Bioavailability, Human Metabolism, and Dietary Interventions of Glucosinolates and Isothiocyanates: Critical Insights and Future Perspectives.

Isothiocyanates (ITCs) exhibit anticancer and antioxidant activity. ITCs are formed from the catalysis of glucosinolates (GLs) by myrosinase (MYR) in Brassicaceae vegetables. This study investigated the GL content, MYR activity, ITC formation, and redox state of rocket upon germination. Banye and Dongsheng were selected from four cultivars by determining growth state and ITC metabolism. In both cultivars, GL levels were the highest on the second day of germination and significantly declined on the eighth day. The 4-day-old sprouts had the highest ITC level. MYR exhibited maximal activity in Banye and Dongsheng on the fourth and the second day of germination, respectively. Regarding the antioxidant system, the sprouts had more chlorophyll, total phenol, and anthocyanin content, and they had higher DPPH scavenging effects compared with raw seeds. Moreover, Dongsheng showed greater scavenging capacity than Banye in general. Practical applications Rocket belongs to the Brassicaceae family, which has been widely considered a good source of bioactive components to prevent tumor formation and reduce the incidence of cardiovascular disease and cancer given the abundance of glucosinolate (GLs). GLs could be catalyzed by myrosinases (MYR) and converted into biologically active products including isothiocyanates (ITCs). After germination, MYR is activated and more ITCs are produced. Using rocket sprouts as a daily diet or raw material is beneficial for human health.

Variation und Vererbung von Glucosinolatgehalt und muster in Grünmasse und Samen von Raps (Brassica napus L.) und deren Zusammenhang zum Befall mit Rapsstängelschädlingen

Glucosinolate (GSL) hydrolysis is mediated by the enzyme myrosinase which together with specifier proteins can give rise to isothiocyanates (ITCs), thiocyanates, and nitriles (NITs) in cruciferous plants. However, little is known about the metabolism of GSLs by the human gut flora. The aim of the work was to investigate the metabolic fates of sinigrin (SNG), glucotropaeolin (GTP), gluconasturtiin (GNT), and their corresponding desulfo-GSLs (DS-GSLs). Three human gut bacterial strains, Enterococcus casseliflavus CP1, Lactobacillus agilis R16, and Escherichia coli VL8, were chosen for this study. GNT was metabolized to completion within 24h to phenethyl ITC and phenethyl NIT (PNIT) by all bacteria, except for L. agilis R16 which produced only PNIT. At least 80% of GTP and SNG were metabolized by all bacteria within 24h to the corresponding ITCs and NITs. The pH of media over time gradually became acidic for both L. agilis R16 and E. coli VL8, while for E. casseliflavus CP1 the media became slightly alkaline with NIT and ITC production occurring between pH 3.0 and 7.5. ITC production peaked between 4 and 10h in most cases and gradually declined while NIT production increased and remained relatively constant over time. The total percentage products accounted for 3-53% of the initial GSL. NITs were produced from DS-GSLs suggesting an alternative metabolism via desulfation for the food based GSLs. The metal ion dependency for NIT production for GNT and its DS form was investigated where it was shown that Fe(2+) increased NIT production, while Mg(2+) stimulated the formation of ITC.

Rocket (Eruca sativa) is a source of health-related metabolites called glucosinolates (GSLs) and isothiocyanates (ITCs) but little is known of the genetic and transcriptomic mechanisms responsible for regulating pre and postharvest accumulations. We present the first de novo reference genome assembly and annotation, with ontogenic and postharvest transcriptome data relating to sulfur assimilation, transport, and utilization. Diverse gene expression patterns related to sulfur metabolism, GSL biosynthesis, and glutathione biosynthesis are present between inbred lines of rocket. A clear pattern of differential expression determines GSL abundance and the formation of hydrolysis products. One breeding line sustained GSL accumulation and hydrolysis product formation throughout storage. Multiple copies of MYB28, SLIM1, SDI1, and ESM1 have increased and differential expression postharvest, and are associated with GSLs and hydrolysis product formation. Two glucosinolate transporter gene (GTR2) copies were found to be associated with increased GSL accumulations in leaves. Monosaccharides (which are essential for primary metabolism and GSL biosynthesis, and contribute to the taste of rocket) were also quantified in leaves, with glucose concentrations significantly correlated with the expression of numerous GSL-related genes. Significant negative correlations were observed between the expression of glutathione synthetase (GSH) genes and those involved in GSL metabolism. Breeding line “B” showed increased GSH gene expression and low GSL content compared to two other lines where the opposite was observed. Co-expression analysis revealed senescence (SEN1) and oxidative stress-related (OXS3) genes have higher expression in line B, suggesting that postharvest deterioration is associated with low GSL concentrations.

