Myrosinase Activity Research Articles

Effect of sucrose and mannitol on the accumulation of health-promoting compounds and the activity of metabolic enzymes in broccoli sprouts

The contents of ascorbic acid, glucosinolates, sulforaphane, anthocyanins, total phenolics, the activity of myrosinase and phenylalanine ammonialyase (PAL) and the antioxidant activity of broccoli sprouts grown under 88 mM and 176 mM of sucrose and mannitol were investigated. The results showed that the contents of sulforaphane, ascorbic acid and anthocyanins in broccoli sprouts were significantly increased after treatment with 88 mM of sucrose compared with the control. The contents of glucosinolates and total phenolics, the activity of PAL and the antioxidant activity in broccoli sprouts treated with 176 mM sucrose were also significantly increased, whereas the activity of myrosinase was significantly reduced. On the other hand, the contents of glucosinolate, sulforaphane and total phenolics in broccoli sprouts were also significantly increased after treatment with 176 mM of mannitol, although the contents of sulforaphane were markedly reduced compared to those treated with 176 mM of sucrose. Sucrose might induce the production of health-promoting compounds through its role of signaling, generating osmotic pressure or serving as a substrate. These results indicate that sucrose treatment could improve the nutritional value of broccoli, and the sprouts growing under adequate concentration of sucrose could benefit our diet by producing more health-promoting compounds.

Glucosinolate content and myrosinase activity evolution in three maca ( Lepidium meyenii Walp .) ecotypes during preharvest, harvest and postharvest drying

Glucosinolate profiles, glucosinolate contents and myrosinase activity were evaluated in yellow, red and black hypocotyls of maca during pre-harvest, at harvest and during post-harvest drying. At harvest, six glucosinolates (GLs) were identified: 5-methylsulfinylpentyl, 4-hydroxybenzyl, benzyl, 3-methoxybenzyl, 4-hydroxy-3-indolylmethyl and 4-methoxy-3-indolylmethyl, of which benzyl glucosinolate was the most abundant in the three ecotypes, representing 80% of the total GLs. A significant increase in GLs was observed for the three ecotypes during the 90 days before harvest and during the 15–30 days of post-harvest drying. This was followed by an important decrease of GLs during the 30–45 day period, which was attributed to cell breakdown, due to fluctuations in temperatures during the drying process, and was correlated with a high myrosinase action. During the last period of post-harvest drying, GLs were much lower and correlated to lower myrosinase activity and lower maca hypocotyl humidity. A combination of artisanal and other processing techniques should be utilised, in order to best preserve maca glucosinolates.

Identification of glucosinolate congeners able to form DNA adducts and to induce mutations upon activation by myrosinase

Juices from Brassicales are mutagenic in Salmonella typhimurium and characteristic adducts are formed with the endogenous DNA in Brassicales homogenates. These effects require myrosinase activity, suggesting an involvement of breakdown products of glucosinolates (GLs). We aimed to identify GLs congeners producing these effects. We investigated twelve individual GLs for mutagenicity in S. typhimurium TA104 and TA100 and for adduct formation with herring sperm DNA using the 32P-postlabelling/thin-layer chromatography method. All bacteriotoxic and mutagenic effects observed required the presence of myrosinase. Neoglucobrassicin, 4-methoxyglucobrassicin and sinalbin showed mutagenicity over wide concentration ranges, with neoglucobrassicin being the most potent congener. Six other GLs led to modest increases in the number of revertants in a small concentration range, before toxicity overshadowed this effect. The remaining three GLs showed some toxicity, but no mutagenicity. However, all twelve GLs formed DNA adducts. Clearly the highest adduct levels were detected with the indole GLs tested. They matched the major adduct spots formed in Brassicales homogenates. The observation that GLs are genotoxic demands follow-up studies on possible genotoxic and carcinogenic effects of these common food compounds in animal models and humans. Our study may be used to prioritize the congeners in further studies.

