Plant Myrosinase Research Articles

Gregarines impact consumption and development but not glucosinolate metabolism in the mustard leaf beetle.

Gregarines are usually classified as parasites, but recent studies suggest that they should be viewed on a parasitism-mutualism spectrum and may even be seen as part of the gut microbiota of host insects. As such, they may also impact the consumption of their hosts and/or be involved in the digestion or detoxification of the host's diet. To study such effects of a gregarine species on those traits in its host, the mustard leaf beetle (Phaedon cochleariae) was used. This beetle species feeds on Brassicaceae plants that contain glucosinolates, which form toxic compounds when hydrolyzed by myrosinases. We cleaned host eggs from gametocysts and spores and reinfected half of the larvae with gregarines, to obtain gregarine-free (G-) and gregarine-infected (G+) larvae. Growth and food consumption parameters of these larvae were assessed by rearing individuals on watercress (Nasturtium officinale, Brassicaceae). A potential involvement of gregarines in the glucosinolate metabolism of P. cochleariae larvae was investigated by offering G- and G+ larvae leaf discs of watercress (containing mainly the benzenic 2-phenylethyl glucosinolate and myrosinases) or pea (Pisum sativum, Fabaceae, lacking glucosinolates and myrosinases) treated with the aliphatic 4-pentenyl glucosinolate or the indole 1-methoxy-3-indolylmethyl glucosinolate. Larval and fecal samples were analyzed via UHPLC-QTOF-MS/MS to search for breakdown metabolites. Larval development, body mass, growth rate and efficiency to convert food into body mass were negatively affected by gregarine infection while the pupal mass remained unaffected. The breakdown metabolites of benzenic and aliphatic glucosinolates were conjugated with aspartic acid, while those of the indole glucosinolate were conjugated with glutamic acid. Gregarine infection did not alter the larvae's ability to metabolize glucosinolates and was independent of plant myrosinases. In summary, some negative effects of gregarines on host performance could be shown, indicating parasitism. Future studies may further disentangle this gregarine-host relationship and investigate the microbiome potentially involved in the glucosinolate metabolism.

Plant glucosinolate biosynthesis and breakdown pathways shape the rhizosphere bacterial/archaeal community.

Rhizosphere microbial community assembly results from microbe-microbe-plant interactions mediated by small molecules of plant and microbial origin. Studies with Arabidopsis thaliana have indicated a critical role of glucosinolates in shaping the root and/or rhizosphere microbial community, likely through breakdown products produced by plant or microbial myrosinases inside or outside of the root. Plant nitrile-specifier proteins (NSPs) promote the formation of nitriles at the expense of isothiocyanates upon glucosinolate hydrolysis with unknown consequences for microbial colonisation of roots and rhizosphere. Here, we generated the A. thaliana triple mutant nsp134 devoid of nitrile formation in root homogenates. Using this line and mutants lacking aliphatic or indole glucosinolate biosynthesis pathways or both, we found bacterial/archaeal alpha-diversity of the rhizosphere to be affected only by the ability to produce aliphatic glucosinolates. In contrast, bacterial/archaeal community composition depended on functional root NSPs as well as on pathways of aliphatic and indole glucosinolate biosynthesis. Effects of NSP deficiency were strikingly distinct from those of impaired glucosinolate biosynthesis. Our results demonstrate that rhizosphere microbial community assembly depends on functional pathways of both glucosinolate biosynthesis and breakdown in support of the hypothesis that glucosinolate hydrolysis by myrosinases and NSPs happens before secretion of products to the rhizosphere.