Effects of CaCl2 on the metabolism of glucosinolates and the formation of isothiocyanates as well as the antioxidant capacity of broccoli sprouts

Light-emitting diode (LED) irradiation can affect postharvest vegetable and fruit shelf life and nutritional quality. In this study, 11 kinds of glucosinolates (GLS) and 6 types of GLS breakdown products were identified in cabbage leaves. To investigate the effects of LED irradiation on quality and GLS metabolism, postharvest cabbage was irradiated with 25 μmol/(m2·s) green LED (GL) light at room temperature for 12 d. The dark condition and natural light were used as controls. GL irradiation inhibited the respiration rate and the accumulation of malondialdehyde, and maintained higher cabbage sensory score, chlorophyll content, and antioxidant enzyme activities, thus extending its postharvest shelf life. Additionally, GL irradiation promoted GLS profile accumulation, including glucoraphanin, glucoiberin, gluconapin, gluconasturtiin, glucobrassicin, and sinigrin, which could have resulted from upregulated expression of the transcription factors MYB28 and MYB51, and the key genes involved in GLS biosynthesis (CYP83A1, CYP83B1, ST5b, and UGT74B1). Furthermore, GLS degradation was promoted by this treatment during the late stage of shelf life, as higher myrosinase activity and relative MYR and ESM expression levels were found in treated cabbage leaves, resulting in higher isothiocyanate (ITC) and indole-3-carbinol contents after day 6. Therefore, GL irradiation was beneficial for cabbage preservation.

The role of beneficial microorganisms, such as mycorrhizas, in improving the resistance to environmental stress of colonized plants is well-known. Plants of Brassicaceae family are of large economic importance, especially for the synthesis of anticarcinogenic compounds such as glucosinolates and their derivatives isothiocyanates. The endophyte fungus Piriformospora indica is able to colonize them and improves their growth and response to environmental stress. However, no information are available on the impact of colonization on glucosinolate metabolism. In this work, colonization of black cabbage (Brassica oleracea cv. Acephala sabellica) is reported as well as the effects on plant growth and on the expression of myrosinase encoding genes, the isothyocianate producing enzymes. Results indicate that P. indica successfully colonized black cabbage as validated by the expression of the marker gene Ptef1. Colonized plants showed increase of biomass weights and shoot length respect to the uncolonized plants and a decrease of myrosinase gene expression. This last finding indicates that P. indica might affect the resistance against biotic stress of black cabbage.

Glucosinolate and isothiocyanate concentration in soil following incorporation of Brassica biofumigants

Isothiocyanates (ITCs), which are derivatives of glucosinolates (GSLs) from Brassica plants, have been investigated as anticancer agents. An extensively studied anticancer ITC is sulforaphane, which is found in low amounts in Chinese cabbage. We aim to investigate the types and content of GSLs (precursors of ITCs with anticancer activity) in Chinese cabbage seeds. GSLs from Chinese cabbage seeds were isolated and purified using acidic Al2O3 column chromatography and preparative HPLC. GSL and ITC profiles were further identified using UHPLC-Q-TOF-MS. The antitumor activities of ITC (produced by exogenous enzymatic hydrolysis of GSLs) were evaluated in vitro. Seventeen GSLs and seven ITCs were identified, and the dominant GSLs were gluconapin, glucobrassicanapin, and progoitrin in Chinese cabbage seeds. High-purity gluconapin (>99%) was purified. The ITCs showed synergistic-, dose-, and time-dependent effects on the inhibition of HepG2 cells, and the key ITCs were 3-butenyl ITC, sulforaphane, and 2-phenylethyl ITC. The corresponding parent GSLs were gluconapin, glucoraphanin, and gluconasturtiin, respectively. 3-Butenyl ITC could significantly induce HepG2 cell proliferation (IC50 = 89.44 μg/mL) and apoptosis (p < 0.05). Our results suggested that Chinese cabbage seed could be a valuable source of natural antitumor ingredients.