Microbial and chemical origins of the bactericidal activity of thermally treated yellow mustard powder toward Escherichia coli O157:H7 during dry sausage ripening

Work examines the origin of bactericidal activity in mustard flour and explores the relative contribution from starter cultures, E. coli O157:H7 itself and other sources. Bacteria can degrade naturally occurring glucosinolates in mustard and form isothiocyanates with antimicrobial activity. In the present work, 24 starter cultures (mostly from commercial mixtures) were screened for their capacity to decompose the glucosinolate, sinalbin. The most active pair, Pediococcus pentosaceus UM 121P and Staphylococcus carnosus UM 123M, were used together for the production of dry fermented sausage contaminated with E. coli O157:H7 (~ 6.5 log CFU/g). They were compared to industrial starters used previously ( P. pentosaceus UM 116P and S. carnosus UM 109M) for their reduction of E. coli O157:H7 viability. Sausage batches containing hot mustard powder (active myrosinase), cold mustard powder (inactivated myrosinase), autoclaved mustard powder (inactivated myrosinase) and no mustard flour (control) were prepared. Interestingly, both pairs of starter cultures yielded similar results. Elimination of E. coli O157:H7 (> 5 log CFU/g) occurred after 31 days in the presence of hot flour and in 38 days when the cold flour was added. Reductions > 5 log CFU/g of the pathogen did not occur (up to 38 days) in the control group. It was found that E. coli O157:H7 itself had a greater effect on sinalbin conversion than either pair of starter cultures, and glucosinolate degradation by the starter cultures was less important in determining E. coli survival. The autoclaved powder caused more rapid bactericidal action against E. coli O157:H7, yielding a > 5 log CFU/g reduction in 18 days. This may have been a result of the formation and/or release of antimicrobial substances by the autoclave treatment. Autoclaved mustard powder could potentially solve an important challenge facing the meat industry as it strives to manufacture safe dry fermented sausages.

Activity of myrosinase from Sinapis alba seeds immobilized into Ca-polygalacturonate as a simplified model of soil-root interface mucigel

Ca-polygalacturonate is a demethoxylated component of pectins which are constitutive of plant root mucigel. In order to define the role of root mucigel in myrosinase immobilization and activity at root level, a myrosinase enzyme which had been isolated from Sinapis alba seeds was immobilized into Ca-polygalacturonate. The activity profile for the immobilized and free enzyme was evaluated using the pH-Stat method as a function of time, temperature, and pH. The Michaelis-Menten kinetic parameters change between the immobilized (V max = 127 ± 13 U mg−1 protein; K M = 6.28 ± 0.09 mM) and free (V max = 17 ± 1 U mg−1 protein; K M = 0.96 ± 0.01 mM) forms of myrosinase, probably due to conformational changes involving the active site as a consequence of enzyme immobilization. Immobilized enzyme activity evaluated as a function of different substrates gave the highest value with nasturtin, the glucosinolate that is typical of several brassicaceae plant roots containing the glucosinolate-myrosinase defensive system. No feedback regulation mechanism was found in the presence of an excess of enzymatic reaction products (i.e. allyl isothiocyanate or sulphate). The high enzyme immobilization yield into Ca-polygalacturonate and its activity preservation under different conditions suggest that the enzyme released by plants at root level could be entrapped in root mucigel in order to preserve its activity.

Induction of Detoxification Enzymes by Feeding Unblanched Brussels Sprouts Containing Active Myrosinase to Mice for 2 Wk

In cruciferous vegetables, myrosinase metabolizes the relatively inactive glucosinolates into isothiocyanates and other products that have the ability to increase detoxification enzyme expression. Thus, maintaining myrosinase activity during food preparation may be critical to receiving the maximum benefit of consumption of Brussels sprouts or other cruciferous vegetables. To test the importance of maintaining myrosinase activity for maximizing bioactivity, experimental diets containing 20% unblanched (active myrosinase) or 20% blanched (inactivated myrosinase) freeze-dried Brussels sprouts and a nutrient-matched control diet were evaluated in vitro and in vivo for their ability to induce detoxification enzymes. Treatment of immortalized HepG2 human hepatoma cells with the unblanched Brussels sprout diet caused a greater increase quinone activity compared to the blanched Brussels sprout diet. C3H/HeJ mice fed the unblanched Brussels sprout diets for 2 wk had significantly higher plasma sulforaphane concentrations. Liver expression of CYP1A1 and epoxide hydrolase, measured using real-time PCR, was correlated with the plasma concentration of sulforaphane. In the lung, expression of epoxide hydrolase, thioredoxin reductase, UDP glucuronosyltransferase, quinone reductase, heme oxygenase, CYP1A1, CYP1A2, and CYP1B1 were also correlated with the plasma concentration of sulforaphane. Together these data demonstrate that, as predicted by the in vitro experiment, in vivo exposure to Brussels sprouts with active myrosinase resulted in greater induction of both phase I and phase II detoxification enzymes in the liver and the lungs that correlated with plasma sulforaphane concentrations.

