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Brassica vegetables are frequently consumed foods of nutritional interest, because they are rich in glucosinolates (GLSs). Among GLS breakdown products, especially isothiocyanates are known for their health-beneficial effects, while nitriles are less beneficial. To increase the understanding of the plant matrix's influence on GLS degradation, differently concentrated vegetable broths were prepared from selected Brassica vegetables (kohlrabi and red cabbage) and subsequently boiled. Altogether, heat stability and conversion of GLSs to the corresponding nitriles were both strongly influenced by vegetable type and plant matrix concentration in the broths. After boiling kohlrabi broths for 120 min, recovery of 4-(methylthio)butyl-GLS as nitrile was 55.5 % in 1 g/mL broth and 8.4 % in 0.25 g/mL broth. In follow-up experiments, a pronounced influence of the matrix's redox status was identified, with H2S being an important factor. A better understanding of these processes will help to preserve health-promoting effects of GLSs in Brassica vegetables in the future.

Glucosinolates are plant secondary metabolites found in cruciferous vegetables (Brassicaceae) that are valued for their potential health benefits. Frequently consumed representatives of these vegetables, for example, are white or red cabbage, which are typically boiled before consumption. Recently, 3-alk(en)yl-4-hydroxythiazolidine-2-thiones were identified as a class of thermal glucosinolate degradation products that are formed during the boiling of cabbage. Since these newly discovered compounds are frequently consumed, this raises questions about their potential uptake and their possible bioactive functions. Therefore, 3-allyl-4-hydroxythiazolidine-2-thione (allyl HTT) and 4-hydroxy-3-(4-(methylsulfinyl) butyl)thiazolidine-2-thione (4-MSOB HTT) as degradation products of the respective glucosinolates sinigrin and glucoraphanin were investigated. After consumption of boiled red cabbage broth, recoveries of consumed amounts of the degradation products in urine collected for 24 h were 18 ± 5% for allyl HTT and 21 ± 4% for 4-MSOB HTT (mean ± SD, n = 3). To investigate the stability of the degradation products during uptake and to elucidate the uptake mechanism, both an in vitro stomach and an in vitro intestinal model were applied. The results indicate that the uptake of allyl HTT and 4-MSOB HTT occurs by passive diffusion. Both compounds show no acute cell toxicity, no antioxidant potential, and no change in NAD(P)H dehydrogenase quinone 1 (NQO1) activity up to 100 μM. However, inhibition of glycogen synthase kinases-3 (GSK-3) in the range of 20% for allyl HTT for the isoform GSK-3β and 29% for 4-MSOB HTT for the isoform GSK-3α at a concentration of 100 μM was found. Neither health-promoting nor toxic effects of 3-alk(en)yl-4-hydroxythiazolidine-2-thiones were found in the four tested assays carried out in this study, which contrasts with the properties of other glucosinolate degradation products, such as isothiocyanates.

Glucosinolates are precursors of bioactive and health-promoting isothiocyanates (ITCs). Upon enzymatic hydrolysis, Brassica vegetables, such as cabbage, also often yield nitriles and epithionitriles as main products next to ITCs. Here, we show that amines can be additional main enzymatic hydrolysis products of glucosinolates in Brassica vegetables. We propose that a plant endogenous ITC hydrolase (ITCase) is responsible for the enzymatic-like conversion of ITCs to amines in cabbage samples. This ITCase seems to have high activity towards alkenyl ITCs like allyl ITC and lower activity towards methylthioalkyl ITCs, and not to converting methylsulfinylalkyl ITCs like sulforaphane. In contrast, during heat treatment of homogenized cabbage material, methylsulfinylalkylamine levels increased by 400 % after 2 h of heating, which is likely due to thermal decomposition of ITCs, whereas alkenyl amine levels did not change due to heat treatment. The results show that amines from glucosinolates are part of the human diet.

Glucosinolates are plant secondary metabolites occurring in Brassicaceae plants. Upon tissue disruption, these compounds can be enzymatically hydrolyzed into isothiocyanates, which are very reactive and can react with nucleophiles during thermal processes such as boiling. Here, a novel type of glucosinolate degradation product was identified resulting from the reaction of thioglucose with the isothiocyanates sulforaphane or allyl isothiocyanate during aqueous heating. The two heterocyclic compounds 4-hydroxy-3-(4-(methylsulfinyl)butyl)thiazolidine-2-thione and 3-allyl-4-hydroxythiazolidine-2-thione were isolated and their structure elucidated by NMR spectroscopy and high-resolution mass spectrometry. Based on a set of chemical experiments, a reaction mechanism was proposed. Finally, the formation of the two 3-alk(en)yl-4-hydroxythiazolidine-2-thiones was quantified in boiled cabbage samples using a standard addition method in which 92 pmol/g and 19 pmol/g fresh weight of the sulforaphane and allyl isothiocyanate derivatives were found, respectively.

