No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Heating decreases epithiospecifier protein activity and increases SF formation in broccoli
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.
... Interestingly, it has been found that heating decreases ESP activity and results in increased SRA formation in broccoli florets and sprouts. Matusheski et al. (2004) showed that heating fresh florets to 60 • C for 5 min markedly reduced ESP activity and increased the extraction yield of SRA. The same effect was seen when fresh sprouts were heated to 70 • C for 10 min [34][35][36]. ...
... Matusheski et al. (2004) showed that heating fresh florets to 60 • C for 5 min markedly reduced ESP activity and increased the extraction yield of SRA. The same effect was seen when fresh sprouts were heated to 70 • C for 10 min [34][35][36]. The difference in heat lability of these two enzymes may be exploited in food product development. ...
... and results in increased SRA formation in broccoli florets and sprouts. Matusheski et al. (2004) showed that heating fresh florets to 60 °C for 5 min markedly reduced ESP activity and increased the extraction yield of SRA. The same effect was seen when fresh sprouts were heated to 70 °C for 10 min [34][35][36]. ...
Our research group previously found that broccoli sprouts possess neuroprotective effects during pregnancy. The active compound has been identified as sulforaphane (SFA), obtained from glucosinolate and glucoraphanin, which are also present in other crucifers, including kale. Sulforaphene (SFE), obtained from glucoraphenin in radish, also has numerous biological benefits, some of which supersede those of sulforaphane. It is likely that other components, such as phenolics, contribute to the biological activity of cruciferous vegetables. Notwithstanding their beneficial phytochemicals, crucifers are known to contain erucic acid, an antinutritional fatty acid. The aim of this research was to phytochemically examine broccoli, kale, and radish sprouts to determine good sources of SFA and SFE to inform future studies of the neuroprotective activity of cruciferous sprouts on the fetal brain, as well as product development. Three broccoli: Johnny’s Sprouting Broccoli (JSB), Gypsy F1 (GYP), and Mumm’s Sprouting Broccoli (MUM), one kale: Johnny’s Toscano Kale (JTK), and three radish cultivars: Black Spanish Round (BSR), Miyashige (MIY), and Nero Tunda (NT), were analyzed. We first quantified the glucosinolate, isothiocyanate, phenolics, and DPPH free radical scavenging activity (AOC) of one-day-old dark- and light-grown sprouts by HPLC. Radish cultivars generally had the highest glucosinolate and isothiocyanate contents, and kale had higher glucoraphanin and significantly higher sulforaphane content than the broccoli cultivars. Lighting conditions did not significantly affect the phytochemistry of the one-day-old sprouts. Based on phytochemistry and economic factors, JSB, JTK, and BSR were chosen for further sprouting for three, five, and seven days and subsequently analyzed. The three-day-old JTK and radish cultivars were identified to be the best sources of SFA and SFE, respectively, both yielding the highest levels of the respective compound while retaining high levels of phenolics and AOC and markedly lower erucic acid levels compared to one-day-old sprouts.
... In raw broccoli, the main product of conversion during such processes is sulforaphane nitrile due to the active ESP present in the broccoli matrix. Several approaches have been assessed to enhance sulforaphane formation in broccoli products such as mild heat treatment to selectively inactivate ESP while maintaining myrosinase activity [9], high pressure thermal processing [10], lactic acid fermentation [11], and a combination of mild heat with lactic acid fermentation [12]. For instance, mild heating to inactivate ESP combined with fermentation enabled a ≈16-time increase in sulforaphane yield in broccoli puree compared to non-treated broccoli puree, which was attributed to the selective inactivation of ESP and fermentation-induced increase in the accessibility of glucoraphanin for myrosinase-catalysed conversion into sulforaphane. ...
... The initial measurable content of GR in the non-processed broccoli floret was 3149.35 µmol/kg DW, which is slightly lower than the GR levels of 4.4-16.4 µmol/g DW reported by Matusheski, Juvik [9]. The measurable GR content dramatically decreased from 3149.35 to 689, 813, and 1241 µmol/kg DW, respectively, in the control, pre-heated, and thermosonicated broccoli puree samples ( Figure 1A), most likely due to myrosinasecatalyzed conversion. ...
... In intact broccoli tissue, myrosinase and its substrate glucoraphanin are in separate compartments, and hence there is no myrosinase-catalyzed conversion in intact broccoli. During pureeing and thermophysical treatments, tissue decompartmentalisation occurs, which leads to myrosinase-glucoraphanin interaction and conversion of glucoraphanin to its metabolites (sulforaphane and sulforaphane nitrile, depending on the presence of active ESP and the pH of the matrix) [6,9,12]. The GR content in thermal or TS pre-treated purees were significantly higher (p < 0.05) than those in control puree ( Figure 1A). ...
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.
... 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). ...
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.
... These conditions guarantee microbiological safety without an increase in temperature during the process (around 35 • C), while maintaining the nutritional value and fresh characteristics of the product [19]. On the other hand, the US can increase the shelf-life of vegetable products through cell disruption without negatively impacting their bioactive compounds [20,21]. The effectiveness of these emerging technologies has been demonstrated in different foods. ...
... Then, carrots were cut into transverse slices, while broccoli stalks were cut into longitudinal portions, taking care to remove browned areas. After that, the cut vegetables were blanched in a microwave oven to inactivate the browning enzymes [2,21,24]. For that, samples of 300 g of each product were exposed twice to 700 W during 2 min, with 1 min between the first exposition and the second. ...
Vegetable beverages are a convenient strategy to enhance the consumption of horticultural commodities, with the possibility of being fortified with plant by-products to increase functional quality. The main objective was to develop a new veggie beverage from broccoli stalks and carrot by-products seasoned with natural antioxidants and antimicrobial ingredients. Pasteurization, Ultrasound (US), and High Hydrostatic Pressure (HHP) and their combinations were used as processing treatments, while no treatment was used as a control (CTRL). A shelf-life study of 28 days at 4 °C was assayed. Microbial load, antioxidant capacity, and bioactive compounds were periodically measured. Non-thermal treatments have successfully preserved antioxidants (~6 mg/L ΣCarotenoids) and sulfur compounds (~1.25 g/L ΣGlucosinolates and ~5.5 mg/L sulforaphane) throughout the refrigerated storage, with a longer shelf life compared to a pasteurized beverage. Total vial count was reduced by 1.5–2 log CFU/mL at day 0 and by 6 log CFU/mL at the end of the storage in HHP treatments. Thus, the product developed in this study could help increase the daily intake of glucosinolates and carotenoids. These beverages can be a good strategy to revitalize broccoli and carrot by-products with high nutritional potential while maintaining a pleasant sensory perception for the final consumer.