Effect of heat processing on bioactive compounds of dehydrated (lyophilized) purple mashua (Tropaeolum tuberosum)

Mashua (Tropaeolum tuberosum) is an Andean tuber rich in phenolic compounds and glucosinolates, known for its consumption that allows the prevention and/or reduction of getting diseases. The present research evaluated the effect of different types of post-harvest management (sunny, bleached, and freeze-dried) on glucosinolate content and its relationship with myrosinase activity. It is also important to note that when plant cells are damaged and glucosinolates meet the plant's myrosinase, which hydrolyzes them and degrades them to other compounds. In the present research, the types of glucosinolates, the content of glucosinolates and the activity of myrosinase of the purple variety of fresh raw mashua and in mashua treated by several types of processing were evaluated, including the previous treatment (sunny and unscrewed), the boiling treatment and the microwave treatment, with whitening times of 3, 4, 5 and 6 minutes, being the condition of the tuber at the time of bleaching in whole form and by cutting, to then be subsequently subjected to lyophilization treatments. Five glucosinolatos (GSL) were identified: 5-methylsulfinilpentile, 4-hydroxybenzyl, 4-hydroxy-3-indolylmethyl, benzyl and 4-methoxybenzyl, of which 4-methoxybenzyl glucosinolate is the most abundant, symbolizing 98% and 95% of the total GSL in the purple ecotype. According to the total concentration of glucosinolates found in the studied variety (purple), it must be 74.22 and 110.60 micromoles per gram of dry base (μmol/g dry weight), so mashua can be considered, the edible plant with the highest existing GSL content.

New Insights into the Plutella xylostella Detoxifying Enzymes: Sequence Evolution, Structural Similarity, Functional Diversity, and Application Prospects of Glucosinolate Sulfatases.

Brassica plants have glucosinolate (GLs)-myrosinase defense mechanisms to deter herbivores. However, Plutella xylostella specifically feeds on Brassica vegetables. The larvae possess three glucosinolate sulfatases (PxGSS1-3) that compete with plant myrosinase for shared GLs substrates and produce nontoxic desulfo-GLs (deGLs). Although PxGSSs are considered potential targets for pest control, the lack of a comprehensive review has hindered the development of PxGSSs-targeted pest control methods. Recent advances in integrative multi-omics analysis, substrate-enzyme kinetics, and molecular biological techniques have elucidated the evolutionary origin and functional diversity of these three PxGSSs. This review summarizes research progress on PxGSSs over the past 20 years, covering sequence properties, evolution, protein modification, enzyme activity, structural variation, substrate specificity, and interaction scenarios based on functional diversity. Finally, we discussed the potential applications of PxGSSs-targeted pest control technologies driven by artificial intelligence, including CRISPR/Cas9-mediated gene drive, transgenic plant-mediated RNAi, small-molecule inhibitors, and peptide inhibitors. These technologies have the potential to overcome current management challenges and promote the development and field application of PxGSSs-targeted pest control.

Deciphering the Myrosinase-like Activity of Lactiplantibacillus plantarum WCFS1 among GH1 Family Glycoside Hydrolases.

The hydrolysis of plant glucosinolates by myrosinases (thioglucosidases) originates metabolites with chemopreventive properties. In this study, the ability to hydrolyze the glucosinolate sinigrin by cultures or protein extracts of Lactiplantibacillus plantarum WCFS1 was assayed. This strain possesses myrosinase-like activity as sinigrin was partly hydrolyzed by induced cultures but not by protein extracts. The 11 glycoside hydrolase GH1 family proteins, annotated as 6-phospho-β-glucosidases, were the proteins most similar to plant myrosinases. The activity of these proteins was assayed against sinigrin and synthetic glucosides. As expected, none of the proteins assayed possessed myrosinase activity against sinigrin or the synthetic β-thio-glucoside derivative or against the β-glucoside. However, all 11 proteins were active on the phosphorylated-β-glucoside derivative. Moreover, only eight of these proteins were active on phospho-β-thioglucose. These results supported that, in L. plantarum WCFS1, glucosinolates may undergo previous phosphorylation, and GH1 proteins are the glycosidases involved in the hydrolysis of phosphorylated glucosinolates.

A steamed broccoli sprout diet preparation that reduces colitis via the gut microbiota

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.