Besides glucosinolates, Brassica vegetables accumulate sulfur-containing (+)-S-methyl-l-cysteine sulfoxide (SMCSO, methiin), mainly known from Allium vegetables. Such (+)-S-alk(en)yl-l-cysteine sulfoxides can degrade to volatile organosulfur compounds (VOSCs), which have been linked to health beneficial effects. In the present study, the accumulation of SMCSO and the formation of VOSCs was investigated in Brassica oleracea vegetables. SMCSO content of commercially available white and red cabbages was monitored over a three-month period and linked with the formation of VOSCs. S-Methyl methanethiosulfinate was the main VOSC released from SMCSO. Upon heating, it degraded to dimethyltrisulfide and dimethyldisulfide, which were less abundant in fresh homogenates. SMCSO made up approximately 1% of the dry matter of cabbages and the overall contents were similar in white and red cabbages (3.2-10.2 and 3.9-10.3 µmol/g fresh weight, respectively). Using proteome profiling it was shown that recovery of VOSCs correlated with abundance of two isoforms of cystine lyase.

Epithionitriles can be main glucosinolate hydrolysis products in Brassica vegetables such as cabbage or pak choi. Here, for the first time, the bioavailability and metabolism of longer-chain epithionitriles (C4-C5) is studied in a human intervention study.After consumption of a white cabbage or pak choi sprouts beverage, rich in either 1-cyano-2,3-epithiopropane (CETP) or 1-cyano-3,4-epithiobutane (CETB) and 1-cyano-4,5-epithiopentane (CETPent), blood and urine samples of nine participants are taken and the metabolites are analyzed. The corresponding N-acetyl-S-(cyano-(methylthio)alkyl)-l-cysteine metabolites are identified and quantified by isotope dilution method using UHPLC-TOF-MS. The standards for N-acetyl-S-(cyano-(methylthio)alkyl)-l-cysteine metabolites from CETB and CETPent are synthesized for the first time and their structure confirmed by NMR spectroscopy. In contrast to the metabolites of CETP and CETPent, the expected metabolite of CETB is not detectable. The recoveries of the CETP and CETPent metabolites are 28 ± 9% for CETP and 12 ± 3% for CETPent in urine within 24 h.CETP and CETPent are quickly uptaken, metabolized via the mercapturic acid pathway, and excreted via urine, while for CETB the corresponding metabolite is not detectable. Therefore, an additional metabolization pathway seems to exist.

Glucosinolates are secondary plant metabolites occurring in Brassicaceae plants. Upon tissue disruption these compounds can be enzymatically hydrolyzed into isothiocyanates. The latter are very reactive and can react with nucleophiles during food processing such as cooking. Here, a novel type of glucosinolate degradation product was identified resulting from the reaction of the isothiocyanates sulforaphane and allyl isothiocyanate with thioglucose during aqueous heat treatment. The cyclic compounds were isolated and their structure elucidated by NMR spectroscopy and high resolution mass spectrometry as 4-hydroxy-3-(4-(methylsulfinyl)butyl)thiazolidine-2-thione and 3-allyl-4-hydroxythiazolidine-2-thione. Based on experiments with isotope-labeled reagents, the determination of the diastereomeric ratio and further reactions, a reaction mechanism was proposed. Finally, the formation of the two 3-alk(en)yl-4-hydroxythiazolidine-2-thiones was quantified in boiled cabbage samples with contents of 92 pmol/g respectively 19 pmol/g fresh weight using standard addition method.

Brassicaceae vegetables are rich in glucosinolates (GLS), which degrade into various breakdown products, including isothiocyanates (ITC), during food processing. ITC are associated with health-promoting properties; therefore, producing food products enriched with a high content of these compounds is of interest for improving and maintaining human health. The present study aimed at evaluating a potential increase in ITC formation in Brassicaceae-enriched bread while minimizing ITC-protein conjugates. The influence of pH on GLS degradation was evaluated in different bread types and pH-adjusted model breads. In all samples, ITC, nitriles, and ITC-amino acid conjugates were analyzed by GC-MS and LC-ESI-MS/MS, respectively. The highest ITC-Lys levels with 33.9 µmol BITC-Lys/g bread could be found in a (more alkaline soda) bread with a pH of 7.2, while "free" BITC content was the lowest. However, this finding could not be directly correlated to pH value, because in model breads no significant relation between the pH and the BITC content could be identified. Especially the baking process impacted the ITC content as it was reduced from dough to the bread by 98%. Therefore, a heated food product is not suitable for an enrichment with GLS-rich vegetables to achieve a high content on BITC. Nevertheless, in the bread matrix itself the degradation products and BITC-Lys conjugates were stable during storage.