... 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. ...
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.
... 14 Being the principal hydrolysis product of ITC, sulforaphane is produced from its precursor glucoraphanin and is considered as one of the most potent inducers of phase II detoxification enzyme quinone reductase (QR). 15 Thus, sulforaphane can reduce DNA damage by capturing the activated carcinogens. 15 Several species of Brassica have been investigated for their bioactive phytochemicals and health-promoting values that support their use in traditional medicine. ...
... 15 Thus, sulforaphane can reduce DNA damage by capturing the activated carcinogens. 15 Several species of Brassica have been investigated for their bioactive phytochemicals and health-promoting values that support their use in traditional medicine. For instance, turnip (Brassica rapa) has been traditionally used for the treatment of human diseases. ...
... 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. ...
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.
... The higher BCN content in the HY isolated using MHG can be explained by the presence of an epithiospecific protein due to its interaction with the enzyme myrosinase, which redirects the reaction toward the formation of epithionitrile or nitrile depending on the glucosinolate structure [25]. The epithiospecific protein is heat sensitive and its activity decreases significantly at high temperatures (>50 • C), which is probably the reason for the lower content of BCN and higher content of BITC in the HY isolated using MAE extraction technique [26]. The obtained results agree with those published so far [2,8,27]. ...
Tropaeolum majus L. is a traditional medicinal plant with a wide range of biological activities due to the degradation products of the glucosinolate glucotropaeolin. Therefore, the goals of this study were to identify volatiles using gas chromatography–mass spectrometry analysis (GC-MS) of the hydrosols (HYs) isolated using microwave-assisted extraction (MAE) and microwave hydrodiffusion and gravity (MHG). Cytotoxic activity was tested against a cervical cancer cell line (HeLa), human colon cancer cell line (HCT116), human osteosarcoma cell line (U2OS), and healthy cell line (RPE1). The effect on wound healing was investigated using human keratinocyte cells (HaCaT), while the antibacterial activity of the HYs was tested against growth and adhesion to a polystyrene surface of Staphylococcus aureus and Escherichia coli. Antiphytoviral activity against tobacco mosaic virus (TMV) was determined. The GC-MS analysis showed that the two main compounds in the HYs of T. majus are benzyl isothiocyanate (BITC) and benzyl cyanide (BCN) using the MAE (62.29% BITC and 15.02% BCN) and MHG (17.89% BITC and 65.33% BCN) extraction techniques. The HYs obtained using MAE showed better cytotoxic activity against the tested cancer cell lines (IC50 value of 472.61–637.07 µg/mL) compared to the HYs obtained using MHG (IC50 value of 719.01–1307.03 μg/mL). Both concentrations (5 and 20 µg/mL) of T. majus HYs using MAE showed a mild but statistically non-significant effect in promoting gap closure compared with untreated cells, whereas the T. majus HY isolated using MHG at a concentration of 15 µg/mL showed a statistically significant negative effect on wound healing. The test showed that the MIC concentration was above 0.5 mg/mL for the HY isolated using MAE, and 2 mg/mL for the HY isolated using MHG. The HY isolated using MHG reduced the adhesion of E. coli at a concentration of 2 mg/mL, while it also reduced the adhesion of S. aureus at a concentration of 1 mg/mL. Both hydrosols showed excellent antiphytoviral activity against TMV, achieving100% inhibition of local lesions on the leaves of infected plants, which is the first time such a result was obtained with a hydrosol treatment. Due to the antiphytoviral activity results, hydrosols of T. majus have a promising future for use in agricultural production.
... A second sample was similarly processed 6 days postharvest. For SFN measurements, samples were heated to 60 • C in water for 10 min to inhibit epithiospecifier protein activity [25], followed by homogenization and 1 h incubation with 2 mg/mL myrosinase (Sinapis alba thioglucosidase, MilliporeSigma, St. Louis, MO, USA) at 60 • C to fully convert GRN to SFN (SFN equivalence). For GRN measurements, samples were heated to 100 • C in water for 10 min to inactivate all enzyme activities, followed by homogenization, and extracted 3 times in water. ...
Cruciferous vegetable consumption is associated with numerous health benefits attributed to the phytochemical sulforaphane (SFN) that exerts antioxidant and chemopreventive properties, among other bioactive compounds. Broccoli sprouts, rich in SFN precursor glucoraphanin (GRN), have been investigated in numerous clinical trials. Broccoli microgreens are similarly rich in GRN but have remained largely unexplored. The goal of this study was to examine SFN bioavailability and the microbiome profile in subjects fed a single serving of fresh broccoli microgreens. Eleven subjects participated in a broccoli microgreens feeding study. Broccoli microgreens GRN and SFN contents and stability were measured. Urine and stool SFN metabolite profiles and microbiome composition were examined. Broccoli microgreens had similar GRN content to values previously reported for broccoli sprouts, which was stable over time. Urine SFN metabolite profiles in broccoli microgreens-fed subjects were similar to those reported previously in broccoli sprouts-fed subjects, including the detection of SFN-nitriles. We also reported the detection of SFN metabolites in stool samples for the first time. A single serving of broccoli microgreens did not significantly alter microbiome composition. We showed in this study that broccoli microgreens are a significant source of SFN. Our work provides the foundation for future studies to establish the health benefits of broccoli microgreens consumption.
... The content of glucosinolates and their breakdown products in Brassica spp. is dependent on several environmental and developmental factors (Velasco et al., 2007). The I3C and SRA HPLC data ( Figure 2) indicate that the kale genotypes used in this work are a potential good source of glucoraphanin, representing the native glucosinolate precursor of the sulphoraphane, well noted for its health promoting properties (Matusheski et al., 2004). ...