Key Role of Fe2+ in Epithiospecifier Protein Activity

The chemical nature of the hydrolysis products from the glucosinolate-myrosinase system depends on the presence or absence of supplementary proteins such as epithiospecifier proteins (ESPs). ESPs promote the formation of epithionitriles from terminal alkenyl glucosinolates and, as recent evidence suggests, simple nitriles at the expense of isothiocyanates. From a human health perspective isothiocyanates are the most important because they are major inducers of carcinogen-detoxifying enzymes. Fe(2+) is an essential factor in ESP activity, although several recent studies have highlighted discrepancies in the understanding of the ESP-iron interaction. To investigate further the role iron species play in regulating ESP activity, four ESP-containing seedpowders were analyzed for ESP and myrosinase activities, endogenous iron content, and glucosinolate degradation products after the addition of iron species, specific chelators, and reducing agents. For the first time this paper shows the effect of these additions on the hydrolysis of individual glucosinolates that constitute the total pool. Aged seeds and 3-day seedlings were also tested to investigate the effects of seed storage and early plant development on iron levels and ESP activity. The four ESP-containing plant systems tested gave two distinctive responses, thus providing strong evidence that ESPs vary markedly in their Fe(2+) requirement for activity. The results also indicated that reduction of ferric to ferrous iron drives variations in ESP activity during early plant development. The reverse oxidation reaction provided a convincing explanation for the loss of ESP activity during seed storage. Aged seeds produced seedlings with substantially lower ESP activity, and there was a concomitant loss in germination rate. It was concluded that manipulation of endogenous iron levels of ESP-containing plants could increase the conversion of glucosinolates to isothiocyanates and enhance potential health benefits.

THE EFFECT OF STEAMING ON THE GLUCOSINOLATE CONTENT IN BROCCOLI

Total and individual glucosinolates were measured after different duration of steaming broccoli (Brassica oleracea L. var. italica). During steaming, the temperature profile, cell lysis and inactivation of myrosinase were assessed as well. Steaming resulted in high retention of total aliphatic and indolyl glucosinolates in the cooked product. Only after extensive steaming of broccoli (30 min) substantial losses of total indolyl glucosinolates of 55% and total aliphatic glucosinolates of 8.5% were observed. Steaming broccoli for more than 6 min result in complete inactivation of the hydrolytic enzyme myrosinase. However, steaming of broccoli for less than 6 min may result in a high intake of glucosinolates, in the presence of a residual active myrosinase, allowing the release of health-protective breakdown products of glucosinolates after consumption.

Removing the mustard oil bomb from seeds: transgenic ablation of myrosin cells in oilseed rape (Brassica napus) produces MINELESS seeds.

Many plant phytochemicals constitute binary enzyme–glucoside systems and function in plant defence. In brassicas, the enzyme myrosinase is confined to specific myrosin cells that separate the enzyme from its substrate; the glucosinolates. The myrosinase-catalysed release of toxic and bioactive compounds such as isothiocyanates, upon activation or tissue damage, has been termed ‘the mustard oil bomb’ and characterized as a ‘toxic mine’ in plant defence. The removal of myrosin cells and the enzyme that triggers the release of phytochemicals have been investigated by genetically modifying Brassica napus plants to remove myrosinase-storing idioblasts. A construct with the seed myrosin cell-specific Myr1.Bn1 promoter was used to express a ribonuclease, barnase. Transgenic plants ectopically expressing barnase were embryo lethal. Co-expressing barnase under the control of the Myr1.Bn1 promoter with the barnase inhibitor, barstar, under the control of the cauliflower mosaic virus 35S promoter enabled a selective and controlled death of myrosin cells without affecting plant viability. Ablation of myrosin cells was confirmed with light and electron microscopy, with immunohistological analysis and immunogold-electron microscopy analysis showing empty holes where myrosin cells normally are localized. Further evidence for a successful myrosin cell ablation comes from immunoblots showing absence of myrosinase and negligible myrosinase activity, and autolysis experiments showing negligible production of glucosinolate hydrolysis products. The plants where the myrosin defence cells have been ablated and named ‘MINELESS plants’. The epithiospecifier protein profile and glucosinolate levels were changed in MINELESS plants, pointing to localization of myrosinases and a 35 kDa epithiospecifier protein in myrosin cells and a reduced turnover of glucosinolates in MINELESS plants.