Vegetables of the plant order Brassicales are believed to have health-promoting properties, as they provide high contents of glucosinolates (GLS) and deriving from these, enzymatically and heat-induced breakdown products, such as isothiocyanates (ITC). Besides their positive physiological effects, ITC are electrophilic and can undergo reactions with food components such as proteins. Following the trend of improving traditional food products with GLS-rich ingredients, interactions of ITC with proteins can diminish the properties of both components—protein’s value and functionality as well as ITC’s bioactivity. In vegetable-enriched bread, where cresses (Lepidium sativum L. or Tropaeolum majus L.) are added to the initial dough, together with benzyl cyanide, benzyl isothiocyanate (BITC) is formed during the baking process. The aim of the present study was to investigate the possible migration behavior of the GLS breakdown products and the formation of ITC-wheat protein conjugates. After the baking process, the breads’ proteins were enzymatically hydrolyzed, and the ITC-amino acid conjugates analyzed using a LC-ESI-MS/MS methodology. In all samples, BITC-protein conjugates were detected as thiourea derivatives, while formation of dithiocarbamates could not be detected. The study showed that GLS and their breakdown products such as ITC migrate into the surrounding food matrix and undergo reactions with proteins, which could in turn lead to modified protein properties and reduce the bioavailability of ITC and lysine.

Understanding individual responses to nutrition and medicine is of growing interest and importance. There is evidence that differences in bitter taste receptor (TAS2R) genes which give rise to two frequent haplotypes, TAS2R38-PAV (functional) and TAS2R38-AVI (non-functional), may impact inter-individual differences in health status. We here analyzed the relevance of the TAS2R38 receptor in the regulation of the human immune response using the TAS2R38 agonist allyl isothiocyanate (AITC) from Brassica plants. A differential response in calcium mobilization upon AITC treatment in leucocytes from healthy humans confirmed a relevance of TAS2R38 functionality, independent from cation channel TRPV1 or TRPA1 activation. We further identified a TAS2R38-dependence of MAPK and AKT signaling activity, bactericidal (toxicity against E. coli) and anti-inflammatory activity (TNF-alpha inhibition upon cell stimulation). These in vitro results were derived at relevant human plasma levels in the low micro molar range as shown here in a human intervention trial with AITC-containing food.

Brassica vegetables contain glucosinolates, which are well-known for their potential to form health-promoting isothiocyanates. Among those crucifers, white and red cabbage are commonly consumed vegetables, exhibiting different glucosinolate and hydrolysis profiles thereof. Regarding the health beneficial effects from these vegetables, more information, especially concerning the seasonal variation of glucosinolate profiles and the formation of their bioactive hydrolysis products in commercial cabbages, is needed. In this study, glucosinolates and glucosinolate hydrolysis product profiles in red and white cabbages from three different food retailers were monitored over six different sampling dates across the selling season in autumn. For the first time, it was shown that, while glucosinolate profiles were similar in each cabbage variety, glucosinolate hydrolysis product profiles and hydrolysis behavior varied considerably over the season. The highest total isothiocyanate concentrations were observed in conventional red (1.66 &mu, mol/g FW) and organic white (0.93 &mu, mol/g FW) cabbages purchased at the first sampling date in September. Here, red cabbage was with up to 1.06 &mu, mol/g FW of 4-(methylsulfinyl)butyl isothiocyanate (sulforaphane), an excellent source for this health-promoting isothiocyanate. Cabbages purchased 11 weeks later in autumn released lower levels of isothiocyanates, but mainly nitriles and epithionitriles. The results indicate that commercial cabbages purchased in early autumn could be healthier options than those purchased later in the year.

Glucosinolates present in

Replant disease is a soil (micro-) biome-based, harmfully-disturbed physiological and morphological reaction of plants to replanting similar cultures on the same sites by demonstrating growth retardation and leading to economic losses especially in Rosaceae plant production. Commonly, replant disease is overcome by soil fumigation with toxic chemicals. With chemical soil fumigation being restricted in many countries, other strategies are needed. Biofumigation, which is characterized by the incorporation of Brassicaceae plant materials into soil, is a promising method. We review the potential of biofumigation in the fight against replant disease. Biofumigation using optimized Brassicaceae seed meal compositions in combination with replant disease tolerant plant genotypes shows promising results, but the efficacy is still soil and site-dependent. Therefore, future studies should address the optimal timing as well as amount and type of incorporated plant material and environmental conditions during incubation in dependence of the soil physical and chemical characteristics.

Isothiocyanates (ITC) play an important role in health promotion and cancer prevention due to their anti-bacterial, anti-inflammatory, and anti-cancerogenic properties. However, ITC are highly reactive so that a reaction with further food components is very likely. For example, a reaction of ITC with nucleophilic amino acid side chains of proteins such as cysteine and lysine can occur, reducing the bioavailability of indispensable amino acids and protein functions may be altered. Therefore, it is of great interest to investigate the fate of ITC in the food matrix. Accordingly, the aim of the present study was to investigate the interaction of milk proteins and the ITC benzyl isothiocyanate (BITC) and allyl isothiocyanate (AITC) forming dithiocarbamates and thioureas in milk and curd. After incubating milk and curd with pure ITC or ITC-containing garden cress (Lepidium sativum L.), proteins were isolated, digested, and analyzed via LC-ESI-MS/MS as amino acid derivatives (“conjugates”). Protein conjugates of AITC and BITC were detected in all samples investigated. Further, the acidic pH value in curd favored the formation of dithiocarbamates over the formation of thioureas. Slightly acidic or neutral conditions like in fresh milk favored the formation of thioureas. The investigations also indicated that AITC shows a higher reactivity and dithiocarbamates are formed preferably, whereas incubation with BITC lead to less protein conjugates and the ratio of thioureas and dithiocarbamates was more balanced. In addition, amino acid modifications were often analyzed with indirect methods like measuring the decline of the amino acid residues. In this study, the modified amino acids were analyzed directly leading to more reliable results concerning the amount of modification.