Unique metabolism of different glucosinolates in larvae and adults of a leaf beetle specialised on Brassicaceae

Brassicaceae plants contain glucosinolates, which are hydrolysed by myrosinases to toxic products such as isothiocyanates and nitriles, acting as defences. Herbivores have evolved various detoxification strategies, which are reviewed here. Larvae of Phaedon cochleariae (Coleoptera: Chrysomelidae) metabolise hydrolysis products of benzenic glucosinolates by conjugation with aspartic acid. In this study, we investigated whether P. cochleariae uses the same metabolic pathway for structurally different glucosinolates, whether the metabolism differs between adults and larvae and which hydrolysis products are formed as intermediates. Feeding experiments were performed with leaves of watercress (Nasturtium officinale, Brassicaceae) and pea (Pisum sativum, non-Brassicaceae), to which glucosinolates with structurally different side chains (benzenic, indole or aliphatic) or their hydrolysis products were applied. Samples were analysed by UHPLC-QTOF-MS/MS or TD–GC–MS. The same aspartic acid conjugates as previously identified in larvae were also detected as major metabolites of benzenic glucosinolates in adults. Indol-3-ylmethyl glucosinolate was mainly metabolised to N-(1H-indol-3-ylcarbonyl) glutamic acid in adults and larvae, while the metabolism of 2-propenyl glucosinolate remains unclear. The metabolism may thus proceed primarily via isothiocyanates rather than via nitriles, while the hydrolysis occurs independently of plant myrosinases. A detoxification by conjugation with these amino acids is not yet known from other Brassicaceae-feeders.

A Specific Broccoli Sprout Preparation Reduces Chemically-Induced Colitis Via Gut Microbiota

ObjectivesSulforaphane is a bioactive metabolite with anti-inflammatory activity that is derived from glucosinolates, which are highly abundant in cruciferous vegetables including broccoli sprouts. There have been limited studies to investigate a whole foods approach to increase sulforaphane levels with therapeutic effect and reduce inflammation. In the current study, using a mouse model of inflammatory bowel disease, we aimed to investigate the ability of steamed broccoli sprouts to ameliorate colitis and the role of the microbiota in mediating any effects. MethodsC57BL/6J male mice were fed base diet or 5% steamed broccoli sprout diet for 2 weeks prior to induction of colitis with 2–3% DSS (5–7 days) and kept on their respective diets until sacrifice. Body weight and disease activity index scores were recorded daily, and histological assessment was performed on H&E sections of colon. Urine, feces, blood, and GI tract tissues and intestinal content were collected for LC-MS/MS quantification of sulforaphane and glucoraphanin. The 16S rRNA sequencing was performed to analyze gut microbiome. Germ-free mice were fed base diet or 5% steamed broccoli sprout diet for 2 weeks followed by treatment with 5 days of DSS to determine the role of gut microbiota in mediating the anti-inflammatory effects of the steamed broccoli sprout diet. ResultsWe 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 gut microbiota, highlighting an important role for the microbiota in the metabolism of cruciferous vegetables to generate bioactive metabolites and promote their anti-inflammatory effects. ConclusionsAltogether, these results suggest that the incorporation of a steamed broccoli sprouts diet is a viable dietary intervention to complement current medical therapies for patients with inflammatory bowel disease. Funding SourcesThis work was supported by USDA-NIFA-AFRI Foundational Program [Grant No. 2018–67,017-27,520].

Improved Production of Recombinant Myrosinase in Pichia pastoris.