Effect of salt stress on phenolic compounds, glucosinolates, myrosinase and antioxidant activity in radish sprouts

The germination, growth, total phenolics, glucosinolate, myrosinase and antioxidant activity of radish sprouts germinated under 0 (control), 10, 50 and 100 mM of NaCl were investigated. The glucoraphasatin (4-methylthio-3-butenyl-glucosinolate), total glucosinolate and total phenolic contents of 5- and 7-day-old sprouts treated with 10 and 50 mM of NaCl were significantly decreased. However, the antioxidant activity of sprouts treated with 10 and 50 mM of NaCl was not affected. The glucoraphasatin and total glucosinolate contents of 5- and 7-day-old sprouts, total phenolic contents of 3- and 5-day-old sprouts were significantly increased and myrosinase activities of 3- and 5-day-old sprouts were inhibited, although the germination was dramatically inhibited by 100 mM of NaCl treatment. These results indicated that salt stress (100 mM of NaCl treatment) could improve the nutritional value of radish sprouts, and germination of sprouts under adequate salt stress could be one useful way to enhance health-promoting compounds of plant food.

Abstract A62: Increased histone acetylation, but no inhibition of HDAC activity, by kale sprout intervention in a human prostate cancer xenograft model

Abstract Cruciferous vegetable consumption has been associated with a reduced risk of prostate cancer. These vegetables such as broccoli and kale are rich in glucosinolates, which are converted to isothiocyanates (ITCs) by myrosinase. ITCs have cancer preventive potential through various mechanisms including anti-proliferative effects. ITC metabolites have been recently identified as inhibitors of histone deacetylases (HDAC), which have the potential to turn on epigenetically silenced genes, leading to cell cycle arrest and apoptosis. In the present study we investigated the effect of kale sprouts commercially available as a food supplement to inhibit the growth of human LNCaP prostate cancer cells in a xenograft model. We were interested whether kale sprout intervention would affect histone acetylation and in studying underlining mechanisms, e.g. HDACs and histone acetyltransferases (HAT). LNCaP human prostate cancer cells were injected to male nude Balb/c mice (10 per group). Mice were fed with either regular rodent chow or chow supplemented with 20% kale sprouts (containing about 60 µmol glucosinolates per 5 g chow consumed daily, together with active myrosinase) from a week before cell injection until sacrifice. 7.5 weeks after injection, tumor tissue was collected. Kale sprout intervention did not significantly inhibit tumor growth in the xenograft model. However, global acetylation levels of histone H4 detected by Western blotting were increased in the kale sprout-treated group, whereas acetylated histone H3 levels were not significantly changed. The increase of H4 lysine 5, 8, 12 and 16 acetylation was individually confirmed. Among them, acetylated H4 K12 was most abundant. Even though global acetylation of H3 did not change, acetylated H3K9 was significantly increased. Unexpectedly, HDAC activity in nuclear extracts of tumor tissue determined by using ZMAL substrate was not significantly inhibited in tumor tissue by kale sprout intervention. Also, protein expression of HDAC family members HDAC1-5 was not changed overall, although HDAC 5 expression was reduced while HDAC2 expression was increased. Instead, kale sprout treatment significantly increased mRNA levels of PCAF and CBP quantified by qRT-PCR, by 36% and 23%, respectively, in tumor tissue (p<0.001), which may contribute to increased H4 acetylation. Taken together, our results indicate that dietary consumption of kale sprouts increased global acetylation of histone H4 in LNCaP prostate cancer xenograft model, possibly by induction of HAT expression rather than inhibition of HDAC activity. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A62.