A new mass spectrometric method for evaluating metabolite formation of the pesticides thiacloprid, azoxystrobin, and difenoconazole was developed for the Brassica species pak choi and broccoli. Both, distribution and transformation kinetics of the active compounds and their metabolites were analyzed by UPLC-TWIMS-QTOF-MS. Additionally, HR-MS analysis and structure elucidation tools such as diagnostic ions, isotopic matches, and collision cross sections were applied for metabolites identification. Following the application of two plant protection products (containing the above-mentioned active compounds) in a greenhouse study plant material was cryo-milled and extracted with water/methanol. The residual levels of active compounds were identified at certain timepoints during pre-harvest intervals and in the final products. Different phase I and phase II metabolites of the pesticides were identified in different plant organs such as leaves, stems, (broccoli) heads, and roots. Three individual degradation pathways and distribution profiles are suggested including eight thiacloprid, eleven azoxystrobin and three difenoconazole metabolites.

The epithionitriles, 1-cyano-2,3-epithiopropane, in particular, and 1-cyano-3,4-epithiobutane, are important, but yet underestimated glucosinolate hydrolysis products that are released instead of the cancer preventative isothiocyanates in Brassica vegetables, such as cabbage, broccoli, or pak choi. Here, we characterized the reactivity of 1-cyano-2,3-epithiopropane under aqueous heat treatment conditions and compared our findings to those of the related epithionitriles 1-cyano-3,4-epithiobutane and 1-cyano-4,5-epithiopentane. In contrast to the other epithionitriles, 1-cyano-2,3-epithiopropane is highly reactive. As a result, 2-aminothiophene and dimeric 1,4-dithiane-2,5-diacetonitrile were identified as main products and a reaction mechanism is proposed. Formation of 2-aminothiophene was also observed in cooked white cabbage samples. Moreover, three novel compounds were identified as derivatives of the related epithionitriles. The results imply that apart from isothiocyanates, process-derived compounds should be considered in regards to cancer preventative Brassica vegetable related bioactivity.

From the order Brassicales, different plant organs, such as inflorescences (e.g. broccoli and cauliflower), leaves (e.g. kale and pak choi), heads (e.g. white and red cabbage), as well as roots and bulbs (e.g. radish and turnip), are frequently consumed brassicaceaous vegetables. The order Brassicales is characterized by a specific group of secondary plant metabolites, namely the glucosinolates. Glucosinolates and their breakdown products the isothiocyanates are linked to conferring beneficial health effects. In addition, some studies have also highlighted the beneficial health effects of phenolic compounds and carotenoids, both well-known as antioxidants. Of interest is that the profiles and concentrations of secondary plant metabolites vary enormously between the species, and genetic factors are thought to affect this the most. Further, environmental and agronomical factors are also known to change concentrations of secondary plant metabolites enormously. The main physiological mechanism to produce secondary plant metabolites is defense. Thus, the intrinsic quality, including color, aroma, taste, and beneficial health properties of brassicaceous vegetables, is remarkably affected by secondary plant metabolite profiles and concentrations.

Mycorrhizal symbiosis is known to be the most prevalent form of fungal symbiosis with plants. Although some studies focus on the importance of mycorrhizal symbiosis for enhanced flavonoids in the host plants, a comprehensive understanding of the relationship still is lacking. Therefore, we studied the effects of mycorrhizal inoculation of onions (Allium cepa L.) regarding flavonol concentration and the genes involved in flavonol biosynthesis when different forms of nitrogen were supplied. We hypothesized that mycorrhizal inoculation can act as a biotic stress and might lead to an increase in flavonols and expression of related genes. The three main quercetin compounds [quercetin-3,4′-di-O-β-d-glucoside (QDG), quercetin-4′-O-β-d-glucoside (QMG), and isorhamnetin-4′-O-β-d-glucoside (IMG)] of onion bulbs were identified and analyzed after inoculating with increasing amounts of mycorrhizal inocula at two time points and supplying either predominantly NO3 − or NH4 + nitrogen. We also quantified plant dry mass, nutrient element uptake, chalcone synthase (CHS), flavonol synthase (FLS), and phenyl alanine lyase (PAL) gene expression as key enzymes for flavonol biosynthesis. Inoculation with arbuscular mycorrhizal fungi (highest amount) and colonization at late development stages (bulb growth) increased QDG and QMG concentrations if plants were additionally supplied with predominantly NH4 +. No differences were observed in the IMG content. RNA accumulation of CHS, FLS, and PAL was affected by the stage of the mycorrhizal symbiosis and the nitrogen form. Accumulation of flavonols was not correlated, however, with either the percentage of myorrhization or the abundance of transcripts of flavonoid biosynthesis genes. We found that in plants at late developmental stages, RNA accumulation as a reflection of a current physiological situation does not necessarily correspond with the content of metabolites that accumulate over a long period. Our findings suggest that nitrogen form can be an important factor determining mycorrhizal development and that both nitrogen form and mycorrhizas interact to influence flavonol biosynthesis.