The effect of the deletion of a 57 bp native signal sequence, which transports the nascent protein through the endoplasmic reticulum membrane in plants, on improved AtTGG1 plant myrosinase production in Pichia pastoris was studied. Myrosinase was extracellularly produced in a 3-liter laboratory fermenter using α-mating factor as the secretion signal. After the deletion of the native signal sequence, both the specific productivity (164.8 U/L/h) and volumetric activity (27 U/mL) increased more than 40-fold compared to the expression of myrosinase containing its native signal sequence in combination with α-mating factor. The deletion of the native signal sequence resulted in slight changes in myrosinase properties: the optimum pH shifted from 6.5 to 7.0 and the maximal activating concentration of ascorbic acid increased from 1 mM to 1.5 mM. Kinetic parameters toward sinigrin were determined: 0.249 mM (Km) and 435.7 U/mg (Vmax). These results could be applied to the expression of other plant enzymes.

Indoles Derived From Glucobrassicin: Cancer Chemoprevention by Indole-3-Carbinol and 3,3'-Diindolylmethane.

Hydrolysis of glucobrassicin by plant or bacterial myrosinase produces multiple indoles predominantly indole-3-carbinol (I3C). I3C and its major in vivo product, 3,3'-diindolylmethane (DIM), are effective cancer chemopreventive agents in pre-clinical models and show promise in clinical trials. The pharmacokinetics/pharmacodynamics of DIM have been studied in both rodents and humans and urinary DIM is a proposed biomarker of dietary intake of cruciferous vegetables. Recent clinical studies at Oregon State University show surprisingly robust metabolism of DIM in vivo with mono- and di-hydroxylation followed by conjugation with sulfate or glucuronic acid. DIM has multiple mechanisms of action, the most well-characterized is modulation of aryl hydrocarbon receptor (AHR) signaling. In rainbow trout dose-dependent cancer chemoprevention by dietary I3C is achieved when given prior to or concurrent with aflatoxin B1, polycyclic aromatic hydrocarbons, nitrosamines or direct acting carcinogens such as N-methyl-N'-nitro-nitrosoguanidine. Feeding pregnant mice I3C inhibits transplacental carcinogenesis. In humans much of the focus has been on chemoprevention of breast and prostate cancer. Alteration of cytochrome P450-dependent estrogen metabolism is hypothesized to be an important driver of DIM-dependent breast cancer prevention. The few studies done to date comparing glucobrassicin-rich crucifers such as Brussels sprouts with I3C/DIM supplements have shown the greater impact of the latter is due to dose. Daily ingestion of kg quantities of Brussels sprouts is required to produce in vivo levels of DIM achievable by supplementation. In clinical trials these supplement doses have elicited few if any adverse effects. Sulforaphane from glucoraphanin can act synergistically with glucobrassicin-derived DIM and this may lead to opportunities for combinatorial approaches (supplement and food-based) in the clinic.

Hijacking the Mustard-Oil Bomb: How a Glucosinolate-Sequestering Flea Beetle Copes With Plant Myrosinases

Myrosinase enzymes play a key role in the chemical defense of plants of the order Brassicales. Upon herbivory, myrosinases hydrolyze the β-S-linked glucose moiety of glucosinolates, the characteristic secondary metabolites of brassicaceous plants, which leads to the formation of different toxic hydrolysis products. The specialist flea beetle, Phyllotreta armoraciae, is capable of accumulating high levels of glucosinolates in the body and can thus at least partially avoid plant myrosinase activity. In feeding experiments with the myrosinase-deficient Arabidopsis thaliana tgg1 × tgg2 (tgg) mutant and the corresponding Arabidopsis Col-0 wild type, we investigated the influence of plant myrosinase activity on the metabolic fate of ingested glucosinolates in adult P. armoraciae beetles. Arabidopsis myrosinases hydrolyzed a fraction of ingested glucosinolates and thereby reduced the glucosinolate sequestration rate by up to 50% in adult beetles. These results show that P. armoraciae cannot fully prevent glucosinolate hydrolysis; however, the exposure of adult beetles to glucosinolate hydrolysis products had no impact on the beetle’s energy budget under our experimental conditions. To understand how P. armoraciae can partially prevent glucosinolate hydrolysis, we analyzed the short-term fate of ingested glucosinolates and found them to be rapidly absorbed from the gut. In addition, we determined the fate of ingested Arabidopsis myrosinase enzymes in P. armoraciae. Although we detected Arabidopsis myrosinase protein in the feces, we found only traces of myrosinase activity, suggesting that P. armoraciae can inactivate plant myrosinases in the gut. Based on our findings, we propose that the ability to tolerate plant myrosinase activity and a fast glucosinolate uptake mechanism represent key adaptations of P. armoraciae to their brassicaceous host plants.