Lepidium draba (hoary cress) is a perennial plant belonging to the Brassicaceae family that produces two dominant glucosinolates (GLSs): glucoraphanin (GRN) and sinalbin (SBN). They represent the stored form, which is converted upon the myrosinase (Myr) hydrolysis activity to active compounds, mainly isothiocyanates (ITCs) such as sulforaphane (SFN) or p-hydroxybenzyl isothiocyanate (pHBITC). Research on ITCs that have proven anticancer, antimicrobial, and chemoprotective properties is usually conducted with pure commercially available compounds. However, these are chemically reactive, making it difficult to use them directly for preventive purposes in dietary supplements. Efforts are currently being made to prepare dietary supplements enriched with GLS and/or Myr. In this study, we report a simple but efficient chromatographic procedure for the isolation and purification of GLSs from MeOH extract from hoary cress based on a combination of ion exchange and gel permeation chromatography on DEAE-Sephadex A-25 and Sephadex LH-20. To obtain the Myr required for efficient hydrolysis of GLSs into antibacterial ITCs, we developed a rapid method for its extraction from the seeds of Lepidium sativum (garden cress). The yields of GLSs were 22.9 ± 1.2 mg GRN (purity 96%) and 10.4 ± 1.1 mg SBN (purity 92%) from 1 g of dry plant material. Both purified GLSs were used as substrates for the Myr. Analysis of the composition of hydrolysis products (HPs) revealed differences in their hydrolysis rates and in the degree of conversion from GLSs to individual ITCs catalyzed by Myr. When GRNs were cleaved, SFNs were formed in an equimolar ratio, but the formation of pHBITCs was only half that of cleaved SBNs. The decrease in pHBITC content is due to its instability compared to SFN. While SFN is stable in aqueous media during the measurement, pHBITC undergoes non-enzymatic hydrolysis to p-hydroxybenzyl alcohol and thiocyanate ions. Testing of the antimicrobial effects of the HPs formed from GRN by Myr under premix or in situ conditions showed inhibition of the growth of model prokaryotic and eukaryotic microorganisms. This observation could serve as the jumping-off point for the design of a two-component mixture, based on purified GLSs and Myr that is, usable in food or the pharmaceutical industry in the future.

While some isothiocyanate (ITCs) are attractive targets for the agricultural and pharmaceutical industries, the presence of goitrin and ITCs has hampered the widespread utilization of rapeseed meal. ITCs are the products of the myrosinase-mediated hydrolysis of glucosinolate (GSLs). As such, a study was conducted in order to gain a better understanding into the identity of the GSLs contained in rapeseed meal. Extraction of the GSLs was carried out with 20% ethanol, affording 3.0% GSL content. The resulting GSL extracts were purified via silica gel column chromatography resulting in the isolation of main three pure GLSs (GSL A, B, and C) and a final GSL content of 39.8%. The indirect-identification of the GSLs in rapeseed meal was also carried out via GC/MS analysis of ITCs. The GSLs, progoitrin and gluconapin, were present in the highest concentration in these extracts. Interestingly, only goitrin was produced when GSL A was the substrate for the defatted rapeseed meal mediated hydrolysis reaction. This indicates GSL A is a progoitrin. Conversely, 3-butenyl ITC was produced only when GSL B was used as substrate, indicating GSL B is gluconapin. These results will be helpful for opening the doors for the use of rapeseed meal in the agricultural or pharmaceutical sectors.