Red cabbage contains glucosinolates, precursors to health-promoting isothiocyanates. However, raw cabbage often releases mainly epithionitriles and nitriles from glucosinolates. To increase isothiocyanate formation, the effect of acid usage in the preparation of red cabbage was evaluated. Moreover, the effects of the chosen boiling method (acidic boiled red cabbage versus neutral boiled blue cabbage) on glucosinolate degradation were investigated using UHPLC-DAD-ToF-MS and GC–MS. The addition of vinegar significantly increased isothiocyanate formation of cabbage salad from 0.09 to 0.21 µmol/g fresh weight, while lemon juice only slightly increased isothiocyanate formation. Acidic boiled red cabbage degraded glucosinolates and increased nitrile formation, while in neutral boiled blue cabbage, glucosinolates were stable. However, shortly boiled blue cabbage (5 min) had the highest isothiocyanate levels (0.08 µmol/g fresh weight). Thus, for a diet rich in isothiocyanates it is recommended to acidify raw cabbage salads and prepare shortly boiled blue cabbage instead of red cabbage.

Glucosinolates present in Brassicaceae play a major role in herbivory defense. Upon tissue disruption, glucosinolates come into contact with myrosinase, which initiates their breakdown to biologically active compounds. Among these, the formation of epithionitriles is triggered by the presence of epithiospecifier protein (ESP) and a terminal double bond in the glucosinolate side chain. One ESP gene is characterized in the model plant Arabidopsis thaliana (AtESP; At1g54040.2). However, Brassica species underwent genome triplication since their divergence from the Arabidopsis lineage. This indicates the presence of multiple ESP isoforms in Brassica crops that are currently poorly characterized. We identified three B. oleracea ESPs, specifically BoESP1 (LOC106296341), BoESP2 (LOC106306810), and BoESP3 (LOC106325105) based on in silico genome analysis. Transcript and protein abundance were assessed in shoots and roots of four B. oleracea vegetables, namely broccoli, kohlrabi, white, and red cabbage, because these genotypes showed a differential pattern for the formation of glucosinolate hydrolysis products as well for their ESP activity. BoESP1 and BoESP2 were expressed mainly in shoots, while BoESP3 was abundant in roots. Biochemical characterization of heterologous expressed BoESP isoforms revealed different substrate specificities towards seven glucosinolates: all isoforms showed epithiospecifier activity on alkenyl glucosinolates, but not on non-alkenyl glucosinolates. The pH-value differently affected BoESP activity: while BoESP1 and BoESP2 activities were optimal at pH 6-7, BoESP3 activity remained relatively stable from pH 4 to 7. In order test their potential for the in vivo modification of glucosinolate breakdown, the three isoforms were expressed in A. thaliana Hi-0, which lacks AtESP expression, and analyzed for the effect on their respective hydrolysis products. The BoESPs altered the hydrolysis of allyl glucosinolate in the A. thaliana transformants to release 1-cyano-2,3-epithiopropane and reduced formation of the corresponding 3-butenenitrile and allyl isothiocyanate. Plants expressing BoESP2 showed the highest percentage of released epithionitriles. Given these results, we propose a model for isoform-specific roles of B. oleracea ESPs in glucosinolate breakdown.

Pak choi ( Brassica rapa subsp. chinensis ) is a leafy vegetable that is widely available in Asia and consumed in rising quantities in Europe. Pak choi contains high levels of secondary plant metabolites, such as carotenoids, chlorophylls, glucosinolates, phenolic compounds, and vitamin K, which are beneficial for humans if consumed on a regular basis. The evaluation of the genotype-induced variation of secondary plant metabolites revealed that the cultivar ‘Amur’ contained the highest concentration of secondary plant metabolites. Furthermore, steaming retained more chlorophylls, glucosinolates, phenolic acids and flavonoid compounds than boiling. In contrast, both domestic cooking methods – boiling, and steaming – reduced the formation of glucosinolate breakdown products, especially the undesired epithionitriles and nitriles but less of the health-beneficial isothiocyanates.

Brassica vegetables such as cabbage or pak choi contain alkenyl glucosinolates which can release epithionitriles and to a lesser degree isothiocyanates upon enzymatic hydrolysis. Here, for the first time, the metabolism of an epithionitrile was investigated in humans, namely 1-cyano-2,3-epithiopropane (CETP). After consumption of Brassica oleracea var. capitata f. alba and Brassica carinata sprouts, the main urinary metabolite of CETP was identified as N-acetyl-S-(3-cyano-2-(methylsulfanyl)propyl-cysteine using an UHPLC-ESI-QToF-MS approach and synthesis of the metabolite. This urinary epithionitrile metabolite is an S-methylated mercapturic acid. No other metabolites were detected. Then, in a preliminary pilot experiment the excretion kinetics of CETP were investigated in three volunteers. After consumption of a B. carinata sprout preparation containing 50.8 µmol of CETP, urinary N-acetyl-S-(3-cyano-2-(methylsulfanyl)propyl-cysteine concentrations were the highest three hours after consumption, ranging from 23.9 to 37.2 µM, and declined thereafter. Thus, epithionitriles are bioavailable compounds that are metabolized similarly to isothiocyanates by the mercapturic acid pathway. In the future, more epithionitrile metabolites should be identified and the pharmacokinetics of these important class of dietary compounds should be assessed in more detail.