... The alkenyl GSLs give rise to epithioalkanes in the presence of epithiospecifier protein (Lambrix et al. 2001). Myrosinase and epithiospecifier proteins, in particular, have a lower thermal tolerance (70 and 60 degrees Celsius, respectively) (Matusheski et al. 2004). Thus, the cooking processes have been extensively researched to maximize the higher recovery of ITCs (Bongoni et al. 2014;Dosz and Jeffery 2013;Rungapamestry et al. 2006). ...
... Sulforaphane and sulforaphane nitrile are byproducts of glucosinolate hydrolysis by cytosolic endogenous myrosinase. 13 The myrosinase enzyme activity, the presence of other proteins like epithiospecifier protein (ESP), and conditions like temperature and pH play a role in this hydrolysis process and compounds that are generated. 14 Today, mineral deficiency, especially of those minerals that are essential for the nourishment of both human beings and animals through various agricultural products, is a determining factor in crop production. ...
Due to the growing world population and increasing environmental stress, improving the production, nutritional quality, and pharmaceutical applications of plants has become an urgent need. Therefore, the current research was designed to investigate the impact of seed priming using plant-growth-promoting bacteria (PGPB) along with selenium nanoparticles (SeNPs) treatment on chemical and biological properties of three Brassica oleracea cultivars [Southern star (VA1), Prominence (VA2), Monotop (VA3)]. With this aim, one out of five morphologically different strains of bacteria, namely, JM18, which was further identified via 16S rRNA gene sequencing as a Nocardiopsis species with strong plant-growth-promoting traits, isolated from soil, was used. To explore the growth-promoting potential of Nocardiopsis species, seeds of three varieties of B. oleracea were primed with JM18 individually or in combination with SeNP treatment. Seed treatments increased sprout growth (fresh and dry weights) and glucosinolate accumulation. The activity of myrosinase was significantly increased through brassica sprouts and consequently enhanced the amino-acid-derived glucosinolate induction. Notably, a reduction in effective sulforaphane nitrile was detected, being positively correlated with a decrease in epithiospecifier protein (EP). Consequently, the antioxidant activities of VA2 and VA3, determined by the ferric reducing antioxidant power (FRAP) assay, were increased by 74 and 79%, respectively. Additionally, the antibacterial activities of JM18-treated cultivars were improved. However, a decrease was observed in SeNP-and JM18 + SeNP-treated VA2 and VA3 against Serratia marcescens and Candida glabrata and VA1 against S. marcescens. In conclusion, seed priming with the JM18 extract is a promising method to enhance the health-promoting activities of B. oleracea sprouts.
... Broccoli, an edible plant in Brassica, is rich in a variety of functional, active ingredients beneficial to human health, such as isothiocyanates (ITCs), sulforaphane (SFN), glucosinolates (GLs), phenolic compounds, etc. [1][2][3]. Shapiro et al. [4] showed that high consumption of broccoli could reduce the incidence of cancer, which is mainly related to ITCs [5]. In plants, ITCs are hydrolysates produced by hydrolysis of GLs under the action of myrosinase (MYR, EC3.2.1.147) ...
Broccoli sprouts have high isothiocyanate and selenium accumulation capacity. This study used a combination of methods, including physiological and biochemical, gene transcription and proteomic, to investigate the isothiocyanate and endogenous selenium accumulation mechanisms in broccoli sprouts under exogenous sodium selenite treatment during germination. Compared with the control, the sprouts length of broccoli sprouts under exogenous selenium treatment was significantly lower, and the contents of total phenol and malondialdehyde in 6-day-old broccoli sprouts were substantially higher. The contents of isothiocyanate and sulforaphane in 4-day-old were increased by up-regulating the relative expression of genes of UGT74B1, OX-1, and ST5b. The relative expression of BoSultr1;1, BoSMT, BoHMT1, and BoCOQ5-2 genes regulating selenium metabolism was significantly up-regulated. In addition, 354 proteins in 4-day-old broccoli sprouts showed different relative abundance compared to the control under selenium treatment. These proteins were classified into 14 functional categories. It was discovered that metabolic pathways and biosynthetic pathways of secondary metabolites were significantly enriched. The above results showed that exogenous selenium was beneficial in inducing the accumulation of isothiocyanate and selenium during the growth of broccoli sprouts.
... Moreover, there was also a significant correlation and effect of EGCG dose on the degree of cataract opacity in diabetic rat models. This illustrates that the higher the administration of EGCG, the milder the lens opacity that occurs [14][15][16] . According to our research hypothesis, EGCG is a compound primarily contained in green tea and has a potent antioxidant effect. ...
Aim:
To evaluate the effect of epigallocatechin gallate (EGCG) in preventing lens opacity and the aggregation of lens αB-crystallin in model rats of diabetes mellitus (DM).
Methods:
This experimental study included Wistar rats for DM as in vivo models and divided into 5 groups. The treatment groups were administered EGCG by orally for 20d and were then assessed for their degree of lens opacity with binocular microscope and lens αB-crystallin expression from Western blot analyze.
Results:
Pearson correlation test and regression analysis on EGCG exposure and final random blood sugar (RBS) obtained a significance level of P<0.05. EGCG exposure can significantly lower RBS with an R 2 of 0.5634 (56.34%). The same analysis on EGCG exposure and the degree of lens opacity obtained a significance level of P<0.05 and increased exposure to EGCG can significantly lower the degree of lens opacity with an R 2 of 0.8577 (85.77%). Correlation analysis between EGCG and the expression of lens αB-crystallin can be concluded that the higher the EGCG exposure administered, the higher the native lens αB-crystallin expression and the lower the aggregate lens αB-crystallin expression. There was also significant effect in which every 1 mg/kg body weight dose of EGCG can increase the native lens αB-crystallin expression by 0.0063 and decrease the aggregate lens αB-crystallin expression by 0.0076.
Conclusion:
The administration of EGCG at a dose of 300, 600, and 1200 mg shows a significant effect on preventing lens opacity and aggregation of αB-crystallin in diabetic rat models and this research could be a biomolecular prevention of cataract.
... The SFN content in pure unprocessed broccoli was 2.05 µmol/g dry weight (dw), which was reduced down to 1.06 µmol/g dw after vacuum cooking [90]. As it is known, broccoli processing at low temperatures (less than 50 • C) is more suitable, as it promotes SFN formation [95], as the epithiospecific protein activity is prominent at this temperature [96]. However, the same authors suggested that a cooking temperature of up to 60 • C allows the formation of SFN. ...
