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The consumption of glucosinolate (GL)-rich foods, including Brassica vegetables, such as mustard, broccoli, and maca, is associated with decreased risk of developing cancer. The GL content in maca, which is recognized as a "superfood", is approximately 100-times higher than that in other brassicas. Although maca is a potential dietary source of GLs, limited studies have examined the bioactivity of maca GLs using the combination of chemical characterization and bioassays. In this study, the fractions (Lm-II and Lm-III) rich in intact GLs (glucotropaeolin and glucolimnanthin) were isolated and characterized from maca ethanolic extracts using chromatography and mass spectrometry. Additionally, the growth-inhibitory effects of Lm-II and Lm-II fractions against hepatocellular carcinoma (HepG2/C3A) and colon adenocarcinoma (HT29) cell lines were examined in the absence or presence of myrosinase (MYR). Fractions lacking low molecular weight sugars dose-dependently exerted cytotoxic effects in the presence of MYR. The half-maximal inhibitory concentration values of Lm-II and Lm-III against HepG2/C3A were 118.8 and 69.9 µg/mL, respectively, while those against HT29 were 102.6 and 71.5 µg/mL, respectively. These results suggest that the anticancer properties of maca can be attributed to GLs and corroborate the categorization of maca as a "superfood."Supplemental data for this article is available online at https://doi.org/10.1080/01635581.2021.1952444.

We determined the physiological effects of glucotropaeolin-rich lyophilized garden cress sprout powder (GC) administered to fasting and nonfasting mice. High-performance liquid chromatography analysis revealed glucotropaeolin (57.4±1.1 mg/g dry weight) as a major phytochemical constituent of GC. Decreasing tendency in body weight and feeding efficiency ratio were detected in the group of mice fed 0.05% (w/w) GC (GC0.05). Nonfasting mice exhibited significantly lower liver weights that were unchanged after fasting. Decreased total lipid (TL) and triglyceride (TG) levels in the liver were detected in the nonfasted GC0.01 and GC0.05 groups, but only in TLs of the fasted groups. The levels of plasma TGs and nonesterified fatty acids of the GC0.05 group, which remained unchanged during nonfasting, decreased after fasting. To determine its effects on the accumulation of lipids in the liver, the glucotropaeolin aglycone, benzyl isothiocyanate (BITC), was added to the liver-derived HepG2 human cell line cultured in a medium containing a high concentration of D-glucose (4,500 mg/L D-glucose) (HG group) or 1 mM oleic acid (SO group). Toxicity was not detected when cells were treated with as much as 5 μM BITC; however, lipid accumulation was inhibited by BITC in a concentration-dependent manner in the HG groups. The same effect was observed when 2 μM BITC was added to the diet of the SO groups. These results suggest that moderate levels of GC or BITC are useful for reducing liver and plasma TGs.

Glucosinolates (GSLs) from Lepidium graminifolium L. were analyzed qualitatively and quantitatively by their desulfo-counterparts using UHPLC-DAD-MS/MS technique and by their volatile breakdown products-isothiocyanates (ITCs) using GC-MS analysis. Thirteen GSLs were identified with arylaliphatic as the major ones in the following order: 3-hydroxybenzyl GSL (glucolepigramin, 7), benzyl GSL (glucotropaeolin, 9), 3,4,5-trimethoxybenzyl GSL (11), 3-methoxybenzyl GSL (glucolimnanthin, 12), 4-hydroxy-3,5-dimethoxybenzyl GSL (3,5-dimethoxysinalbin, 8), 4-hydroxybenzyl GSL (glucosinalbin, 6), 3,4-dimethoxybenzyl GSL (10) and 2-phenylethyl GSL (gluconasturtiin, 13). GSL breakdown products obtained by hydrodistillation (HD) and CH2Cl2 extraction after hydrolysis by myrosinase for 24 h (EXT) as well as benzyl ITC were tested for their cytotoxic activity using MTT assay. Generally, EXT showed noticeable antiproliferative activity against human bladder cancer cell line UM-UC-3 and human glioblastoma cell line LN229, and can be considered as moderately active, while IC50 of benzyl ITC was 12.3 μg/mL, which can be considered as highly active.

The identification and quantification of Glucosinolates (Gls) via UPLC® MS‐MS/PDA/qToF in ten mashua cultivars was carried out. Gls total contents were within the 4.9–54.2 μmol g⁻¹ dry matter range, of which 96–99% corresponded to glucoaubrietin. Other less abundant Gls were glucotropaeolin and tentatively two isomers of hydroxybenzyl Gls. Postharvest refrigeration and shade storage conditions increased the content of Gls up to day 6 in 39.7% and 51.5% respectively. Sun exposure increased the Gls content in 40% up to day 3 but from day 6 considerable losses were attained (92% at day 15). Low correlation levels (R²) between the Gls and myrosinase (MYR) activity of 0.57, 0.28 and 0.39 for the refrigeration, shade and sun exposure treatments were obtained. The cooking regimes tested, boiling, microwaving and baking totally inactivated MYR without affecting the Gls content.