Abstract A148: Concomitant activation of pro- and antiproliferative mechanisms abrogate tumor growth inhibition by kale sprout intervention in a human prostate cancer xenograft model

Abstract Cruciferous vegetables are rich in glucosinolates, which are converted to isothiocyanates (ITCs) and indole-based compounds by the enzymatic activity of myrosinase. Tumor-growth inhibitory efficacy of purified constituents derived from cruciferous vegetables has been demonstrated in animal models at various organ sites including the prostate. However, except induction of detoxifying enzymes, relatively little is known on mechanisms affected by dietary intervention with cruciferous vegetables, which represent a mixture of various constituents potentially enhancing or blocking their activities (Pappa et al., 2007). In the present study, we tested the tumor growth inhibitory potential of kale sprouts in a human prostate cancer xenograft model. LNCaP human prostate cancer cells were injected to male nude Balb/c mice (10 per group). Mice were fed with either regular rodent chow or chow supplemented with 20% kale sprouts (containing about 60 µmol glucosinolates per 5 g chow consumed daily, together with active myrosinase) from a week before cell injection until sacrifice after 7.5 weeks of intervention. Compared with normal diet, kale sprout intervention did not significantly inhibit average tumor weights. Secreted prostate specific antigen (PSA), which is used as a biomarker for prostate cancer, was highly correlated with tumor size in both intervention groups. Interestingly however, plasma PSA levels in the kale sprout group were 2.5-fold higher (p<0.05) than in the control group. In tumor tissue, mRNA expression of hormone-regulated genes such as PSA, androgen receptor, insulin-like growth factor-1 and its receptor were significantly increased. We also observed an increase in histone H4 acetylation, which was not linked to inhibition of HDAC activity. As a consequence however, p21 mRNA and protein expression was upregulated by kale sprout intervention. In addition, although there was no difference in the expression of PCNA as a proliferation marker, phosphorylated histone H3 staining as an indication of mitotic arrest was enhance in the kale sprout group. Interestingly, hemoglobin levels in tumor cell lysate, and vessels in tumor sections were significantly reduced by consuming kale sprout powder, indicating anti-angiogenic potential. This was associated with downregulated VEGF-c mRNA expression. Overall, our in vivo study indicates that components in kale sprouts or their metabolites concomitantly affect both anti-proliferative (histone H4 acetylation, cell cycle arrest, anti-angiogenesis) and pro-proliferative mechanisms (hormone signaling), which in combination may abrogate inhibitory effects on tumor growth. These data may contribute to a better understanding of the effects of cruciferous vegetable consumption in cancer prevention. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A148.

MODIFIED VACUOLE PHENOTYPE1 Is an Arabidopsis Myrosinase-Associated Protein Involved in Endomembrane Protein Trafficking

We identified an Arabidopsis (Arabidopsis thaliana) ethyl methanesulfonate mutant, modified vacuole phenotype1-1 (mvp1-1), in a fluorescent confocal microscopy screen for plants with mislocalization of a green fluorescent protein-delta tonoplast intrinsic protein fusion. The mvp1-1 mutant displayed static perinuclear aggregates of the reporter protein. mvp1 mutants also exhibited a number of vacuole-related phenotypes, as demonstrated by defects in growth, utilization of stored carbon, gravitropic response, salt sensitivity, and specific susceptibility to the fungal necrotroph Alternaria brassicicola. Similarly, crosses with other endomembrane marker fusions identified mislocalization to aggregate structures, indicating a general defect in protein trafficking. Map-based cloning showed that the mvp1-1 mutation altered a gene encoding a putative myrosinase-associated protein, and glutathione S-transferase pull-down assays demonstrated that MVP1 interacted specifically with the Arabidopsis myrosinase protein, THIOGLUCOSIDE GLUCOHYDROLASE2 (TGG2), but not TGG1. Moreover, the mvp1-1 mutant showed increased nitrile production during glucosinolate hydrolysis, suggesting that MVP1 may play a role in modulation of myrosinase activity. We propose that MVP1 is a myrosinase-associated protein that functions, in part, to correctly localize the myrosinase TGG2 and prevent inappropriate glucosinolate hydrolysis that could generate cytotoxic molecules.