In the last decade, most of the evidence on the clinical benefits of including cruciferous foods in the diet has been focused on the content of glucosinolates (GSL) and their corresponding isothiocyanates (ITC), and mercapturic acid pathway metabolites, based on their capacity to modulate clinical, biochemical, and molecular parameters. The present systematic review summarizes findings of human studies regarding the metabolism and bioavailability of GSL and ITC, providing a comprehensive analysis that will help guide future research studies and facilitate the consultation of the latest advances in this booming and less profusely researched area of GSL for food and health. The literature search was carried out in Scopus, PubMed and the Web of Science, under the criteria of including publications centered on human subjects and the use of Brassicaceae foods in different formulations (including extracts, beverages, and tablets), as significant sources of bioactive compounds, in different types of subjects, and against certain diseases. Twenty-eight human intervention studies met inclusion criteria, which were classified into three groups depending on the dietary source. This review summarizes recent studies that provided interesting contributions, but also uncovered the many potential venues for future research on the benefits of consuming cruciferous foods in our health and well-being. The research will continue to support the inclusion of GSL-rich foods and products for multiple preventive and active programs in nutrition and well-being.
... It is well-known that the formation of these nitriles is affected by the presence of epithiospecifier protein. Our results are in accordance with the findings of Matusheski et al. (2004) who reported that this protein is heat-sensitive. In our previous paper, two approaches were used, autolysis followed by hydrodistillation and exogenous myrosinase hydrolysis followed by solvent extraction (Bla zevi c et al. 2011). ...
Two wild-growing Brassicaceae plants of Croatian origin, Aurinia leucadea (Guss.) K. Koch and Lepidium draba L., were investigated to uncover glucosinolates via GC/MS analysis of their degradation products. The main constituents of Aurinia leucadea (Guss.) K.Koch distillate were hex-5-enenitrile (28.8%) and but-3-enyl isothiocyanate (18.8%), while 4,5-epithiopentanenitrile (50%) and 5,6-epithiohexanenitrile (18.5%) were the main volatile compounds in autolysate. 4-(Methylsulfanyl)butyl isothiocyanate (96.4%) constituted almost the entire Lepidium draba L. distillate, while the autolysate was characterized by 4-(methylsulfinyl)butyl isothiocyanate (57.3%). So, regarding the glucosinolate degradation products, the main glucosinolates of A. leucadea were glucobrassicanapin and gluconapin, and of L. draba glucoerucin and glucoraphanin.
... The interaction of the ESP with the enzyme myrosinase diverts the reaction towards the production of epithionitriles or nitriles, depending on the glucosinolate structure (33). Given that ESP is thermally sensitive, it is known that its activity decreases significantly at 50 °C or higher (34), which is the main reason for the low presence of BCN in the essential oil of T. majus. On the other hand, soaking for 1 h in water before MHG technique enabled the formation of BCN, which explains its high percentage in the the seed extract. ...
Research background. Plant Tropaeolum majus L. (garden nasturtium) belongs to the family Tropaeolaceae and contains benzyl glucosinolate. The breakdown product of benzyl glucosinolate, benzyl isothiocyanate (BITC), exhibits various biological activities such as antiproliferative, antibacterial and antiinflammatory. In order to optimize the content of biologically active volatile compounds in plant extract and essential oil, the use of appropriate extraction technique has a crucial role.
Experimental approach. The current study investigates the effect of two modern extraction methods, microwave-assisted distillation (MAD) and microwave hydrodiffusion and gravity (MHG), on the chemical composition of volatile components present in the essential oil and extract of garden nasturtium (T. majus L. var. altum) seeds. Investigation of the biological activity of samples (essential oil, extract and pure compounds) was focused on the antiproliferative effect against different cancer cell lines: cervical cancer cell line (HeLa), human colon cancer cell line (HCT116) and human osteosarcoma cell line (U2OS), and the antibacterial activity which was evaluated against the growth and adhesion of Staphylococcus aureus and Escherichia coli to polystyrene surface.
Results and conclusions. Essential oil and extract of garden nasturtium (T. majus) seeds were isolated by two extraction techniques: MAD and MHG. BITC and benzyl cyanide (BCN) present in the extract were identified by gas chromatography-mass spectrometry. Essential oil of T. majus showed higher antiproliferative activity (IC50
... Our results are similar to the results of previous studies that have indicated a significant increase in the sulforaphane content of broccoli subjected to heating [34]. e study of Matusheski et al. [35] also reported that preheating broccoli florets and sprouts to 60°C significantly increased the sulforaphane in vegetable tissue extracts following crushing. In addition, we observed that the contents of two types of isothiocyanates (erucin and PEITC) also increased in the hot air-dried samples (Figure 3). ...
Cabbage (Brassica oleracea var. capitata f. alba), a cruciferous vegetable, is one of the most widely consumed vegetables worldwide. However, research on whether there are differences in its contents of phytochemicals and biological activities according to the drying method for each portion of the vegetable remains insufficient. The present study investigated the contents of representative polyphenols and isothiocyanates, the antioxidant capacity, and the antiproliferative effects among six leaf layers of cabbage subjected to hot air and freeze-drying. High-performance liquid chromatography analysis showed that most phenolic and flavonoid bioactive compounds were significantly accumulated in the outer cabbage leaf layer (P1), whereas isothiocyanates were most abundant in the leaf layer close to the core of the head (P5). The contents of isothiocyanates, gallic acid, epicatechin, p-coumaric acid, sinapic acid, and myricetin were significantly higher in the hot air-dried sample than in the freeze-dried sample, whereas the contents of catechin hydrate, chlorogenic acid, 4-hydroxybenzoic acid, and rutin hydrate were significantly higher in the freeze-dried sample. Compared to other leaf layers, P1 exhibited high antiproliferative efficacy against pancreatic, breast, and gastric cancer cells. P1 also showed excellent DPPH·(EC 50–4.208 ± 0.033 and 4.611 ± 0.053 mg/mL for hot air and freeze-dried samples, respectively) and ABTS· (2.422 ± 0.068 and 2.224 ± 0.070 mg/mL for hot air and freeze-dried samples, respectively) radical-scavenging effects. These results indicate that the contents of polyphenols and isothiocyanates in cabbage may vary depending on the leaf layer and the drying method. Our findings provide insight for applying appropriate food drying methods that can be used to produce cabbage leaf-based products with enhanced bioactivity.
