Intact Glucosinolates Research Articles

Cultivated Winter-Type Lunaria annua L. Seed: Deciphering the Glucosinolate Profile Integrating HPLC, LC-MS and GC-MS Analyses, and Determination of Fatty Acid Composition.

Lunaria annua L. (Brassicaceae) is an ornamental plant newly identified in Europe as a promising industrial oilseed crop for its valuable very-long-chain monounsaturated fatty acids (MUFAs), especially erucic acid (EA) and nervonic acid (NA). L. annua seeds were obtained from annual winter-type plants selected and cultivated in Northern France. Using a systematic multiple-method approach, we set out to determine the profile and content of glucosinolates (GSLs), which are the relevant chemical tag of Brassicaceae. Intact GSLs were analyzed through a well-established LC-MS method. Identification and quantification were performed by HPLC-PDA of desulfo-GSLs (dGLs) according to the official EU ISO method. Moreover, GSL structures were confirmed by GC-MS analysis of the related isothiocyanates (ITCs). Seven GSLs were identified, directly or indirectly, as follows: 1-methylethyl GSL, (1S)-1-methylpropyl GSL, (Rs)-5-(methylsulfinyl)pentyl GSL, (Rs)-6-(methylsulfinyl)hexyl GSL, (2S)-2-hydroxy-4-pentenyl GSL, 2-phenylethyl GSL, and 1-methoxyindol-3-ylmethyl GSL. In other respects, the FA composition of the seed oil was determined. Results revealed cultivated L. annua seed to be a source of NA-rich oil, and presscake as a valuable coproduct. This presscake is indeed rich in GSLs (4.3% w/w), precursors of promising bioactive molecules for agricultural and nutraceutical applications.

Characterization and quantification of intact glucosinolates in Catozza rapeseeds: A promising food matrix for nutraceuticals development as a source of hydrogen sulfide

Glucosinolates (GSLs), anionic sulfur-containing secondary metabolites of Brassicales order, have shown beneficial effects for the prevention of cardiovascular diseases. This study has focused on HPLC-HESI-MS/MS analysis of intact GSLs in a poorly studied food matrix, Catozza rapeseeds (Brassica rapa L. var. rapa DC.), providing the first qualitative profile of this matrix by the identification of fifteen GSLs and the quantification of the nine most abundant compounds. After the evaluation of different powder granulometries, the best myrosinase inactivation treatment condition was achieved with a 30-minute heating treatment at 80 °C, resulting in the highest GSLs content. Two extraction methods were performed on the rapeseed meal to obtain hydroalcoholic Catozza rapeseed extracts (HCRE) and aqueous Catozza rapeseed extracts (ACRE) showing a concentration-dependent and endothelium-independent relaxing effect in mice aorta by releasing hydrogen sulfide (H2S). This study represents the starting point for further investigation of Catozza rapeseed use for GSLs-based formulations for nutraceuticals purpose.

Leaf Mustard (Brassica juncea) Germplasm Resources Showed Diverse Characteristics in Agro-Morphological Traits and Glucosinolate Levels.

Leaf mustard, characterized by its purple/red/green leaves with a green/white midrib, is known for its thick, tender, and spicy leaves with a unique taste and flavor. There were only a few studies reported on leaf mustard for its morphological and biochemical traits from Korea. A total of 355 leaf mustard accessions stored at the GenBank of the National Agrobiodiversity Center were evaluated for 25 agro-morphological traits and seven intact glucosinolates (GSLs). The accessions showed a wide variation in terms of most of the traits. The quantitative agro-morphological traits varied from 16.0 (leaf length) to 48.7% (petiole width) of the coefficient of variation (CV). The highest variation was observed in glucoiberin (299.5%, CV), while the total GSL showed a CV of 66.1%. Sinigrin, followed by gluconapin and gluconasturtiin, was the most abundant GSL, accounting for as high as 75% of the total GSLs, while glucobrassicanapin and glucoiberin were the least abundant, contributing 0.7% and 0.1% on average, respectively. Sinigrin had a positive significant correlation with all GSLs but gluconasturtiin, while glucobarbarin and gluconasturtiin were highly positively correlated to each other, but least correlated with other GSLs. The leaf length was negatively correlated with sinigrin and glucoiberin. The width of the petiole showed a positive correlation with gluconapin, glucobrassicanapin, and glucobrassicin, while the length of the petiole had a negative correlation with sinigrin, glucobrassicanapin, glucoiberin, glucobrassicin, and the total GSLs. A higher width of the midrib was associated with higher contents of gluconapin, glucobrassicanapin, and glucobrassicin. A PCA analysis based on the agro-morphological traits showed that the first and second principal components accounted for 65.2% of the overall variability. Accessions that form a head tend to exhibit a longer leaf length, a larger plant weight, a thicker midrib, and higher widths of the midrib, petiole, and leaf. The GSLs showed inconsistent inter-and intra-leaf variation. Accessions that identified for various traits in their performance, such as, for example, Yeosu66 and IT259487 (highest total glucosinolates) and IT228984 (highest plant weight), would be promising lines for developing new varieties.