A method to determine 15 intact glucosinolates (epiprogoitrin; glucoalyssin; glucobrassicanapin; glucobrassicin; glucoerucin; glucoiberin; gluconapin; gluconasturtiin; glucoraphanin; glucotropaeolin; 4-hydroxyglucobrassicin; 4-methoxyglucobrassicin; neoglucobrassicin; progoitrin; sinigrin) in bee pollen has been proposed, by means of ultra-high liquid chromatography coupled to a quadrupole time-of-flight mass detector. Efficient sample treatment (with average analyte recoveries between 80 and 96%) involving a solid-liquid extraction with hot water, followed by solid-phase extraction with a weak anion exchange sorbent (NH2), was employed to analyze those intact glucosinolates in bee pollen. Chromatographic analysis (15 min) was performed on a fully porous silica–based column (Luna® Omega C18) with a mobile phase composed of a mixture of acetonitrile (0.1% formic acid) and water (0.1% formic acid) in gradient elution mode at a flow rate of 0.2 mL/min. Accordingly, the analytical performance of the proposed method was evaluated, and it was then applied to determine intact glucosinolates in commercial bee pollen samples from different Spanish regions. Ten intact glucosinolates were identified in certain samples over a wide concentration range.

Maca is a Peruvian tuberous root of the Brassicaceae family grown in the central Andes between altitudes of 4000 and 4500 m. The medicinal plant is a nutraceutical with important biological activities and health effects. In this study, we report a rapid high-performance thin layer chromatography (HPTLC)-(-)desorption electrospray ionization (DESI)-mass spectrometry (MS) method to profile and separate intact glucosinolates without prior biochemical modifications from the hydromethanolic extracts of two phenotypes, red and black Maca (Lepidium peruvianum) seeds. In the first stage of the plant's life cycle, aromatic glucosinolates were the main chemical constituents whereby six aromatic, three indole, and one aliphatic glucosinolate were tentatively identified. At the seedling stage, glucolepigramin/Glucosinalbin was the most predominant precursor, rather than Glucotropaeolin, which is mainly found in hypocotyls and roots. These findings lead us to suggest that glucolepigramin/glucosinalbin play a major role as active precursors in the biosynthetic pathways of other secondary metabolites in the early stages of plant development. Between red and black Maca seeds, only minor differences in the relative abundances of glucosinolates were observed rather than different plant metabolites. For the first time, we report six potential plant antibiotics, phytoanticipins: glycosylated ascorbigens and dihydroascorbigens from Maca seeds. We also investigated a targeted reverse phase C18 functionalized TLC-DESI-MS method with high sensitivity and specificity for Brassicaceae fatty acids in Maca seeds and health supplements such as black Maca root lyophilized powder and tinctures. The investigation of secondary metabolites by normal and reverse phase TLC-DESI-MS methods, described in this study, can aid in their identification as they begin to emerge in later stages of development in plant tissues such as leaves, hypocotyls, and roots.

The present study focuses on the utilization of moringa sprout powder (MSP) for development of healthy and ready-to-eat snack bars. Different MSP proportions (9–26%) were evaluated for snack bar formulation and compared with control formulation (0% MSP). 18% MSP-containing prototype (18MSP) was selected as the optimal formulation based on the results of the fracturability assay. 18MSP bar exhibited higher protein, ash and fat and lower total carbohydrate contents than control, and was a good source of B1 and B2 vitamins (983.0 and 94.1 μg/100 g dry matter, dm, respectively) and minerals. 18% MSP inclusion significantly (p ≤ 0.05) enhanced the contents of total amino acid (80.8 mg/g dm), the relative content of unsaturated fatty acids (FA) (64.0 g/100 g of total FA), γ-aminobutyric acid (GABA, 43.3 mg/100 g dm), and total and individual glucosinolates (GLS) (29.6, 1.1, 0.21 and 30.9 μmol/g dm for glucomoringin, glucosinalbin, glucotropaeolin and total GLS, respectively), as well as the antioxidant activity (647.2 mg TE/100 g dm). 18MSP had good sensorial acceptability, mainly due to its appearance, texture and odour. Questionnaires performed to potential Ecuadorian consumers indicated the favourable intention of purchasing the novel MSP-supplemented snack bar., This study was funded by grants AGL2015-67598-R (MINECO/FEDER), AGL2017-83718-R (AEI/FEDER, UE), and intramural project grant number PI201870I097 (CSIC).

Bunias erucago belongs to the Brassicaceae family, which represents a forgotten crop of the Euro-Mediterranean area. The aim of the present study was to determine the glucosinolate profile in different plant parts and biological properties (antioxidant, anticholinesterase, and cytotoxic activities) of the isolates containing glucosinolate breakdown products. The chemical profiles were determined by using HPLC-PDA-MS/MS of desulfoglucosinolates and GC-MS of glucosinolate degradation products. The analysis of B. erucago showed the presence of seven glucosinolates: gluconapin (1), glucoraphasatin (2), glucoraphenin (3), glucoerucin (4), glucoraphanin (5), glucotropaeolin (6), and glucosinalbin (7). The total glucosinolate content ranged from 7.0 to 14.6 &micro, mol/g of dry weight, with the major glucosinolate glucosinalbin in all parts. The antioxidant activity of all volatile isolates was not notable. At a tested concentration of 227 &mu, g/mL, flower hydro-distillate (FH) showed good AChE inhibition, i.e., 40.9%, while root hydro-distillate (RH) had good activity against BChE, i.e., 54.3%. FH showed the best activity against both tested human bladder cancer cell lines, i.e., against T24 after 72 h, which have IC50 of 16.0 &mu, g/mL, and against TCCSUP after 48 h with IC50 of 7.8 &mu, g/mL, and can be considered as highly active. On the other hand, RH showed weak activity against tested cancer cells.