The Challenges of Designing and Implementing Clinical Trials With Broccoli Sprouts… and Turning Evidence Into Public Health Action.

Broccoli sprouts are a convenient and rich source of the glucosinolate glucoraphanin, which can generate the chemopreventive agent sulforaphane through the catalytic actions of plant myrosinase or β-thioglucosidases in the gut microflora. Sulforaphane, in turn, is an inducer of cytoprotective enzymes through activation of Nrf2 signaling, and a potent inhibitor of carcinogenesis in multiple murine models. Sulforaphane is also protective in models of diabetes, neurodegenerative disease, and other inflammatory processes, likely reflecting additional actions of Nrf2 and interactions with other signaling pathways. Translating this efficacy into the design and implementation of clinical chemoprevention trials, especially food-based trials, faces numerous challenges including the selection of the source, placebo, and dose as well as standardization of the formulation of the intervention material. Unlike in animals, purified sulforaphane has had very limited use in clinical studies. We have conducted a series of clinical studies and randomized clinical trials to evaluate the effects of composition (glucoraphanin-rich [± myrosinase] vs. sulforaphane-rich or mixture beverages), formulation (beverage vs. tablet) and dose, on the efficacy of these broccoli sprout-based preparations to evaluate safety, pharmacokinetics, pharmacodynamic action, and clinical benefit. While the challenges for the evaluation of broccoli sprouts in clinical trials are themselves formidable, further hurdles must be overcome to bring this science to public health action.

BMI Is Associated With Increased Plasma and Urine Appearance of Glucosinolate Metabolites After Consumption of Cooked Broccoli.

Introduction: Preclinical studies suggest that brassica vegetable diets decrease cancer risk, but epidemiological studies show varied effects, resulting in uncertainty about any health impact of brassicas. Factors controlling absorption of glucosinolate metabolites may relate to inconsistent results. We reported previously that subjects with BMI > 26 kg/m2 (HiBMI), given cooked broccoli plus raw daikon radish (as a source of plant myrosinase) daily for 17 days, had lower glucosinolate metabolite absorption than subjects given a single broccoli meal. This difference was not seen in subjects with BMI < 26 kg/m2 (LoBMI). Our objective in this current study was to determine whether a similar response occurred when cooked broccoli was consumed without a source of plant myrosinase.Methods: In a randomized crossover study (n = 18), subjects consumed no broccoli for 16 days or the same diet with 200 g of cooked broccoli daily for 15 days and 100 g of broccoli on day 16. On day 17, all subjects consumed 200 g of cooked broccoli. Plasma and urine were collected for 24 h and analyzed for glucosinolate metabolites by LC-MS.Results: There was no effect of diet alone or interaction of diet with BMI. However, absorption doubled in HiBMI subjects (AUC 219%, plasma mass of metabolites 202% compared to values for LoBMI subjects) and time to peak plasma metabolite values and 24-h urinary metabolites also increased, to 127 and 177% of LoBMI values, respectively.Conclusion: BMI impacts absorption and metabolism of glucosinolates from cooked broccoli, and this association must be further elucidated for more efficacious dietary recommendations.Clinical Trial Registration: This trial was registered at clinicaltrials.gov (NCT03013465).