The effects of biofumigation with Brassica juncea ‘Terra Plus’ and Raphanus sativus ‘Defender’ in comparison to Basamid on apple plant growth and on soil microbial communities were studied at three sites affected by replant disease under field conditions. Apple rootstocks were planted on differently treated plots to evaluate the effect of the treatments on plant growth under field and greenhouse conditions. The glucosinolates in biofumigant plant organs and their breakdown products in soils were determined. Denaturing gradient gel electrophoresis fingerprints were performed with 16S rRNA gene and ITS fragments amplified from total community DNA extracted from different soils. The highest glucosinolate concentrations were found in inflorescences of both biofumigant plant species with no differences between sites. The most abundant degradation product in soil biofumigated with B. juncea was 2-propenyl isothiocyanate, while in soil treated with R. sativus only 4-(methylthio)-3-butenyl isothiocyanate was detected. Effects of biofumigation were recorded to be stronger on fungi than on bacteria. Growth of apple rootstocks was positively affected by the treatments in a site-dependent manner. The effects of biofumigation evaluated by the apple plant growth were site-dependent and might result from suppression of soil-borne pests and pathogens, changes in soil microbial community compositions, and additional nutrients from the incorporated biomass.

Consumption of Brassica vegetables is linked to health benefits, as they contain high concentrations of the following secondary plant metabolites (SPMs): glucosinolate breakdown products, carotenoids, chlorophylls, and phenolic compounds. Especially Brassica vegetables are consumed as microgreens (developed cotyledons). It was investigated how different ontogenetic stages (microgreens or leaves) of pak choi (Brassica rapa subsp. chinensis) and kale (Brassica oleracea var. sabellica) differ in their SPM concentration. The impact of breadmaking on SPMs in microgreens (7 days) and leaves (14 days) in pak choi and kale as a supplement in mixed wheat bread was assessed. In leaves, carotenoids, chlorophylls, and phenolic compounds were higher compared to those of microgreens. Breadmaking caused a decrease of SPMs. Chlorophyll degradation was observed, leading to pheophytin and pyropheophytin formation. In kale, sinapoylgentiobiose, a hydroxycinnamic acid derivative, concentration increased. Thus, leaves of Brassica species are suitable as natural ingredients for enhancing bioactive SPM concentrations in bread.

The present human intervention trial investigated the health-promoting potential of B. carinata, with a focus on effects of thermal processing on bioactivity. Twenty-two healthy subjects consumed a B. carinata preparation from raw (allyl isothiocyanate-containing) or cooked (no allyl isothiocyanate) leaves for five days in a randomized crossover design. Peripheral blood mononuclear cells were exposed to aflatoxin B1 (AFB1), with or without metabolic activation using human S9 mix, and subsequently analyzed for DNA damage using the comet assay. Plasma was analyzed for total antioxidant capacity and prostaglandin E2 (PGE2) levels. Cooked B. carinata significantly reduced DNA damage induced by AFB1 as compared to baseline levels (+S9 mix: 35%, &minus, S9 mix: 33%, p &le, 0.01, respectively). Raw B. carinata only reduced DNA damage by S9-activated AFB1 by 21% (p = 0.08). PGE2 plasma levels were significantly reduced in subjects after consuming raw B. carinata. No changes in plasma antioxidant capacity were detectable. A balanced diet, including raw and cooked Brassica vegetables, might be suited to fully exploit the health-promoting potential. These results also advocate the promotion of B. carinata cultivation in Eastern Africa as a measure to combat effects of unavoidable aflatoxin exposure.

The present human intervention trial investigated the health-promoting potential of B. carinata, with a focus on effects of thermal processing on bioactivity. Twenty-two healthy subjects consumed a B. carinata preparation from raw (allyl isothiocyanate-containing) or cooked (no allyl isothiocyanate) leaves for five days in a randomized crossover design. Peripheral blood mononuclear cells were exposed to aflatoxin B1 (AFB1), with or without metabolic activation using human S9 mix, and subsequently analyzed for DNA damage using the comet assay. Plasma was analyzed for total antioxidant capacity and prostaglandin E2 (PGE2) levels. Cooked B. carinata significantly reduced DNA damage induced by AFB1 as compared to baseline levels (+S9 mix: 35%, −S9 mix: 33%, p ≤ 0.01, respectively). Raw B. carinata only reduced DNA damage by S9-activated AFB1 by 21% (p = 0.08). PGE2 plasma levels were significantly reduced in subjects after consuming raw B. carinata. No changes in plasma antioxidant capacity were detectable. A balanced diet, including raw and cooked Brassica vegetables, might be suited to fully exploit the health-promoting potential. These results also advocate the promotion of B. carinata cultivation in Eastern Africa as a measure to combat effects of unavoidable aflatoxin exposure.