Here we describe the structure elucidation and quantification of six glucosinolates (GSLs) from the roots of the desert plant Ochradenus baccatus, Delile 1813 (family Resedaceae; order Brassicales). The structure elucidation was established on the corresponding enzymatically desulfated derivatives of the native GSLs of the plant. Among these GSLs we describe the previously undescribed 2″-O-(α-L-arabinopyranosyloxy)benzylglucosinolate (1a), for which we propose the name glucoochradenin. The other five glucosinolates (2a-6a) were (2S)-2-hydroxy-2-phenylethylglucosinolate (2a; glucobarbarin), 2″-O-(α-L-rhamnopyranosyloxy)benzylglucosinolate (3a), benzylglucosinolate (4a; glucotropaeolin), indol-3-ylmethylglucosinolate (5a; glucobrassicin) and phenethylglucosinolate (6a; gluconasturtiin), all elucidated as their desulfo-derivatives, 2b-6b respectively). Structures were elucidated by MS and 1D and 2D-NMR techniques, the identity of the arabinose verified by ion chromatography, and the absolute configuration of the sugar units determined by hydrolysis, coupling to cysteine methyl-ester and phenyl isothiocyanate followed by HPLC-MS analysis of the resulted diastereomers. Response factors were generated for desulfo-2″-O-(α-L-arabinopyranosyloxy)benzylglucosinolate and for desulfo-2″-O-(α-L-rhamnopyranosyloxy)benzylglucosinolate and all six GSLs were quantified, indicating that the root of O. baccatus is rich in GSLs (Avg. 61.3 ± 10.0 μmol/g DW and up to 337.2 μmol/g DW).

The demand and sales of dietary supplements derived from maca (Lepidium meyenii) have skyrocketed in the last decade and a variety of related nutritional and healthcare products have mushroomed into a business with market prominence. However, the lack of standard testing protocols for quality control could jeopardize the immediate benefits of these products for public health. We describe herein the development of analytical procedures for the determination of glucosinolates (GLs), the biologically active ingredients in maca. Because of the high polarity and instability caused by enzymatic hydrolysis, GLs in maca have been exclusively analyzed using desulfated GLs. This indirect analysis requires additional sample preparation steps, which is labor-intensive, and may lose the original GLs and introduce artificial compounds. Furthermore, the reported GL profiles of maca are inconsistent and incomplete, some GLs may be structurally misidentified. In this context, we focused on direct analysis of intact GLs in maca without the enzymatic desulfation. Four GLs (sinalbin, glucolepigramin, glucolimnanthin, and glucotropaeolin) were identified as the major GLs in maca root powder. An HPLC method based on ion pair chromatography was developed to determine individual and total GLs; chromatographic separations were achieved on a Luna column (C18, 4.6 × 100 mm,3 mm) using 0.1 % TFA in water and in methanol as mobile phase in a gradient elution mode. The developed procedures were validated within the calibration range of 10–500 μg/mL. Inter- and intra-day precision were shown to be lower than 3% at all concentrations levels with recovery between 100.2 % and 103.3 %. The procedures were applied to a total of 42 maca root powder products from 11 manufacturers. Sample analysis revealed a consistent correlation of glucotropaeolin: glucolimnanthin (1: 0.19) across all products with a correlation coefficient of 0.994. The correlation in combination with total GL contents for each product could be used for authentication and GL content determination. Incorporation of the developed procedures into USP monographs will strengthen the public standards for maca powder dietary supplements.

In order to evaluate the quality of maca samples, 15 batches of maca hypocotyls from different geographical origins were analyzed by using HPLC and liquid chromatography tandem mass spectrometry (LC-MS). Their glucosinolates (GLs) were identified, profiled, and quantified. Three aromatic GLs, glucosinalbin (GSB), glucotropaeolin (GTL), and glucolimnanthin (GLH), were identified from maca roots. It was found that the HPLC profiles of maca samples showed a similar qualitative pattern except for some differences in their contents. The amount ranges (μmol g−1, DW) of GSB, GTL, and GLH were 2.62–5.55, 12.99–57.12, and 3.15–12.12, respectively. Furthermore, a HCA (hierarchical clustering analysis) was used to classify the quality of maca samples according to their cultivated sources and GLs contents. The results showed that the combination of GSB, GTL, and GLH could be a marker for accurate determination and quality control of maca samples. It was concluded that the multi-component analysis in combination with HCA could be a supplement to assess the quality of maca materials.