... ESP hinders the synthesis of ITCs but it has been observed that cooking at about 60 o C denatures the ESP and favors the formation of ITCs rather than epithionitriles. 12,13 Nowadays, researchers are keen to study these compounds as they have been linked to prevention and cure (to some extent), some of the most cataclysmic diseases for human health. For instance, isothiocyanates are reported to react with phase I and phase II enzymes to detoxify carcinogens and excrete them out of the body, inhibit cytochrome p450, and suppress cancer cell proliferation thus preventing cancer. ...
Isothiocyanates are the highly reactive organo-sulphur phytochemicals and are product of hydrolysis of glucosinolates which are present mainly in the cruciferous vegetables. These compounds due to their unique chemical reactivity possess anti-cancer, anti-inflammatory, and neuroprotective properties. Epidemiological and experimental evidences suggest that isothiocyanates reduce oxidative stress and act as indirect antioxidants as well as antimicrobials, therefore, have received attention from the researchers for their possible application in pharmacological and food industry. However, due to high volatility and heat sensitivity of these bioactive compounds, their extraction is very challenging and requires the application of various innovative technologies. In addition to that, their fate during the processing conditions also needs to be considered as these processes tend to affect their bioavailability. Isothiocyanates exhibit wide range of antimicrobial activity due to their ability of reducing oxygen consumption and depolarizing the mitochondrial membrane in bacterial cells. They are generally regarded as safe (GRAS) compounds and hence are allowed to be added to the food as preservatives. Due to their antimicrobial properties, isothiocyanates incorporated food packaging films have become popular in the last decade. They are known to act as substrates to activate lactoperoxidases (LPO) for extension of shelf life of dairy products due to its bactericidal and bacteriostatic properties. This review addresses the detailed evidences supporting the biological activities, bioavailability and stability, methods of extraction, and explanation for their taste perception, as well as utilization of these isothiocyanates in food packaging as natural antimicrobials or natural preservatives to improve shelf life of foodstuffs.
... Freezing at -20°C increased the ITCs formation in broccoli sprouts . Heating at 60°C prior to homogenisation improved the sulforaphane formation but decreased sulforaphane nitrile formation in broccoli florets (Matusheski et al., 2004a). It has been observed that there was higher epithiospecifier protein (ESP) activity resulted in the formation of nitrile during GLs hydrolysis, while there was a decrease in the formation of ITCs . ...
The present work investigated the effects of soaking followed by heating on the metabolism of glucosinolates (GLs) in rocket seeds and sprouts. Soaking for 1 h increased myrosinases (MYR) activity of seeds. Heating at 70°C for 10 min after soaking resulted in the highest isothiocyanates (ITCs) formation in seeds. Both immerse-heating and steam-heating decreased the total GLs content in seeds and 2-day old sprouts. However, steam-heating showed minor effect on total GLs content when compared with immerse-heating. Both methods showed a decreased MYR activity in seeds and sprouts. Nevertheless, heating had a positive impact on ITCs formation. Under immerse-heating, ITCs formation was the highest in seeds and sprouts at 70°C heating. However, under steam-heating, ITCs formation in seeds and sprouts was higher at 70 and 60°C, respectively. The results indicated that steam-heating treatment was effective for improving ITCs formation in rocket seeds and 2-day old sprouts.
... Especially, broccoli seeds contain the highest glucoraphanin; hence, they are a good source of preparation of sulforaphane. However, the conversion rate of glucoraphanin to sulforaphane is very low due to the presence of epithiospecifier protein (ESP) [11]. It has been reported that the heat sensitivity of ESP protein is higher than that of myrosinase. ...
In this study, microwave pretreatment and grinding treatment were used to enhance sulforaphane formation, then ultrasonic-assisted extraction (UAE) was applied to extract sulforaphane using simultaneous hydrolysis and extraction method. The effects of various parameters, which were ultrasonic time,ultrasonic power, solid-water ratio and solid-ethyl acetate ratio on the extraction rate of sulforaphane were investigated. The results showed that microwave pretreatment enhanced sulforaphane formation. Excessive size reduction did not increase or even reduced extraction rate of sulforaphane. Simultaneous hydrolysis and extraction significantly increased extraction rate of sulforaphane compared to hydrolysis followed by extraction. UAE accelerated mass transfer and the solubilization of the targeted compounds due to the acoustic cavitation effect, thus enhanced enzymatic hydrolysis of glucoraphanin and the extraction rate of sulforaphane. The extraction rate of sulforaphane using UAE with simultaneous hydrolysis and extraction was 4.07-fold of the conventional extraction method. UAE was an effective method to extract sulforaphane from broccoli seeds since it led to higher yield of sulforaphane in a much shorter extraction time.
... The presence of epithiospecifier protein (ESP) disrupts the process of glucoraphanin hydrolysis, reducing the bioavailability of sulforaphane and sulforaphane nitrile, with the nitrile form being less active in its binding to pharmacological targets [102] . Since ESP is temperature insensitive, heating the broccoli at 60 ºC decreases the formation of sulforaphane nitrile [103] . Once absorbed, sulforaphane is conjugated with glutathione and metabolized by the meracaptopurine pathway; it is then excreted as N-acetylcysteine conjugates [104] . ...
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.
... Penelitian menunjukkan bahwa peningkatan konsumsi brokoli dapat menurunkan risiko kanker payudara (Pledgie-Tracy et al., 2007), kulit (Tahata et al., 2018) (Matusheski et al., 2004), Gas Chromatography (GC) (Wieczorek & Jelen, 2019), GC dengan Mass Spectrometry (GC-MS) (Rahmatia, 2016), Solid Phase Extraction (Han & Row, 2011), Column Chromatography (Hafezian et al., 2019), Low-Speed Countercurrent Chromatography (LPCC) (Hao Liang et al., 2005), dan Macroporous Resin Adsorption Chromatography (Li et al., 2008). Karena metode yang ada mahal, elaboratif dan tidak sesuai untuk pemurnian pada skala industri. ...