Novel glucosinolate metabolism in larvae of the leaf beetle Phaedon cochleariae

Plants of the Brassicales are defended by a binary system, in which glucosinolates are degraded by myrosinases, forming toxic breakdown products such as isothiocyanates and nitriles. Various detoxification pathways and avoidance strategies have been found that allow different herbivorous insect taxa to deal with the glucosinolate-myrosinase system of their host plants. Here, we investigated how larvae of the leaf beetle species Phaedon cochleariae (Coleoptera: Chrysomelidae), a feeding specialist on Brassicaceae, cope with this binary defence. We performed feeding experiments using leaves of watercress (Nasturtium officinale, containing 2-phenylethyl glucosinolate as major glucosinolate and myrosinases) and pea (Pisum sativum, lacking glucosinolates and myrosinases), to which benzenic glucosinolates (benzyl- or 4-hydroxybenzyl glucosinolate) were applied. Performing comparative metabolomics using UHPLC-QTOF-MS/MS, N-(phenylacetyl) aspartic acid, N-(benzoyl) aspartic acid and N-(4-hydroxybenzoyl) aspartic acid were identified as major metabolites of 2-phenylethyl-, benzyl- and 4-hydroxybenzyl glucosinolate, respectively, in larvae and faeces. This suggests that larvae of P. cochleariae metabolise isothiocyanates or nitriles to aspartic acid conjugates of aromatic acids derived from the ingested benzenic glucosinolates. Myrosinase measurements revealed activity only in second-instar larvae that were fed with watercress, but not in freshly moulted and starved second-instar larvae fed with pea leaves. Our results indicate that the predicted pathway can occur independently of the presence of plant myrosinases, because the same major glucosinolate-breakdown metabolites were found in the larvae feeding on treated watercress and pea leaves. A conjugation of glucosinolate-derived compounds with aspartic acid is a novel metabolic pathway that has not been described for other herbivores.

Ontogenetic differences in the chemical defence of flea beetles influence their predation risk

Abstract Several insect species have evolved two‐component chemical defences that enable the rapid release of deterrent or toxic metabolites upon predator attack. However, whether these chemical defences vary across insect ontogeny and how this affects the predation risk of different life stages has rarely been addressed. The horseradish flea beetle Phyllotreta armoraciae possesses a two‐component chemical defence that consists of sequestered glucosinolates and an insect myrosinase capable of converting the non‐toxic glucosinolates to deterrent isothiocyanates. Here, we show that the levels of sequestered glucosinolates only varied 2‐fold across beetle ontogeny, but that insect myrosinase activity differed up to 43‐fold among ontogenetic stages. Specifically, glucosinolate levels were 1.5‐fold lower in the larvae of P. armoraciae than in pupae, but they showed 43.4‐fold higher levels of myrosinase activity. Consistent with the distinct levels of myrosinase activity in larvae and pupae, only larvae released high amounts of toxic isothiocyanates when they were attacked by the generalist predator Harmonia axyridis. P. armoraciae larvae deterred the predator and survived one attack, whereas pupae were killed. Feeding of P. armoraciae larvae on plants that differed in glucosinolates and plant myrosinase activity influenced the accumulation of glucosinolates in larvae and their subsequent interaction with H. axyridis. Larvae with low levels of sequestered glucosinolates were much more susceptible to predation than larvae containing high glucosinolate levels. Our results demonstrate that sequestered plant defence metabolites selectively protect specific ontogenetic stages of P. armoraciae from predation. The strong influence of plant defensive chemistry on sequestration indicates that predators have played an important role in the evolution of host use in this specialist herbivore. The distinct life styles of flea beetle life stages and their strategies to prevent predation by biologically relevant predator communities deserve further investigations. A free Plain Language Summary can be found within the Supporting Information of this article.

Cooking has the potential to decrease the antitumor effect of fresh Betong watercress.