We aimed to characterize and quantify glucosinolate compounds and contents in broccoli, and a total of 80 genotypes and eight developmental organs were analyzed with UHPLC-Triple-TOF-MS. The method was validated in terms of performance, and the coefficients of determination (R2) were 0.97 and 0.99 for glucoraphanin and gluconapin, respectively. In 80 genotypes, twelve glucosinolates were found in broccoli florets ranging from 0.467 to 57.156 µmol/g DW, with the highest glucosinolate content being approximately 122-fold higher than the lowest value. The principal component of glucobrassicin, neoglucobrassicin and glucoraphanin explained 60.53% of the total variance. There were positive correlations among hydroxyglucobrassicin, methoxyglucobrassicin, glucobrassicin, glucoerucin, gluconasturtiin, glucoraphanin, and glucotropaeolin (P

Glucosinolates (GSLs) are sulfur-containing secondary metabolites naturally occurring in Brassica species. The purpose of this study was to identify the GSLs, determine their content, and study their accumulation patterns within and between leaves of kimchi cabbage (Brassica rapa L.) cultivars. GSLs were analyzed using UPLC-MS/MS in negative electron-spray ionization (ESI-) and multiple reaction monitoring (MRM) mode. The total GSL content determined in this study ranged from 621.15 to 42434.21 &mu, molkg-1 DW. Aliphatic GSLs predominated, representing from 4.44% to 96.20% of the total GSL content among the entire samples. Glucobrassicanapin (GBN) contributed the greatest proportion while other GSLs such as glucoerucin (ERU) and glucotropaeolin (TRO) were found in relatively low concentrations. Principal component analysis (PCA) yielded three principal components (PCs) with eigenvalues &ge, 1, altogether representing 74.83% of the total variation across the entire dataset. Three kimchi cabbage (S/No. 20, 4, and 2), one leaf mustard (S/No. 26), and one turnip (S/No. 8) genetic resources were well distinguished from other samples. The GSL content varied significantly among the different positions (outer, middle, and inner) of the leaves and sections (top, middle, bottom, green/red, and white) within the leaves. In most of the samples, higher GSL content was observed in the proximal half and white sections and the middle layers of the leaves. GSLs are regarded as allelochemicals, hence, the data related to the patterns of GSLs within the leaf and between leaves at a different position could be useful to understand the defense mechanism of Brassica plants. The observed variability could be useful for breeders to develop Brassica cultivars with high GSL content or specific profiles of GSLs.

Here, accumulation of glucosinolates and expression of glucosinolates biosynthesis genes in green and red mustard hairy roots were identified and quantified by HPLC and qRT-PCR analyses. The total glucosinolates content of green mustard hairy root (10.09 µg/g dry weight) was 3.88 times higher than that of red mustard hairy root. Indolic glucosinolates (glucobrassicin, 4-methoxyglucobrassicin, and neoglucobrassicin) in green mustard were found at 30.92, 6.95, and 5.29 times higher than in red mustard hairy root, respectively. Conversely, levels of glucotropaeolin (aromatic glucosinolate) was significantly higher in red mustard than in green mustard. Accumulation of glucoraphasatin, an aliphatic glucosinolate, was only observed only in red mustard hairy roots. Quantitative real-time PCR analysis showed that the expression level of genes related to aliphatic and aromatic glucosinolate biosynthesis were higher in red mustard, exception BjCYP83B. The expression of BjCYP79B2, which encodes a key enzyme involved in the indolic glucosinolate biosynthetic pathway, was higher in green mustard than in red mustard. Additionally, to further distinguish between green mustard and red mustard hairy roots, hydrophilic and lipophilic compounds were identified by gas chromatography-mass spectrometry and subjected to principal component analysis. The results indicated that core primary metabolites and glucosinolate levels were higher in the hairy roots of green mustard than in those of red mustard.

Biofumigation is an integrated pest-management method involving the mulching of a glucosinolate-containing cover crop into a field in order to generate toxic isothiocyanates (ITCs), which are effective soil-borne-pest-control compounds. Variation in biofumigation efficacy demonstrates a need to better understand the factors affecting pest-control outcomes and develop best practices for choosing biofumigants, growth conditions, and mulching methods that allow the greatest potential isothiocyanate release. We measured the glucosinolate concentrations of six different commercial varieties of three biofumigant plant species: Brassica juncea (ISCI99, Vitasso, and Scala) Raphanus sativus (Diablo and Bento), and Sinapis alba (Ida Gold). The plants were grown in the range of commercially appropriate seeding rates and sampled at three growth stages (early development, mature, and 50% flowering). Within biofumigant species, the highest ITC-release potentials were achieved with B. juncea cv. ISCI99 and R. sativus cv. Bento. The highest ITC-release potential occurred at the 50% flowering growth stage across the species. The seeding rate had a minor impact on the ITC-release potential of R. sativus but had no significant effects on the ITC-release potentials of the B. juncea or S. alba cultivars.