Brokoli (Brassica oleracea L.) merupakan sayuran dalam keluarga Brassicaceae (Cruciferae) yang kaya akan senyawa bioaktif, salah satunya adalah sulforaphane. Penelitian menunjukan bahwa senyawa sulforaphane dalam brokoli memiliki potensi sebagai agen anti kanker. Metode isolasi dan analisa sulforaphane secara sederhana dan murah dibutuhkan untuk mendapatkan sulforaphane murni dengan mengoptimalkan jenis pelarut dan fase gerak dalam proses ekstraksi dan pemisahannya. Senyawa sulforaphane diperoleh melalui proses ekstraksi, fraksinasi dan purifikasi serta proses identifikasi menggunakan NMR dan FT-IR. Isolat hasil purifikasi diuji aktivitas antikanker dengan metode 3-(4,5-dimethyl thiazolyl-2 - yl)-2, 5- diphenyl tetrazolium bromide (MTT) terhadap sel T47D. Hasil penelitian menunjukan sulforaphane dapat di isolasi dan di analisis dengan FTIR serta H-NMR dengan metode ekstraksi yang sederhana. Hasil isolasi dan identifikasi senyawa diperoleh satu isolat, yaitu sulforaphane yang merupakan senyawa golongan isothiocyanate. Nilai IC50 terhadap sel T47D dari ekstrak brokoli, fraksi etil esetat brokoli, dan isolat masing-masing sebesar 46.853,00; 25.559,00; dan 109.950,00 µg/ml.
... 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]. ...
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.
... Chemical composition of glucosinolate degradation products are affected by parent glucosinolate type (substrate) and reaction conditions including pH values, epithiospecifier protein (ESP; a non-catalytic cofactor of myrosinase), and availability of ferrous ions (Cartea and Velasco, 2008;Agerbirk et al., 2009). The ESP is susceptible to heat and is denatured by heat at 60 • C (Matusheski et al., 2004), implying that the ESP in canola co-products is mostly inactivated during the process of oil extraction. Conventional diets for pigs contain iron (Liu et al., 2014). ...
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.
... 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. ...
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.
... 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]. ...
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.
... 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]. ...
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. ...
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.
... 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. ...
... 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. ...
... 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 . ...
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). ...
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.
Microgreens contain an abundance of antioxidants (glucosinolates, carotenoids, phenolic compounds), vitamins (C, E, K), minerals (iron, magnesium), and other nutrients (fibre, omega‐3 fatty acids). They have been shown to promote cardiovascular health, reduce inflammation, combat cancer, manage diabetes, protect the nervous system, and improve digestion. This review investigates the antioxidant content and health implications of microgreens, providing current insights and prospective outlooks. The types of antioxidants found in microgreens, as well as the factors that influence their content, such as species, growing conditions, and harvesting time, are discussed. The review highlights antioxidants' role in cardiovascular health, inflammation reduction, cancer defence, diabetes management, and nervous system protection. The antioxidant properties of microgreen varieties are discussed. The bioavailability of antioxidants from microgreens, culinary applications, and incorporation into the diet are elaborated. The section of future perspectives covers the commercial potential of microgreens, optimal harvest timing, research directions, and potential functional food and nutraceutical uses. The aim and scope of this review are to provide a comprehensive understanding of the antioxidant content in microgreens and their associated health benefits. It also seeks to identify gaps in current research and suggest avenues for future studies, especially concerning their relevance in human health and nutrition.
Sulforaphane (SFN) is an isothiocyanate commonly found in cruciferous vegetables. It is formed via the enzymatic hydrolysis of glucoraphanin by myrosinase. SFN exerts various biological effects, including anti-cancer, anti-oxidation, anti-obesity, and anti-inflammatory effects, and is widely used in functional foods and clinical medicine. However, the structure of SFN is unstable and easily degradable, and its production is easily affected by temperature, pH, and enzyme activity, which limit its application. Hence, several studies are investigating its physicochemical properties, stability, and biological activity to identify methods to increase its content. This article provides a comprehensive review of the plant sources, extraction and analysis techniques, in vitro and in vivo biological activities, and bioavailability of SFN. This article highlights the importance and provides a reference for the research and application of SFN in the future.
The health-beneficial effects of Brassica vegetables are mainly attributed to their high contents of glucosinolates and the products of their hydrolysis, especially isothiocyanates. Distribution of glucosinolates across plant organs can strongly vary. Here, we investigated the effect of leaf age on glucosinolate accumulation and hydrolysis in two leafy Brassica vegetables, pak choi and giant red mustard. We also evaluated the activity of the hydrolyzing enzyme myrosinase across the leaves. Finally, we assessed whether glucosinolates are transported from older leaves to younger leaves. Young leaves of both species contained more than 3-fold more glucosinolates than older ones. Accordingly, more isothiocyanates were released in the young leaves. Myrosinases fully hydrolyzed all of the amounts of glucosinolates regardless of the leaf age. Moreover, older leaves were observed to supply younger leaves with glucosinolates. Thus, this study suggests that consumers can improve the nutritional value of food by incorporating young leaves of leafy Brassicas in their diet.
Introduction:
Folic acid (FA) is a critical metabolite in all living organisms and an important nutritional component of broccoli. Few studies have been conducted on the impact of an exogenous application of FA on the postharvest physiology of fruits and vegetables during storage. In this regard, the mechanism by which an exogenous application of FA extends the postharvest quality of broccoli is unclear.
Objective:
This study utilized a multicomponent analysis to investigate how an exogenous application of FA effects the postharvest quality of broccoli.
Methods:
Broccoli was soaked in 5 mg L-1 FA for 10 min and the effect of the treatment on the appearance and nutritional quality of broccoli was evaluated. These data were combined with transcriptomic, metabolomic, and DNA methylation data to provide insight into the potential mechanism by which FA delays senescence.
Results:
The FA treatment inhibited the yellowing of broccoli during storage. CHH methylation was identified as the main type of methylation that occurs in broccoli and the FA treatment was found to inhibit DNA methylation, promote the accumulation of endogenous FA and chlorophyl, and inhibit ethylene biosynthesis in stored broccoli. The FA treatment also prevented the formation of off-odors by inhibiting the degradation of glucosinolate.