Glucosinolates and their breakdown products, especially isothiocyanates (ITCs), are hypothesized to exert a broad range of bioactivities. However, physiological mechanisms are not yet completely understood. In this study, formation of protein conjugates after incubation with benzyl isothiocyanate (BITC) was investigated in vitro. A survey of protein conjugates was done by determining BITC cysteine and lysine amino acid conjugates after protein digestion. Therefore, a liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) method was developed and validated. Stability studies showed that cysteine conjugates are not stable under alkaline conditions, and lysine conjugates did not show any correlation to pH values, although stability increased at low temperatures. Lysine conjugates were the preferred form of protein conjugates, and longer BITC exposure times led to higher amounts. Knowledge about the reaction sites of ITCs in eukaryotic cells may help to understand the mode of action of ITCs leading to health promoting as well as toxicological effects in humans.

An in-depth glucosinolate (GLS) profiling was performed on a core collection of 91 Brassica rapa accessions, representing diverse morphotypes of heterogeneous geographical origin, to better understand the natural variation in GLS accumulation and GLS breakdown product formation. Leaves of the 91 B. rapa accessions were analyzed for their GLS composition by UHPLC-DAD and the corresponding breakdown products by GC-MS. Fifteen different GLSs were identified, and aliphatic GLSs prevailed regarding diversity and concentration. Twenty-three GLS breakdown products were identified, among them nine isothiocyanates, ten nitriles, and four epithionitriles. Epithionitriles were the prevailing breakdown products due to the high abundance of alkenyl GLSs. The large scale data set allowed the identification of correlations in abundance of specific GLSs or of GLS breakdown products. Discriminant function analysis identified subspecies with high levels of similarity in the acquired metabolite profiles. In general, the five main subspecies grouped significantly in terms of their GLS profiles.

Pak choi (Brassica rapa subsp. chinensis) is rich in secondary metabolites and contains numerous antioxidants, including flavonoids; hydroxycinnamic acids; carotenoids; chlorophylls; and glucosinolates, which can be hydrolyzed to epithionitriles, nitriles, or isothiocyanates. Here, we investigate the effect of reduced exposure to ultraviolet B (UVB) and UV (UVA and UVB) light at four different developmental stages of pak choi. We found that both the plant morphology and secondary metabolite profiles were affected by reduced exposure to UVB and UV, depending on the plant’s developmental stage. In detail, mature 15- and 30-leaf plants had higher concentrations of flavonoids, hydroxycinnamic acids, carotenoids, and chlorophylls, whereas sprouts contained high concentrations of glucosinolates and their hydrolysis products. Dry weights and leaf areas increased as a result of reduced UVB and low UV. For the flavonoids and hydroxycinnamic acids in 30-leaf plants, less complex compounds were favored, for example, sinapic acid acylated kaempferol triglycoside instead of the corresponding tetraglycoside. Moreover, also in 30-leaf plants, zeaxanthin, a carotenoid linked to protection during photosynthesis, was increased under low UV conditions. Interestingly, most glucosinolates were not affected by reduced UVB and low UV conditions. However, this study underlines the importance of 4-(methylsulfinyl)butyl glucosinolate in response to UVA and UVB exposure. Further, reduced UVB and low UV conditions resulted in higher concentrations of glucosinolate-derived nitriles. In conclusion, exposure to low doses of UVB and UV from the early to late developmental stages did not result in overall lower concentrations of plant secondary metabolites.

Rocket is rich in glucosinolates and valued for its hot and spicy taste. Here we report the structure elucidation, bioactivity, and stability of the mainly formed glucosinolate hydrolysis product, namely sativin, which was formerly thought to be 4-mercaptobutyl isothiocyanate. However, by NMR characterization we revealed that sativin is in fact 1,3-thiazepane-2-thione, a tautomer of 4-mercaptobutyl isothiocyanate with 7-membered ring structure and so far unknown. This finding was further substantiated by conformation sampling using molecular modeling and total enthalpy calculation with density functional theory. During aqueous heat treatment sativin in general was quite stable, while the isothiocyanates erucin and sulforaphane were labile, having half-lives of 132 min and 56 min (pH 5, 100 °C), respectively. Moreover, using a WST-1 assay, we found that sativin did not reduce cell viability of HepG2 cells in a range of 0.3–30 µM, and, therefore, exhibited no cytotoxic effects in this cell line.