SCOPE Benzyl isothiocyanate (BITC), which occurs in Brassicales, has demonstrated chemopreventive potency and cancer treatment properties in cell and animal studies. However, fate of BITC in human body is not comprehensively studied. Therefore, the present human intervention study investigates the metabolism of the glucosinolate (GSL) glucotropaeolin and its corresponding BITC metabolites. Analyzing BITC metabolites in plasma and urine should reveal insights about resorption, metabolism, and excretion. METHODS AND RESULTS Fifteen healthy men were randomly recruited for a cross-over study and consumed 10 g freeze-dried Indian cress as a liquid preparation containing 1000 μmol glucotropaeolin. Blood and urine samples were taken at several time points and investigated by LC-ESI-MS/MS after sample preparation using SPE. Plasma contained high levels of BITC-glutathione (BITC-GSH), BITC-cysteinylglycine (BITC-CysGly), and BITC-N-acetyl-L-cysteine (BITC-NAC) 1-5 h after ingestion, with BITC-CysGly appearing as the main metabolite. Compared to human plasma, the main urinary metabolites were BITC-NAC and BITC-Cys, determined 4-6 h after ingestion. CONCLUSION This study confirms that consumption of Indian cress increases the concentration of BITC metabolites in human plasma and urine. The outcome of this human intervention study supports clinical research dealing with GSL-containing innovative food products or pharmaceutical preparations.

A new method was developed to determine twelve intact-glucosinolates (GLSs) (glucoiberin, GIB; glucoraphanin, GRA; glucoerucin GER; gluconapin, GNA; glucotropaeolin, GTL; glucobrassicin, GBC; gluconasturtiin, NAS; glucoalyssin, ALY; 4-hydroxyglucobrassicin, 4OH; 4-methoxyglucobrassicin, 4ME; neoglucobrassicin, NEO; sinigrin, SIN) in bee pollen, by means of liquid chromatography tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI). An efficient extraction procedure was proposed (average analyte recoveries were between 85% and 96%); this involved a solid-liquid extraction (SLE) with heated water, followed by a solid phase extraction (SPE) with a weak anion exchange (NH2) sorbent. Chromatography was performed on a Gemini(®) C18 analytical column with a mobile phase of formic acid in water (0.5%,v/v) and formic acid in acetonitrile (0.5%,v/v), in gradient elution mode at 1mL/min, resulted in baseline-separated peaks and a run time of 30min. The method was fully validated in terms of selectivity, limits of detection (LOD) and quantification (LOQ), linearity, carry-over effect, reinjection reproducibility, precision and accuracy. A good selectivity, low LODs and LOQs, ranging from 1 to 16μg/kg, wide linear ranges from LOQ to 1000μg/kg, and satisfactory reinjection reproducibility, precision and accuracy with relative standard deviation and relative error values lower than or equal to 9%; meanwhile, results indicates a negligible carry-over effect. The proposed method was applied to analyze intact-GLSs in bee pollen. Nine of the GLSs studied were identified in certain samples analyzed over a wide concentration range (LOQ-2226μg/kg), and significant differences in GLS content were observed among the samples.

A new method was developed to determine 12 intact glucosinolates (glucoiberin, glucoraphanin, glucoerucin, gluconapin, glucotropaeolin, glucobrassicin, gluconasturtiin, glucoalyssin, 4-hydroxyglucobrassicin, 4-methoxyglucobrassicin, neoglucobrassicin, sinigrin) in beeswax, which is an authorized food additive in the European Union, by means of liquid chromatography tandem mass spectrometry with electrospray ionization. An efficient extraction procedure was proposed (average analyte recoveries were between 88 and 101 %); this involved the dissolution of melted beeswax with hexane, a solvent extraction using a mixture of ethanol and water (80:20, v/v), and a concentration step in a rotary evaporator. Chromatography was performed on a C18 analytical column with a mobile phase of formic acid in water (0.5 %, v/v) and formic acid in acetonitrile (0.5 %, v/v), at a flow rate of 1 mL/min. The method was fully validated in terms of selectivity, limits of detection and quantification, linearity, precision, and accuracy. The limits of detection and quantification values were below 7 and 23 μg/kg, respectively. The proposed method was applied to analyze beeswax samples collected from organic apiaries.

The effect of different doses of supplemental ultraviolet-B (UV-B) illumination on yield, plant growth, biochemical changes and antioxidant activity of broccoli florets was evaluated. The broccoli plants were grown under three different supplemental UV-B illumination doses (2.2, 8.8 and 16.4 kJ m−2 d−1) in the glasshouse. Plant height decreased with increasing supplementary UV-B illumination dose. However, leaf thickness increased with increasing UV-B dose. Chlorophyll content in the leaves also increased during the growing period. The lowest chlorophyll content was found at 16.4 kJ m−2 d−1 UV-B dose. Total yield decreased with supplemental illumination, especially at 16.4 kJ m−2 d−1 illuminated dose. Total dry matter, total soluble solids, carotenoids, chlorophyll a and b content in broccoli florets decreased with increasing UV-B illumination dose. Conversley, ascorbic acid, sinigrin, total phenolic, and flavonoid content and antioxidant activity increased in UV-B illuminated florets. Surprisingly, glucotropaeolin content, one of the forms of glucosinolate in broccoli, was not affected significantly by UV-B illumination or enhanced by UV-B illumination doses.