Folivory versus florivory—adaptiveness of flower feeding

The distribution of resources and defence is heterogeneous within plants. Specialist insects may prefer tissue with high concentrations of the plant's characteristic defence compounds. Most herbivorous butterfly or sawfly larvae are considered to be folivores, so also the turnip sawfly Athalia rosae (Hymenoptera: Tenthredinidae), a specialist herbivore on Brassicaceae. We investigated which tissue larvae choose to feed upon and how they perform on flowers, young or old leaves of Sinapis alba. Furthermore, constitutive and inducible levels of glucosinolates and myrosinases were investigated and nutrients analysed. Larvae moved from leaves to flowers for feeding from the third larval instar on. Flowers were not actively chosen, but larvae moved upwards on the plant, regardless of how plants were orientated (upright or inverted). Flower-feeding larvae were heavier and developed faster than larvae feeding on young leaves, and adults laid more eggs. Old leaves as food source resulted in the lowest growth rates. Flowers contained three and ten times higher myrosinase activities than young and old leaves, respectively, whereas glucosinolate concentrations and nitrogen levels of flowers and young leaves were comparable. Glucosinolate concentrations of old leaves were very low. Changes in tissue chemistry caused by larval feeding were tissue specific. Defence levels did not change in flowers and old leaves after A. rosae feeding in contrast to young leaves. The high insect performance on flowers cannot be explained by differences in chemical defence. Instead, the lack of mechanical defence (trichomes) is probably responsible. Movement to the flowers and folivory is overall highly adaptive for this sawfly species.

Myrosinase activity and quality parameters on fresh-cut Chinese cabbage stored at two different temperatures

Myrosinase activity and quality parameters on fresh-cut Chinese cabbage stored at two different temperatures

Myrosinases from root and leaves of Arabidopsis thaliana have different catalytic properties

Myrosinases (EC 3.2.1.147) are β-thioglucoside glucosidases present in Brassicaceae plants. These enzymes serve to protect plants against pathogens and insect pests by initiating breakdown of the secondary metabolites glucosinolates into toxic products. Several forms of myrosinases are present in plants but the properties and role of different isoenzymes are not well understood. The dicot plant model organism Arabidopsis thaliana seems to contain six myrosinase genes ( TGG1– TGG6). In order to compare the different myrosinases, cDNAs corresponding to TGG1 from leaves and TGG4 and TGG5 from roots were cloned and overexpressed in Pichia pastoris. The His-tagged recombinant proteins were purified using affinity chromatography and the preparations were homogenous according to SDS–PAGE analysis. Myrosinase activity was confirmed for all forms and compared with respect to catalytic activity towards the allyl-glucosinolate sinigrin. There was a 22-fold difference in basal activity among the myrosinases. The enzymes were active in a broad pH range, are rather thermostable and active in a wide range of salt concentrations but sensitive to high salt concentrations. The myrosinases showed different activation–inhibition responses towards ascorbic acid with maximal activity around 0.7–1 mM. No activity was registered towards desulphosinigrin and this compound did not inhibit myrosinase activity towards sinigrin. All myrosinases also displayed O-β-glucosidase activity, although with lower efficiency compared to the myrosinase activity. The differences in catalytic properties among myrosinase isozymes for function in planta are discussed.

Differing mechanisms of simple nitrile formation on glucosinolate degradation in Lepidium sativum and Nasturtium officinale seeds

Glucosinolates are sulphur-containing glycosides found in brassicaceous plants that can be hydrolysed enzymatically by plant myrosinase or non-enzymatically to form primarily isothiocyanates and/or simple nitriles. From a human health perspective, isothiocyanates are quite important because they are major inducers of carcinogen-detoxifying enzymes. Two of the most potent inducers are benzyl isothiocyanate (BITC) present in garden cress ( Lepidium sativum), and phenylethyl isothiocyanate (PEITC) present in watercress ( Nasturtium officinale). Previous studies on these salad crops have indicated that significant amounts of simple nitriles are produced at the expense of the isothiocyanates. These studies also suggested that nitrile formation may occur by different pathways: (1) under the control of specifier protein in garden cress and (2) by an unspecified, non-enzymatic path in watercress. In an effort to understand more about the mechanisms involved in simple nitrile formation in these species, we analysed their seeds for specifier protein and myrosinase activities, endogenous iron content and glucosinolate degradation products after addition of different iron species, specific chelators and various heat treatments. We confirmed that simple nitrile formation was predominantly under specifier protein control (thiocyanate-forming protein) in garden cress seeds. Limited thermal degradation of the major glucosinolate, glucotropaeolin (benzyl glucosinolate), occurred when seed material was heated to >120 °C. In the watercress seeds, however, we show for the first time that gluconasturtiin (phenylethyl glucosinolate) undergoes a non-enzymatic, iron-dependent degradation to a simple nitrile. On heating the seeds to 120 °C or greater, thermal degradation of this heat-labile glucosinolate increased simple nitrile levels many fold.