Domestic processing methods, such as boiling, significantly affect the glucosinolate content and the formation of breakdown products in Brassica vegetables. Here, we comprehensively describe the effect of aqueous heat treatment on the degradation and leaching kinetics of glucosinolates on the formation of their enzymatic and non-enzymatic hydrolysis and breakdown products. The results were correlated with the inactivation kinetics of myrosinase and epithiospecifier protein activity in the Brassica oleracea vegetables kohlrabi, white cabbage, and red cabbage. Short-term heating increased isothiocyanate formation due to inactivation of the epithiospecifier protein. Myrosinase was inactivated shortly after that. Boiling led to leaching of glucosinolates and their hydrolysis products into the boiling water. Heating to 99 °C resulted in thermally-induced glucosinolate breakdown and nitrile formation, both in vegetables and boiling water. Finally, kinetic modeling not only revealed differences in myrosinase inactivation among the vegetables, but also glucosinolate leaching and degradation kinetics differed between individual glucosinolates and vegetables.

Plants of the order Brassicales produce glucosinolates (GS), a group of secondary metabolites that are part of an elaborate defense system. But it is not the GS itself rather its enzymatic hydrolysis products that cause the bioactive effects protecting the plants against pests and pathogens. Thus the enzymatic hydrolysis and a variety of additional influential factors determine the structural outcome of the GS degradation process. To evaluate the possible diversity of defense metabolites a range of 19 Arabidopsis thaliana accessions were selected showing divergence in their geographical origin, in their phenotype, and in their GS profile. These particular accessions accumulate several alkenyl GS, hydroxyalkyl GS, methylthioalkyl GS, and methylsulfinylalkyl GS in their rosette leaves whereas the indole GS contents are relatively invariant, as analyzed by UHPLC-DAD. After tissue disruption the enzymatic formation of GS hydrolysis products was examined and breakdown products were identified and quantified by GC-MS. Great differences in the amount and structure of volatile enzymatic degradation products could be observed in the different accessions, with strong variation in formation of epithionitriles, nitriles, and isothiocyanates. The occurrence of specific GS hydrolysis products was put in relation to relative gene expression profiles of myrosinases and specifier proteins as measured by RT-qPCR, and in relation to relative protein abundance of epithiospecifier protein. Dependent on the different GS profiles and reliant on degradation protein abundance and composition the ecotypes strongly varied in their ability to form isothiocyanates, nitriles and epithionitriles, thus increasing the plants' equipment of defense metabolites.

Vegetables of the Brassica oleracea group, such as broccoli, cauliflower, and cabbage, play an important role for glucosinolate consumption in the human diet. Upon maceration of the vegetable tissue, glucosinolates are degraded enzymatically to form volatile isothiocyanates, nitriles, and epithionitriles. However, only the uptake of isothiocyanates is linked to the cancer-preventive effects. Thus, it is of great interest to evaluate especially the isothiocyanate formation. Here, we studied the formation of glucosinolates and their respective hydrolysis products in sprouts and fully developed vegetable heads of different genotypes of the five B. oleracea varieties: broccoli, cauliflower as well as white, red, and savoy cabbages. Further, the effect of ontogeny (developmental stages) during the head development on the formation of glucosinolates and their respective hydrolysis products was evaluated at three different developmental stages (mini, fully developed, and over-mature head). Broccoli and red cabbage were mainly rich in 4-(methylsulfinyl)butyl glucosinolate (glucoraphanin), whereas cauliflower, savoy cabbage and white cabbage contained mainly 2-propenyl (sinigrin) and 3-(methylsulfinyl)propyl glucosinolate (glucoiberin). Upon hydrolysis, epithionitriles or nitriles were often observed to be the main hydrolysis products, with 1-cyano-2,3-epithiopropane being most abundant with up to 5.7 μmol/g fresh weight in white cabbage sprouts. Notably, sprouts often contained more than 10 times more glucosinolates or their hydrolysis products compared to fully developed vegetables. Moreover, during head development, both glucosinolate concentrations as well as hydrolysis product concentrations changed and mini heads contained the highest isothiocyanate concentrations. Thus, from a cancer-preventive point of view, consumption of mini heads of the B. oleracea varieties is recommended.

Selenium (Se) is an essential micronutrient for human health. Se deficiency affects hundreds of millions of people worldwide, particularly in developing countries, and there is increasing awareness that suboptimal supply of selenium can also negatively affect human health. Selenium enters the diet primarily through the ingestion of plant and animal products. Although plants are not dependent on Se they take it up from the soil through the sulfur (S) uptake and assimilation pathways. Therefore, geographic differences in the availability of soil Se and agricultural practices have a profound influence on the Se content of many foods, and there are increasing efforts to biofortify crop plants with Se. Plants from the Brassicales are of particular interest as they accumulate and synthesize Se into forms with additional health benefits, such as methylselenocysteine (MeSeCys). The Brassicaceae are also well known to produce the glucosinolates; S-containing compounds with demonstrated human health value. Furthermore, the recent discovery of the selenoglucosinolates in the Brassicaceae raises questions regarding their potential bioefficacy. In this review we focus on Se uptake and metabolism in the Brassicaceae in the context of human health, particularly cancer prevention and immunity. We investigate the close relationship between Se and S metabolism in this plant family, with particular emphasis on the selenoglucosinolates, and consider the methodologies available for identifying and quantifying further novel Se-containing compounds in plants. Finally, we summarise the research of multiple groups investigating biofortification of the Brassicaeae and discuss which approaches might be most successful for supplying Se deficient populations in the future.