Background Glucosinolates, anionic sulfur rich secondary metabolites, have been extensively studied because of their occurrence in the agriculturally important brassicaceae and their impact on human and animal health. There is also increasing interest in the biofumigant properties of toxic glucosinolate hydrolysis products as a method to control agricultural pests. Evaluating biofumigation potential requires rapid and accurate quantification of glucosinolates, but current commonly used methods of extraction prior to analysis involve a number of time consuming and hazardous steps; this study aimed to develop an improved method for glucosinolate extraction. Results Three methods previously used to extract glucosinolates from brassicaceae tissues, namely extraction in cold methanol, extraction in boiling methanol, and extraction in boiling water were compared across tissue type (root, stem leaf) and four brassicaceae species (B. juncea, S. alba, R. sativus, and E. sativa). Cold methanol extraction was shown to perform as well or better than all other tested methods for extraction of glucosinolates with the exception of glucoraphasatin in R. sativus shoots. It was also demonstrated that lyophilisation methods, routinely used during extraction to allow tissue disruption, can reduce final glucosinolate concentrations and that extracting from frozen wet tissue samples in cold 80% methanol is more effective. Conclusions We present a simplified method for extracting glucosinolates from plant tissues which does not require the use of a freeze drier or boiling methanol, and is therefore less hazardous, and more time and cost effective. The presented method has been shown to have comparable or improved glucosinolate extraction efficiency relative to the commonly used ISO method for major glucosinolates in the Brassicaceae species studied: sinigrin and gluconasturtiin in B. juncea; sinalbin, glucotropaeolin, and gluconasturtiin in S. alba; glucoraphenin and glucoraphasatin in R. sativus; and glucosatavin, glucoerucin and glucoraphanin in E. sativa. Electronic supplementary material The online version of this article (doi:10.1186/s13007-017-0164-8) contains supplementary material, which is available to authorized users.

International audience; Plants from the Brassicaceae family are known to contain secondary metabolites called glucosinolates. Our goal was to establish by LC/MS the glucosinolate profile of seeds of three Brassicaceae species known to hyperaccumulate heavy metals. We investigated Alyssum fallacinum auct. non Hausskn., Iberis intermedia Guers., and Noccaea caerulescens (J. Presl & C. Presl) F. K. Mey. Our results indicate that A. fallacinum seeds contain glucoiberin and glucoibervirin, which had not been previously identified in this plant. Furthermore, we report for the first time the presence of glucoiberin, glucoibervirin, glucotropaeolin, and sinigrin in I. intermedia. We have detected for the first time glucoconringiin in N. caerulescens. In addition, glucosinalbin, 4-hydroxyglucobrassicin, and glucomoringin were also detected.

Glucosinolates (GLSs) are abundant in cruciferous vegetables and reported to have anti thyroidal effects. Four GLSs (sinigrin, progoitrin, glucoerucin, and glucotropaeolin) were administered orally to rats, and the breakdown products of GLSs (GLS-BPs: thiocyanate ions, cyanide ions, organic isothiocyanates, organic nitriles, and organic thiocyanates) were measured in serum. Thiocyanate ions were measured by colorimetric method, and cyanide ions were measured with CI-GC-MS. Organic isothiocyanates and their metabolites were measured with the cyclocondensation assay. Organic nitriles and organic thiocyanates were measured with EI-GC-MS. In all treatment groups except for progoitrin, thiocyanate ions were the highest among the five GLS-BPs. In the progoitrin treated group, a high concentration of organic isothiocyanates (goitrin) was detected. In the glucoerucin treated group, a relatively low amount of goitrogenic substances was observed. The metabolism to thiocyanate ions happened within five hours of the administration, and the distribution of GLSs varied with the side chain. GLSs with side chains that can form stable carbocation seemed to facilitate the degradation reaction and produce a large amount of goitrogenic thiocyanate ions. Because goitrogenic metabolites can be formed without myrosinase, the inactivation of myrosinase during cooking would have no effect on the anti-nutritional effect of GLSs in cruciferous vegetables.

There has been much interest in obtaining crystals for crystallographic analysis of biologically active glucosinolates. Crystals of potassium (2,3-dichlorophenyl)glucosinolate were obtained as a dual solvate, containing one methanol and one ethanol molecule of crystallization, K+·C13H14Cl2NO9S2−·CH3OH·C2H5OH. The three-dimensional polymeric network consists of chains containing the potassium ions coordinated and bridged by sugar O atoms, which run parallel to theaaxis and are further crosslinked through the sugar molecules. The channels of this network are occupied by the dichlorophenyl substituents and the ethanol and methanol solvent molecules. The structure of theS-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-2,3-dichlorophenylacetothiohydroxymate, C21H23Cl2NO10S, precursor has also been determined and the β-configuration andZisomer of the thiohydroximate substituent is confirmed.