Betong watercress (Nasturtium officinale R.Br.) contains phenethyl isothiocyanate (PEITC), derived from myrosinase-mediated hydrolysis of glucosinolates. Effects of fresh and cooked Betong watercress (FBW & CBW) on N-demethylation and C-8-hydroxylation of caffeine (CF) in rats were investigated. Wistar rats received a single dose of CF before and after pretreatments with a single or five-day administration of PEITC, FBW, and CBW dry powders. Plasma CF metabolic ratios (CMRs) were compared between before and after pretreatments. Single pretreatment with PEITC, FBW, but not CBW, significantly decreased CMRs. Five-day pretreatment with PEITC, FBW, and CBW significantly decreased CMRs. The decreases in CMRs after multiple doses of PEITC, FBW, and CBW were significantly higher than after a single dose. The decrease in CMRs caused by CBW was significantly lower than those by FBW, both single- and multiple doses. Cooking decreases the activity of FBW in inhibition of CYP1A2 mediating CF metabolism. PRACTICAL APPLICATIONS: PEITC and fresh watercress possess chemoprotective effects due to the inhibitory activity of PEITC on cytochrome P450s mediated bioactivation of carcinogens. Several clinical trials of the therapeutic uses of PEITC for cancer and other diseases are still in the pipeline. Betong watercress is a common ingredient in hot soup and stir-fried Thai recipes. Cooking heat inactivates plant myrosinase involving the production of PEITC. Consumption of watercress in cooked form may contribute less chemoprotective benefit. More appropriate preparation to deliver PEITC is needed to be evaluated.

One Pathway Is Not Enough: The Cabbage Stem Flea Beetle Psylliodes chrysocephala Uses Multiple Strategies to Overcome the Glucosinolate-Myrosinase Defense in Its Host Plants.

The cabbage stem flea beetle (Psylliodes chrysocephala) is a key pest of oilseed rape in Europe, and is specialized to feed on Brassicaceae plants armed with the glucosinolate-myrosinase defense system. Upon tissue damage, the β-thioglucosidase enzyme myrosinase hydrolyzes glucosinolates (GLS) to form toxic isothiocyanates (ITCs) which deter non-adapted herbivores. Here, we show that P. chrysocephala selectively sequester GLS from their host plants and store these throughout their life cycle. In addition, P. chrysocephala metabolize GLS to desulfo-GLS, which implies the evolution of GLS sulfatase activity in this specialist. To assess whether P. chrysocephala can largely prevent GLS hydrolysis in ingested plant tissue by sequestration and desulfation, we analyzed the metabolic fate of 4-methylsulfinylbutyl (4MSOB) GLS in adults. Surprisingly, intact and desulfo-GLS together accounted for the metabolic fate of only 26% of the total ingested GLS in P. chrysocephala, indicating that most ingested GLS are nevertheless activated by the plant myrosinase. The presence of 4MSOB-ITC and the corresponding nitrile in feces extracts confirmed the activation of ingested GLS, but the detected amounts of unmetabolized ITCs were low. P. chrysocephala partially detoxifies ITCs by conjugation with glutathione via the conserved mercapturic acid pathway. In addition to known products of the mercapturic acid pathway, we identified two previously unknown cyclic metabolites derived from the cysteine-conjugate of 4MSOB-ITC. In summary, the cabbage stem flea beetle avoids ITC formation by specialized strategies, but also relies on and extends the conserved mercapturic acid pathway to prevent toxicity of formed ITCs.

Validity and reproducibility of isothiocyanate intake assesses by food frequency questionnaire in the JPHC-next validation study: Comparison with 12-day weighed food records