Conclusions:
FA treatment inhibited the loss of nutrients during the storage of broccoli, delayed its yellowing, and inhibited the generation of off-odors. Our study provides deeper insight into the mechanism by which the postharvest application of FA delays postharvest senescence in broccoli and provides the foundation for further studies of postharvest metabolism in broccoli.
Broccoli sprouts have a strong ability to accumulate isothiocyanate and selenium. In this study, the isothiocyanate content increased significantly as a result of ZnSO4 stress. Particularly, based on the isothiocyanate content is not affected, the combined ZnSO4 and Na2SeO3 treatment alleviated the inhibition of ZnSO4 and induced selenium content. Gene transcription and protein expression analyses revealed the changes in isothiocyanate and selenium metabolite levels in broccoli sprouts. ZnSO4 combined with Na2SeO3 was proven to activate a series of isothiocyanate metabolite genes (UGT74B1, OX1, and ST5b) and selenium metabolite genes (BoSultr1;1, BoCOQ5-2, and BoHMT1). The relative abundance of the total 317 and 203 proteins, respectively, in 4-day-old broccoli sprouts varied, and the metabolic and biosynthetic pathways for secondary metabolites were significantly enriched in ZnSO4/control and ZnSO4 combined Na2SeO3/ZnSO4 comparisons. The findings demonstrated how ZnSO4 combined with Na2SeO3 treatment reduced stress inhibition and the accumulation of encouraged selenium and isothiocyanates during the growth of broccoli sprouts.
The health-beneficial effects of Brassica vegetables are mainly attributed to their high contents of glucosinolates and the products of their enzymatic hydrolysis, especially isothiocyanates. Distribution of glucosinolates across plant organs can strongly vary. Consequently, the effect of leaf age on glucosinolate accumulation as well as hydrolysis was investigated in two leafy Brassica vegetables, pak choi and giant red mustard. Furthermore, activity of the hydrolyzing enzyme, myrosinase, was evaluated across the leaves. Additionally, a possible glucosinolate transport from older to younger leaves was monitored. Young leaves of both species contained of more glucosinolates than old ones. Accordingly, more isothiocyanates were released upon glucosinolate hydrolysis in young leaves. Myrosinases fully hydrolyzed the whole glucosinolates regardless of the leaf age. It was confirmed that older leaves can supply younger leaves with glucosinolates. The results can help to improve the health-beneficial value of Brassicas in the diet by an increased formation of isothiocyanates.
Broccoli sprouts have been considered as functional foods which have received increasing attention because they have been highly prized for glucosinolates, phenolics, and vitamins in particular glucosinolates. One of hydrolysates-sulforaphane from glucoraphanin is positively associated with the attenuation of inflammatory, which could reduce diabetes, cardiovascular and cancer risk. In recent decades, the great interest in natural bioactive components especially for sulforaphane promotes numerous researchers to investigate the methods to enhance glucoraphanin levels in broccoli sprouts and evaluate the immunomodulatory activities of sulforaphane. Therefore, glucosinolates profiles are different in broccoli sprouts varied with genotypes and inducers. Physicochemical, biological elicitors, and storage conditions were widely studied to promote the accumulation of glucosinolates and sulforaphane in broccoli sprouts. These inducers would stimulate the biosynthesis pathway gene expression and enzyme activities of glucosinolates and sulforaphane to increase the concentration in broccoli sprouts. The immunomodulatory activity of sulforaphane was summarized to be a new therapy for diseases with immune dysregulation. The perspective of this review served as a potential reference for customers and industries by application of broccoli sprouts as a functional food and clinical medicine.
Sulforaphane is a bioactive metabolite with anti-inflammatory activity and is derived from the glucosinolate glucoraphanin, which is highly abundant in broccoli sprouts. However, due to its inherent instability its use as a therapeutic against inflammatory diseases has been limited. There are few studies to investigate a whole food approach to increase sulforaphane levels with therapeutic effect and reduce inflammation. In the current study, using a mouse model of inflammatory bowel disease, we investigated the ability of steamed broccoli sprouts to ameliorate colitis and the role of the gut microbiota in mediating any effects. We observed that despite inactivation of the plant myrosinase enzyme responsible for the generation of sulforaphane via steaming, measurable levels of sulforaphane were detectable in the colon tissue and feces of mice after ingestion of steamed broccoli sprouts. In addition, this preparation of broccoli sprouts was also capable of reducing chemically-induced colitis. This protective effect was dependent on the presence of an intact microbiota, highlighting an important role for the gut microbiota in the metabolism of cruciferous vegetables to generate bioactive metabolites and promote their anti-inflammatory effects.
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.
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.
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.
Chinese broccoli [ Brassica oleracea var. alboglabra (L. H. Bailey) Musil] is an annual herbaceous healthy vegetable plant mainly cultivated in southern China. Previous studies have been primarily focused on investigating the nutritional ingredients of this plant, little is known about its medicinal potentials. Here we report the profiling of sulforaphane, an active ingredient with potent anticancer activities, in 47 Chinese broccoli germplasms collected from China and some southeast‐Asian countries. High‐performance liquid chromatography technology was used to systemically quantify the sulforaphane content in leaf, stem, and flower bud clusters of all 47 Chinese broccoli germplasms. The results indicated a diverse distribution pattern of sulforaphane among different tissues. Flower bud contained the highest average of sulforaphane, roughly twice as much as in leaf and stem tissues. Interestingly, different germplasms exhibited great differences in terms of sulforaphane abundance. Germplasm BOK15‐20 is extremely rich in sulforaphane in all tissues tested, ∼2,500 times of BOK15‐15 in leaf, 66 times of BOK15‐42 in the stem, and 77 times of BOK15‐40 in flower bud. The tremendous disparity of sulforaphane content among different germplasms suggests considerable variations in germplasms’ medicinal potentials. In addition, we systemically characterized a series of morphological and phenotypic traits of all 47 Chinese broccoli germplasms. Cluster analysis through NTSYSpc v2.2 showed that the 47 germplasms were classified into five different groups, respectively, based on sulforaphane content and qualitative phenotypic traits. Our results not only confirmed the medicinal values of Chinese broccoli but also provided crucial information that will eventually benefit future germplasm development and new variety breeding.
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.
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.
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.
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.
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.
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.
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.
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.