BackgroundRadish (Raphanus sativus L.), is an important root vegetable crop worldwide. Glucosinolates in the fleshy taproot significantly affect the flavor and nutritional quality of radish. However, little is known about the molecular mechanisms underlying glucosinolate metabolism in radish taproots. The limited availability of radish genomic information has greatly hindered functional genomic analysis and molecular breeding in radish.ResultsIn this study, a high-throughput, large-scale RNA sequencing technology was employed to characterize the de novo transcriptome of radish roots at different stages of development. Approximately 66.11 million paired-end reads representing 73,084 unigenes with a N50 length of 1,095 bp, and a total length of 55.73 Mb were obtained. Comparison with the publicly available protein database indicates that a total of 67,305 (about 92.09% of the assembled unigenes) unigenes exhibit similarity (e –value ≤ 1.0e-5) to known proteins. The functional annotation and classification including Gene Ontology (GO), Clusters of Orthologous Group (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the main activated genes in radish taproots are predominately involved in basic physiological and metabolic processes, biosynthesis of secondary metabolite pathways, signal transduction mechanisms and other cellular components and molecular function related terms. The majority of the genes encoding enzymes involved in glucosinolate (GS) metabolism and regulation pathways were identified in the unigene dataset by targeted searches of their annotations. A number of candidate radish genes in the glucosinolate metabolism related pathways were also discovered, from which, eight genes were validated by T-A cloning and sequencing while four were validated by quantitative RT-PCR expression profiling.ConclusionsThe ensuing transcriptome dataset provides a comprehensive sequence resource for molecular genetics research in radish. It will serve as an important public information platform to further understanding of the molecular mechanisms involved in biosynthesis and metabolism of the related nutritional and flavor components during taproot formation in radish.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-14-836) contains supplementary material, which is available to authorized users.

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.

Glucosinolates (GSLs) are produced by plants of the Capparales order. Upon enzymatic hydrolysis the GSLs can be transformed to the toxic isothiocyanates (ITCs), which can be used as biofumigants for the control of soil-borne pests. The rates of ITC formation and degradation are critical to both biofumigation and the toxicity and leaching of GSLs and ITCs in soil. Degradation kinetics of benzyl GSL and benzyl ITC in a sandy and clayey surface and subsoil at 8-9 degrees C at natural moisture contents were investigated, as was the rate of formation of ITC from the GSL. Degradation of GSL followed logistic kinetics with t 1/2 = 0.7-9.1 days. Degradation was faster in clayey soil compared to sandy soil, and faster in surface soil compared to subsoil. In surface soils, up to 25% of added GSL was detected as ITC, while only 1-6% were detected in the subsoils. ITC degradation followed first-order kinetics with t 1/2 = 0.3-1.7 days, with faster degradation in subsoils than in surface soils. Based on the data for GSL hydrolysis and ITC degradation, the concentration of ITC following GSL application was successfully modeled assuming complete conversion of glucosinolate to isothiocyanate and first-order degradation of isothiocyanate.

Although previous studies have investigated the association of cruciferous vegetable consumption with breast cancer risk, few studies focused on the association between bioactive components in cruciferous vegetables, glucosinolates (GSL) and isothiocyanates (ITC), and breast cancer risk. This study aimed to examine the association between consumption of cruciferous vegetables and breast cancer risk according to GSL and ITC contents in a Chinese population. A total of 1485 cases and 1506 controls were recruited into this case-control study from June 2007 to March 2017. Consumption of cruciferous vegetables was assessed using a validated FFQ. Dietary GSL and ITC were computed by using two food composition databases linking GSL and ITC contents in cruciferous vegetables with responses to the FFQ. The OR and 95 % CI were assessed by unconditional logistic regression after adjusting for the potential confounders. Significant inverse associations were found between consumption of cruciferous vegetables, GSL and ITC and breast cancer risk. The adjusted OR comparing the highest with the lowest quartile were 0·51 (95 % CI 0·41, 0·63) for cruciferous vegetables, 0·54 (95 % CI 0·44, 0·67) for GSL and 0·62 (95 % CI 0·50, 0·76) for ITC, respectively. These inverse associations were also observed in both premenopausal and postmenopausal women. Subgroup analysis by hormone receptor status found inverse associations between cruciferous vegetables, GSL and ITC and both hormone-receptor-positive or hormone-receptor-negative breast cancer. This study indicated that consumption of cruciferous vegetables, GSL and ITC was inversely associated with breast cancer risk among Chinese women.