Introduction Isothiocyanate (ITC) found in cruciferous vegetables are known for its chemopreventive properties that inhibits phase-I enzyme and activates phase-II enzymes. In epidemiologic studies, dietary ITC has been indicated in prevention of various types of cancer, such as lungs and bladder. Food frequency questionnaire (FFQ) is commonly used to assess ITC intake, which combines with laboratory data on the ITC content of specific cruciferous vegetables. Although availability of ITC depends on the contribution of plant myrosinase in the hydrolysis of glucosinolates, ITC content is measured on the basis of raw cruciferous vegetables in general. To our knowledge, no prospective studies have considered the effect of cooking. Using such databases could limit the study outcome of dietary ITC and disease relationships due to overestimation of ITC intake. Here, we aim to evaluate the validity of FFQ in the estimation of isothiocyanate using 12-day weighed food record (WFR) as the reference method by taking cooking methods into account. Methods Two hundred and forty participants (98 men and 142 women) aged 40–74 years from five areas in the JPHC-NEXT validation study were asked to respond to the 172-item FFQ and provide 12-day WFR as reference. Dietary ITC was calculated by combining the newly developed ITC database after consideration of popular cooking methods used in Japan. As we calculated the percentage of both raw and cooked cruciferous vegetable intake from WFR, cooked ITC intake was estimated from FFQ by weighing the average percentage of raw and cooked cruciferous vegetable on the ITC value. Spearman's correlation coefficients between estimates from FFQ and WFR were calculated and corrected for intra-individual variation of the WFR. Reproducibility of ITC intake was also assessed between 2 FFQs administered at an interval of 1-year. Results The mean intake of isothiocyanate according to FFQ was slightly lower than intake by WFR in men and higher in women. Percentage of difference of ITC were −3% for men and +19% in women. Deattenuated Spearman's correlation coefficient for ITC was 0.36 for men and 0.60 for women. Results were virtually unchanged when we applied the sex-unique percentage of raw and cooked cruciferous vegetable intake from WFR to derive correlation coefficients. Conclusions In conclusion, the validity of FFQ in estimating ITC was moderate. The reason for slightly poor correlation of ITC in men, is probably due to larger variance as a results of smaller sample size than in women. Additional confirmation may be warranted by using urinary isothiocyanate, which reflects the true intake of ITC.

Bioavailability of Glucoraphanin and Sulforaphane from High-Glucoraphanin Broccoli.

ScopeBroccoli accumulates 4‐methylsulphinylbutyl glucosinolate (glucoraphanin) which is hydrolyzed to the isothiocyanate sulforaphane. Through the introgression of novel alleles of the Myb28 transcription factor from Brassica villosa, broccoli genotypes have been developed that have enhanced levels of glucoraphanin. This study seeks to quantify the exposure of human tissues to glucoraphanin and sulforaphane following consumption of broccoli with contrasting Myb28 genotypes.Methods and resultsTen participants are recruited into a three‐phase, double‐blinded, randomized crossover trial (NCT02300324), with each phase comprising consumption of 300 g of a soup made from broccoli of one of three Myb28 genotypes (Myb28B/B, Myb28B/V, Myb28V/V). Plant myrosinases are intentionally denatured during soup manufacture. Threefold and fivefold higher levels of sulforaphane occur in the circulation following consumption of Myb28V/B and Myb28V/V broccoli soups, respectively. The percentage of sulforaphane excreted in 24 h relative to the amount of glucoraphanin consumed varies among volunteers from 2 to 15%, but does not depend on the broccoli genotype.ConclusionThis is the first study to report the bioavailability of glucoraphanin and sulforaphane from soups made with novel broccoli varieties. The presence of one or two Myb28V alleles results in enhanced delivery of sulforaphane to the systemic circulation.

Back cover: Allyl Isothiocyanate Ameliorates Obesity by Inhibiting Galectin-12

Mol. Nutr. Food Res. 2018, 62, 1700616 DOI: 10.1002/mnfr.201700616 Allyl isothiocyanate (AITC) is an organosulfur compound formed through the hydrolysis of glucosinolates by plant myrosinase. Glucosinolates are present in many cruciferous vegetables such as radish, cabbage, kale, broccoli, horseradish, and wasabi, which are widely and often consumed by humans. AITC inhibits adipocyte differentiation by suppressing galectin-12 expression and has an anti-obesity effect in HFD-fed mice. Further details can be found in article number 1700616 by Lei Wan and co-workers.