Identification and Characterization of the Glucosinolate–Myrosinase System in Caper (Capparis ovata Desf.)

Myrosinase (EC 3.2.1.147) catalyzes cleavage of glucosinolates, which consist of a thioglucoside moiety linked to amino acid-derived side chains. Myrosinase activity and expression profiles were investigated together with glucosinolate contents in Capparis ovata (caper) in order to characterize the glucosinolate–myrosinase system. The desulfoglucosinolates—glucocapparin, glucoiberin, progoitrin, epiprogoitrin, sinigrin, gluconapin, glucosinalbin, and glucobrassicin—were extracted and quantified from leaves, seeds, flowers, flower buds, and young shoots. The major desulfoglucosinolate was glucocapparin, which accumulated to values of 39.35 ± 0.09 and 25.56 ± 0.11 μmol g−1 dry weight in seed and leaf extracts, respectively. Myrosinase has high activity in caper seeds, leaves, flowers, and flower bud tissues having the highest total activities in seed extracts (79.23 ± 0.18 U). However, specific activities were the highest in flower bud extracts (200.44 ± 0.09 U mg−1 protein). The myrosinase protein migrated as a single band with a molecular weight of 65 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis and on Western blots probed with the myrosinase-specific 3D7 antibodies. Native gel electrophoresis revealed two putative myrosinase isoenzymes in seeds, leaves, and flower tissues. The caper homolog of the Arabidopsis thaliana TGG1 gene was differentially expressed in seeds, leaves, flowers, and flower buds with the highest expression levels in leaves and flower bud tissues.

Characterization of glucosinolate—myrosinase system in developing salt cress Thellungiella halophila

Glucosinolates are specialized plant metabolites derived from amino acids. They can be hydrolyzed by myrosinases into different degradation products, which have a variety of biological activities. In this study, the compositions and contents of glucosinolates in salt cress (Thellungiella halophila) at different developmental stages were analyzed by high performance liquid chromatography and mass spectrometry (HPLC-MS). Myrosinase activities were also measured. Seven glucosinolates were identified in T. halophila throughout its life cycle. The glucosinolate profiles varied significantly among different tissues. The roots at stage 4 contained the highest concentrations of total, aromatic and indole glucosinolates among all tissues. Whereas roots, flowers and siliques contained all seven glucosinolates, seeds contained only four aliphatic glucosinolates. During development, the concentrations also displayed significant changes. From seeds to cotyledons and from stage 4 roots to stage 5 roots, there were dramatic declines of glucosinolates, which correlated well with changes in myrosinase activities. In other tissues, myrosinase activity alone could not explain the glucosinolate concentration changes. Certain tissues of T. halophila contained Arabidopsis myrosinase TGG1 and TGG2 orthologs. The molecular basis and functional significance of our findings are discussed here.

Oven‐dried Brussels sprouts with undetectable levels of glucosinolates or isothiocyanates increased expression of detoxification enzymes in vitro and in vivo

Many individuals use supplementation to increase exposure to crucifers in order to decrease their risk of chronic disease. However supplements require extensive processing including grinding and drying that can affect their ability to induce detoxification enzymes. This study measured the ability of oven‐dried (OD) (without glucosinolates (GLS); without myrosinase activity (MYR)), freeze‐dried (FD) (with GLS; with MYR), and blanched, freeze‐dried (BFD) (with GLS; without MYR) Brussels sprouts to increase detoxification enzyme expression in vitro and in vivo. A 48 hour treatment with OD and FD, but not BFD, Brussels dose‐dependently induced quinone reductase (QR) activity and the expression of RNA for cytochrome P450 (CYP) 1A1, CYP1A2, heme oxygenase, and QR in HepG2 human carcinoma cells. In vivo, mRNA expression of detoxification enzymes was measured in the livers of mice fed diets containing BFD or OD Brussels (20%, 10% or 5%) for 14 days. Mice fed diet containing OD Brussels demonstrated a dose‐dependent induction of CYP1A1, CYP1B1, and QR in the liver, while BFD Brussels had no effect. Despite lacking the phytochemicals typically thought to be responsible, the OD Brussels sprouts induced detoxification enzymes. Additionally BFD Brussels failed to induce detoxification enzymes in vitro and in vivo, suggesting that endogenous myrosinase activity is required for the bioactivity of FD Brussels sprouts.