Glucoraphanin in Brassica vegetables breaks down to either sulforaphane or sulforaphane nitrile depending on the conditions, and sulforaphane can be further conjugated with glutathione. Using a high-throughput microtitre plate assay and TaqMan real time quantitative RT-PCR to measure mRNA, we show that sulforaphane and its glutathione conjugate, but not the nitrile, increased significantly (P < 0.05) both UGT1A1 and GSTA1 mRNA levels in HepG2 and HT29 cells. These changes were accompanied by an increase in UGT1A1 protein, as assessed by immunoblotting, and a 2-8-fold increase in bilirubin glucuronidation. When treated together, the nitrile derivative did not affect sulforaphane induction. The induction of UGT1A1 and GSTA1 mRNA by sulforaphane was time and concentration dependent. The results show a functional induction of glucuronidation by sulforaphane but not sulforaphane nitrile, and show that the pathway of metabolism of glucosinolates in Brassica vegetables is important in determining the resulting biological and anticarcinogenic activities.
For the first time the human intestinal effective permeability, estimated from the luminal disappearance and intestinal metabolism of phytochemicals, sulforaphane and quercetin-3,4'-glucoside, as well as the simultaneous changes in gene expression in vivo in enterocytes, has been studied in the human jejunum in vivo (Loc-I-Gut). Both compounds as components of an onion and broccoli extract could readily permeate the enterocytes in the perfused jejunal segment. At the physiologically relevant, dietary concentration tested, the average effective jejunal permeability (Peff) and percentage absorbed (+/- S.D.) were 18.7 +/- 12.6 x 10-4 cm/s and 74 +/- 29% for sulforaphane and 8.9 +/- 7.1 x 10-4 cm/s and 60 +/- 31% for quercetin-3,4'-diglucoside, respectively. Furthermore, a proportion of each compound was conjugated and excreted back into the lumen as sulforaphane-glutathione and quercetin-3'-glucuronide. The capacity of the isolated segment to deconjugate quercetin from quercetin-3,4'-diglucoside during the perfusion was much higher than the beta-glucosidase activity of the preperfusion jejunal contents, indicating that the majority (79-100%) of the beta-glucosidase capacity derives from the enterocytes in situ. Simultaneously, we determined short-term changes in gene expression in exfoliated enterocytes, which showed 2.0 +/- 0.4-fold induction of glutathione transferase A1 (GSTA1) mRNA (p < 0.002) and 2.4 +/- 1.2-fold induction of UDP-glucuronosyl transferase 1A1 (UGT1A1) mRNA (p < 0.02). The changes in gene expression were also seen in differentiated Caco-2 cells, where sulforaphane was responsible for induction of GSTA1 and quercetin for induction of UGT1A1. These results show that food components have the potential to modify drug metabolism in the human enterocyte in vivo very rapidly.
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).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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
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.
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.
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.
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.
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.
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.
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.
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.
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.
We describe a rapid and direct assay of NAD(P)H:(quinone-acceptor) oxidoreductase (EC 1.6.99.2) activity in cultured cells suitable for identifying and purifying inducers of this detoxication enzyme. Hepa 1c1c7 murine hepatoma cells are plated in 96-well microtiter plates, grown for 24 h, and exposed to inducing agents for another 24 h. The cells are then lysed and quinone reductase activity is assayed by the addition of a reaction mixture containing an NADPH-generating system, menadione (2-methyl-1,4-naphthoquinone), and MTT [3-(4,-5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide]. Quinone reductase catalyzes the reduction of menadione to menadiol by NADPH, and MTT is reduced nonenzymatically by menadiol resulting in the formation of a blue color which can be quantitated on a microtiter plate absorbance reader. The reaction is more than 90% dicoumarol inhibitable and menadione dependent. The results are comparable to those obtained by harvesting cells from larger plates, preparing cytosols, and carrying out spectrophotometric measurements.
Sonicated extract of crambe seed meal prepared in the presence of ferrous ion and dithiothreitol enzymatically converts epi-progoitrin to glucose, HSO4−, and a mixture of 1-cyano-2-hydroxy-3,4-epithiobutanes (50–70%) and 1-cyano-2-hydroxy-3-butene (30–50%). A fraction of the extract precipitating between 60 and 70% saturated ammonium sulfate contains thioglucosidase that converts epi-progoitrin essentially to 1-cyano-2-hydroxy-3-butene. Chromatography (on cross-linked dextran) of a 40–60% ammonium sulfate fraction leads to separation of a proteinaceous material (s20 = 2.6 S) that does not hydrolyze epi-progoitrin but, in the presence of thioglucosidase, promotes the formation of 1-cyano-2-hydroxy-3,4-epithiobutanes in amounts proportional to those from crude seed meal extract.
Sulforaphane [1-isothiocyanato-4-(methyl-sulfinyl)butane] was recently isolated from one variety of broccoli as the major and very potent inducer of phase 2 detoxication enzymes in murine hepatoma cells in culture. Since phase 2 enzyme induction is often associated with reduced susceptibility of animals and their cells to the toxic and neoplastic effects of carcinogens and other electrophiles, it was important to establish whether sulforaphane could block chemical carcinogenesis. In this paper we report that sulforaphane and three synthetic analogues, designed as potent phase 2 enzyme inducers, block the formation of mammary tumors in Sprague-Dawley rats treated with single doses of 9,10-dimethyl-1,2-benzanthracene. The analogues are exo-2-acetyl-exo-6-isothiocyanatonorbornane, endo-2-acetyl-exo-6-isothiocyanatonorbornane, and exo-2-acetyl-exo-5-isothiocyanatonorbornane. When sulforaphane and exo-2-acetyl-exo-6-isothiocyanatonorbornane were administered by gavage (75 or 150 mumol per day for 5 days) around the time of exposure to the carcinogen, the incidence, multiplicity, and weight of mammary tumors were significantly reduced, and their development was delayed. The analogues endo-2-acetyl-exo-6-isothiocyanatonorbornane and exo-2-acetyl-exo-5-isothiocyanatonorbornane were less potent protectors. Thus, a class of functionalized isothiocyanates with anticarcinogenic properties has been identified. These results validate the thesis that inducers of phase 2 enzymes in cultured cells are likely to protect against carcinogenesis.