitriles from SECM-derived glucosinolates, but did not affect production of thiocyanate from SECM-derived glucosinolates. Glucosinolates in CPCC were less degradable than those in SECM, and were less affected by the fermentation medium pH used in the current study. It appears that there are other factors other than pH that affect the degradation of canola-derived glucosinolates by microorganisms from hindgut of pigs.
Chapter
Isothiocyanates are derived from their naturally-occurring glucosinolate precursors, which are abundant in cruciferous vegetables. Numerous scientific studies beginning more than half a century ago have documented the chemoprotective activities of these compounds. Isothiocyanates have numerous protein targets through which they exert protection in the context of various diseases such as cancer, neurodegeneration, inflammatory disease, metabolic disease and infection. The major mechanisms by which the isothiocyanates confer protection involve induction of stress response pathways that restore the cellular redox and protein homeostasis, and contribute to resolution of inflammation. However, high concentrations of isothiocyanates cause cell cycle arrest and selectively kill cancer cells by inducing apoptosis, autophagy or necrosis. In this review, we present readers with a detailed overview of isothiocyanates functions and discuss their molecular targets and antineoplastic effects. Furthermore, we provide an up-to-date summary of the evidence on the chemoprotective activities of the most widely-studied isothiocyanates: sulforaphane, phenethyl isothiocyanate (PEITC) and benzyl isothiocyanate (BITC).
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The list of known health benefits from inclusion of brassica vegetables in the diet is long and growing. Once limited to cancer prevention, a role for brassica in prevention of oxidative stress and anti-inflammation has aided in our understanding that brassica provide far broader benefits. These include prevention and treatment of chronic diseases of aging such as diabetes, neurological deterioration, and heart disease. Although animal and cell culture studies are consistent, clinical studies often show too great a variation to confirm these benefits in humans. In this review, we discuss causes of variation in clinical studies, focusing on the impact of the wide variation across humans in commensal bacterial composition, which potentially result in variations in microbial metabolism of glucosinolates. In addition, as research into host–microbiome interactions develops, a role for bitter-tasting receptors, termed T2Rs, in the gastrointestinal tract and their role in entero-endocrine hormone regulation is developing. Here, we summarize the growing literature on mechanisms of health benefits by brassica-derived isothiocyanates and the potential for extra-oral T2Rs as a novel mechanism that may in part describe the variability in response to brassica among free-living humans, not seen in research animal and cell culture studies.
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Metabolic profiling of glucosinolates and their breakdown products in sprouts of 22 Chinese kale (Brassica oleracea var. alboglabra, BOA) varieties were investigated by using high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). Relationships between glucosinolate metabolites and flavor of Chinese kale sprouts were also analyzed. Results showed that compositions and contents of both glucosinolates and their breakdown products varied greatly among different varieties of Chinese kale sprouts. Gluconapin and 4,5-Epithio-pentanenitrile were the dominant glucosinolate and glucosinolate breakdown product in Chinese kale sprouts, respectively. Gluconapin and glucobrassicin were significantly related to bitterness (r = 0.577, 0.648, respectively; p < 0.05). BOA 1 and BOA 13, BOA 3 and BOA 10 are good candidates for future breeding programs since the former two varieties have light bitterness and pungency, and the latter two varieties contain high levels of glucosinolate breakdown products such as isothiocyanates and epithionitriles in sprouts.
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Innate immunity, particularly macrophages, is critical for intestinal homeostasis. Sulforaphane, a dietary isothiocyanate from cruciferous vegetables, has been reported to protect against intestinal inflammation. However, the role of macrophages in sulforaphane mediated intestinal inflammation and the underlying molecular mechanisms have not been studied yet. In this study, sulforaphane effectively attenuated dextran sodium sulphate (DSS) induced intestinal inflammation in murine model. Of note, sulforaphane skewed the switching from classically (M1) to alternatively (M2) activated phenotype both in intestinal and bone marrow-derived macrophages (BMDMs). The expression levels of M1 associated maker genes induced by DSS or lipopolysaccharide (LPS) plus interferon gamma-γ (IFN-γ) were suppressed by sulforaphane while M2 marker gene expression levels were improved. This resulted in alteration of inflammatory mediators, particularly interleukin-10 (IL-10), both in colon tissues and culture medium of BMDMs. Subsequently, IL-10 was found to mediate the sulforaphane induced M2 phenotype switching of BMDMs through the activation of STAT3 signaling. This was confirmed by immunofluorescence analysis with increased number of p-STAT3-positive cells in the colon sections. Moreover, anti-IL-10 neutralizing antibody significantly interfered M2 phenotyping of BMDMs induced by sulforaphane with reduced STAT3 phosphorylation. Findings here introduced a potential utilization of sulforaphane for intestinal inflammation treatment with macrophages as the therapeutic targets.
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The isothiocyanate sulforaphane (SF) is one of the most potent naturally occurring Phase 2 enzymes inducers derived from brassica vegetables like broccoli, cabbage, brussel sprouts, etc. Ingestion of broccoli releases SF via hydrolysis of glucoraphanin (GRP) by plant myrosinase and/or intestinal microbiota. However, both SF and plant myrosinase are thermal-labile, and the epithiospecifier protein (ESP) directs the hydrolysis of GRP toward formation of sulforaphane nitrile instead of SF. In addition, bacterial myrosinase has low hydrolyzing efficiency. In this review, we discuss strategies that could be employed to improve the stability of SF, increase SF formation during thermal and non-thermal processing of broccoli, and enhance the myrosinase-like activity of the gut microbiota. Furthermore, new cooking methods or blanching technologies should be developed to maintain myrosinase activity, and novel thermostable myrosinase and/or microbes with high SF producing abilities should also be developed.
Chapter
The Himalayan region is home to diverse ecological systems covering many important flora and fauna that are indispensable for human livelihood. Himalayan medicinal plants have great value in the traditional systems of medicine such as Ayurveda, Chinese traditional medicine, and Tibetan traditional medicine systems and other indigenous medicine systems. Due to extreme environmental conditions, climate change, and other anthropogenic factors, there is huge biodiversity loss, and various medicinal plants are endangered. Similarly, these environmental challenges also affect the content of bioactive chemical constituents in medicinal plants. This chapter summarizes some of these aspects of Himalayan medicinal plants and their conservation and environmental challenges with a focus on the bioactive chemical constituents.
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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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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).
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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 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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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
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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.
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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.
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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.
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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.
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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.
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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.
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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.