Tropaeolum majus L. is widely known as a medicinal plant in human medicine. It belongs to the Tropaeolaceae which contains mustard oil glycosides like cruciferous plants. In the case of T. majus, the intact glucosinolate glucotropaeolin showed no biological activity, but their degradation products, the isothiocyanates, did. The substances are thought to be enriched in the urinary bladder and the lungs, the active sites to develop their antimicrobial effectiveness. In animal nutrition these effects are of interest in sow management and piglet rearing. Therefore, the kinetics of benzyl-isothiocyanat (BITC) in plasma and the excretion with pig urine in response to nasturtium supplementation at different dosing regimens and galenic forms were examined. Four different groups with catheterized pigs were studied. The animals received the T. majus in different dosages (2.3, 6.9 and 13.4 mg GTL/kg BW) and different galenic forms (enteric coated tablets, pulverized tablets and powder) admixed to the feed ration as single bolus to the morning feeding. Blood and urine samples were collected within 24 h after nasturtium intake and analyzed for free BITC. The results indicated that the enteric coating was not effective in animal feeding. The concentration of BITC in tablet fed pigs was lower compared to animals fed powder or pulverized tablets. The bio-availability of the tablets was only 45% within 24 h relative to pulverized tablets. Nevertheless, the tablets could have some advantages in terms of the handling, the stability of the active substance and the dosing. Furthermore, it could be shown that all dosages yielded concentrations in urine and plasma, which can be considered to have an antimicrobial effect.

Myrosinase (EC 3.2.3.1) found in Brassicaceae plants, is the enzyme responsible for hydrolysis of glucosinolates. As a result a variety of biologically active metabolites are liberated, whose importance in crop protection and especially in cancer chemoprevention is rapidly gaining recognition. The growing practical application of glucosinolate degradation products requires that sensitive and reliable methods of myrosinase activity determination in different types of plant samples are established. With the use of commercial myrosinase prep, we systematically optimised conditions of measurement of this enzyme activity by spectrophotometric and pH-stat methods. The parameters evaluated included: sample preparation, choice of substrate, its concentration, reaction temperature and detection wavelength. Two substrates with different spectral properties were chosen: sinigrin (SIN) and glucotropaeolin (GTL). For both substrates, the best reliability was achieved at reaction temperature of 37 °C and substrate concentration of 0.2 mM and 5 mM for spectrophotometric and pH-stat methods, respectively. GTL exhibiting higher absorption at the recommended detection wavelength of 230 nm ensured greater sensitivity of spectrophotometric determination of myrosinase activity in the case of transparent plant samples. GTL seemed to increase also the sensitivity of pH-stat method, however, in this case homogenisation of plant samples turned out to be most important. The optimised conditions were then verified for a range of plant samples. Based on these results, the optimised protocols of myrosinase activity determination for both methods are proposed.

Maca is a highly appreciated Andean crop with multiple attributed health claims due to its assortment of bioactive compounds. The extraction parameters of glucosinolates (GLs), total phenolic compounds (TPC) and antioxidant capacity (AC) of maca (Lepidium meyenii) hypocotyls were optimized using response surface methodology (RSM). The effect of different extraction parameters: temperature (°C), liquid-to-solid ratio (ml/g), time (min) and ethanol concentration (%) on GLs, TPC and AC extraction yields were investigated using a Box–Behnken design (BBD) based on response surface methodology (RSM). Maximum yields of 16.4 μmol/g of dried matter (DM), 9.89 mg of gallic acid equivalents (GAE)/g of DM and 61.4 μmol of trolox equivalents (TE)/g were obtained at conditions of 47, 70 and 70 °C, liquid-to-solid ratio of 10, 16 and 10 ml/g, extraction time of 90, 30 and 48 min and ethanol concentration of 60%, 60% and 50%, for GLs, TPC and AC respectively. Optimal extraction conditions for the simultaneous extraction of GLs, TPC and AC determined by RSM were 70 °C, 10 ml/g, 90 min and 58% ethanol with extraction yields of 14.2 μmol/g of DM, 9.3 mg of GAE/g of DM and 56.9 μmol TE/g for GLs, TPC and AC respectively. Under these conditions, glucotropaeolin represented 79% of total GLs and catechins and gallocatechin derivatives represented 97% of total phenolics. The optimized conditions could be successfully employed by the Nutraceutical industry to extract bioactive compounds from maca flour.

Glucosinolates are plant secondary metabolites abundant in Brassica vegetables that are substrates for the enzyme myrosinase, a thioglucoside hydrolase. Enzyme-mediated hydrolysis of glucosinolates forms several organic products, including isothiocyanates (ITCs) that have been explored for their beneficial effects in humans. Myrosinase has been shown to be tolerant of non-natural glucosinolates, such as 2,2-diphenylethyl glucosinolate, and can facilitate their conversion to non-natural ITCs, some of which are leads for drug development. An HPLC-based method capable of analyzing this transformation for non-natural systems has been described. This current study describes (1) the Michaelis-Menten characterization of 2,2-diphenyethyl glucosinolate and (2) a parallel evaluation of this analogue and the natural analogue glucotropaeolin to evaluate effects of pH and temperature on rates of hydrolysis and product(s) formed. Methods described in this study provide the ability to simultaneously and independently analyze the kinetics of multiple reaction components. An unintended outcome of this work was the development of a modified Lambert W(x) which includes a parameter to account for the thermal denaturation of enzyme. The results of this study demonstrate that the action of Sinapis alba myrosinase on natural and non-natural glucosinolates is consistent under the explored range of experimental conditions and in relation to previous accounts.

Benzyl isothiocyanate (BITC) is a hydrolysis product of glucotropaeolin, a compound found in cruciferous vegetables, and has been shown to have anti-tumor properties. In the present study, we investigated whether BITC inhibits the development of prostate cancer in the transgenic adenocarcinoma mouse prostate (TRAMP) mice. Five-week old, male TRAMP mice and their nontransgenic littermates were gavage-fed with 0, 5, or 10 mg/kg of BITC every day for 19 weeks. The weight of the genitourinary tract increased markedly in TRAMP mice and this increase was suppressed significantly by BITC feeding. H and E staining of the dorsolateral lobes of the prostate demonstrated that well-differentiated carcinoma (WDC) was a predominant feature in the TRAMP mice. The number of lobes with WDC was reduced by BITC feeding while that of lobes with prostatic intraepithelial neoplasia was increased. BITC feeding reduced the number of cells expressing Ki67 (a proliferation marker), cyclin A, cyclin D1, and cyclin-dependent kinase (CDK)2 in the prostatic tissue. In vitro cell culture results revealed that BITC decreased DNA synthesis, as well as CDK2 and CDK4 activity in TRAMP-C2 mouse prostate cancer cells. These results indicate that inhibition of cell cycle progression contributes to the inhibition of prostate cancer development in TRAMP mice treated with BITC.

Plant samples of Hornungia petraea were analyzed for glucosinolate (GLS) autolysis metabolites for the first time. GC–MS analysis of the autolysate and the synthesis of a series (12 compounds) of possible glucosinolate breakdown products revealed/corroborated the presence of glucoaubrietin, glucolimnanthin, glucolepigramin and glucotropaeolin in this species as the most likely “mustard oil” precursors. GLS degradation products identified in the autolysate of H. petraea, benzyl isothiocyanate, 3- and 4-methoxybenzyl isothiocyanate, along with several other structurally related compounds were evaluated for antimicrobial activity in order to possibly pinpoint the role of the latter secondary metabolites in the plant tissues. The assays showed a very high antibacterial activity of the tested isothiocyanates against Sarcina lutea and an antifungal effect against Aspergillus fumigatus and Candida albicans with MIC values in the order of 1 μg/ml value.

Glucosinolates are phytochemicals with health promoting properties. Determination as desulpho-glucosinolates is widely used and a desulphation in microtiter plates has been applied to reach high throughput. The use of various sulphatase concentrations and volumes throughout literature necessitates the identification of an appropriate desulphation procedure in microtiter plates. High sulphatase concentrations (⩾15 mg/ml) decreased the concentration of the internal standard glucotropaeolin, whereas the other glucosinolates were less affected. Due to the calculation based on the recovery of glucotropaeolin, this leads to an overestimation of GL concentrations after desulphation with high sulphatase concentrations. A glucosidase side-activity, present in the crude sulphatase powder, is likely causing this phenomenon. At lower sulphatase concentrations (1 mg/ml) glucoiberin and glucoraphanin were insufficiently desulphated. Combining these effects results in a small range of applicable sulphatase concentrations. A purified sulphatase preparation resulted in good recoveries for a diversity of samples and is hence recommended for high throughput desulphation in microtiter plates.

International audience; Glucosinolates (GSLs) are a class of phytochemicals found in all calciferous plants (Brassicaceae) family as well as in the whole order of Brassicales (syn. Capparales). GSLs and their hydrolysis products (e.g. isothiocyanates) are known to play a defensive role by protecting the plant against exterior aggressions and may be potent antitumor, anticancer, antioxidant and antibiotic agents. In order to obtain pure GSLs standards for developing research, an efficient two-step method was developed to preparatively isolate and separate GSLs from papaya (Carica papaya), upland cress (Barbarea verna), cauliflower (Brassica oleracea L. var. botrytis L.), and broccoletti seeds (Brassica rapa ruvo). In this process, solvent extraction was followed by a strong anion-exchange centrifugal partition chromatography protocol. TLC, HPLC-PDA, ESI-MS and NMR analysis of the collected fractions, demonstrated that the GSLs from papaya and upland cress seeds (i.e. glucotropaeolin and gluconasturtiin, respectively) as well as all the GSLs from cauliflower and broccoletti seeds (i.e. sinigrin, glucoiberin, glucoiberverin, gluconapin, glucobrassicanapin and gluconasturtiin) can be purified in a single chromatographic step. Gram amounts of pure reference standards were obtained. These results will facilitate investigation of the biological activities and the isolation of such compounds in other crucifers. (C) 2011 Published by Elsevier B.V.

Wild radish (Raphanus raphanistrum L.) produces glucosinolates (GSL), which are important for its use as a biofumigation or allelopathic plant for weed management. Total GSL concentrations and individual GSLs were quantified in different plant parts at different developmental stages. Eight GSLs were found in various plant tissues but glucoerucin, glucoraphenin, and glucotropaeolin comprised90% of the total GSLs. All three are degraded to isothiocyanates, which are associated with weed suppression. Maximum GSL concentration (1942.2 micromol/plant) occurred at 50% flowering stage prior to the time of maximum biomass production, when GSL concentration was 1246.65 mumol/plant. Roots contributed15% of the total GSL. The highest concentration of GSLs was in flowers at flowering stage, but due to the low biomass they contributed only 11.83% to the total GSL. On the basis of these results, wild radish should be incorporated into soil at 50% flowering to provide the most GSLs for weed suppression.