Dynamic profiling of intact glucosinolates in radish by combining UHPLC-HRMS/MS and UHPLC-QqQ-MS/MS.

Glucosinolates (GSLs) and their degradation products in radish confer plant defense, promote human health, and generate pungent flavor. However, the intact GSLs in radish have not been investigated comprehensively yet. Here, an accurate qualitative and quantitative analyses of 15 intact GSLs from radish, including four major GSLs of glucoraphasatin (GRH), glucoerucin (GER), glucoraphenin (GRE), and 4-methoxyglucobrassicin (4MGBS), were conducted using UHPLC-HRMS/MS in combination with UHPLC-QqQ-MS/MS. Simultaneously, three isomers of hexyl GSL, 3-methylpentyl GSL, and 4-methylpentyl GSL were identified in radish. The highest content of GSLs was up to 232.46 μmol/g DW at the 42 DAG stage in the 'SQY' taproot, with an approximately 184.49-fold increase compared to the lowest content in another sample. That the GSLs content in the taproots of two radishes fluctuated in a similar pattern throughout the five vegetative growth stages according to the metabolic profiling, whereas the GSLs content in the '55' leaf steadily decreased over the same period. Additionally, the proposed biosynthetic pathways of radish-specific GSLs were elucidated in this study. Our findings will provide an abundance of qualitative and quantitative data on intact GSLs, as well as a method for detecting GSLs, thus providing direction for the scientific progress and practical utilization of GSLs in radish.

Glucosinolates, a natural chemical arsenal: More to tell than the myrosinase story.

Glucosinolates are a group of thioglucosides that belong to the class of plant nitrogen-containing natural products. So far, very little biological activity has been associated with intact glucosinolates. The hydrolysis of glucosinolates has, for long, attracted attention because of the potent biological activity of the hydrolysis products. From allelopathic to antiparasitic, antimicrobial and antineoplastic effects, the activity spectrum of the degradation products of typical glucosinolates has been the subject of much research. The present review seeks to address the various means of glucosinolate degradation (thermal, enzymatic, or chemical degradation) and the ensuing products. It also aims to draw a comparative profile of the various antimicrobial effects of these degradation products to provide a further understanding of the biological function of these important compounds.

Fresh Brassicaceae sprouting broccoli (Bimi®) glucosinolates profile characterization and bioaccessibility through an in vitro dynamic digestion study

Bimi® (Brassica oleracea Italica x Alboglabra) glucosinolate content was determined as 83.64 mg/100 g of fresh sample, mainly characterised by aliphatic (61 %) and indolic (39 %) glucosinolates. Glucoraphanin was the main compound (36.75 ± 1.96 mg/100 g) followed by glucosbrassicin (22.46 ± 6.37 mg/100 g) and progoitrin (11.95 ± 1.31 mg/100 g). Other remarkable compounds were neoglucobrassicin, 4-methoxyglucobrassicin and gluconapin. An in vitro Dynamic Gastrointestinal Digester system (DGD) was used to simulate the gastrointestinal digestion. Important reductions of glucosinolates were observed (bioaccessibility of 23 %), where aliphatic compounds were the most bioaccesibles. The main glucosinolate in the bioaccesible fraction was the glucoraphanin (13.20 ± 3.66 mg/100 g of fresh sample). Other bioaccesible compounds detected in a lesser extent were progoitrin, glucobrassicin, 4-methoxy-glucobrassicin, gluconapin, glucoalisin and neoglucobrassicin. Therefore, diverse intact glucosinolates (together with well-known derived isothiocyanate generated during digestion process) are available after the digestion process. Compared to the literature data of its constitutive plant (broccoli), Bimi® showed a similar trend in glucosinolate profile but, in general, higher amounts. Close bioaccessibility values were also determined compared broccoli. This study suggests that a chronic intake of Bimi® may allow a chronic intake of bioaccesible glucosinolates such as glucoraphanin, generaly recognized as an anticancer compound.

The Yeast Fermentation Effect on Content of Bioactive, Nutritional and Anti-Nutritional Factors in Rapeseed Meal.

The aim of this study was to evaluate the changes in the content of bioactive, nutritional and anti-nutritional factors in rapeseed meal that was fermented with Saccharomyces cerevisiae or Saccharomyces boulardii yeasts at two different periods of time, for improvement of nutritional characteristics in piglets’ feeding. The fermentation has reduced the content of two anti-nutritional factors, intact glucosinolates and 3-butyl isothiocyanate, by 51.60–66.04% and 55.21–63.39%, respectively, by fermentation with either Saccharomyces cerevisiae or Saccharomyces boulardii for 24 h. The fermentation by these yeasts also lowered the content of total polyphenolic compounds by 21.58–23.55% and antioxidant activity (DPPH) by 17.03–21.07%. Furthermore, the content of carbohydrates and organic acids has dramatically decreased between 89.20 and 98.35% and between 31.48 and 77.18%, respectively. However, the content of some individual phenolic acids (gallic, p-coumaric, sinapic) and crude protein content (10–13%) has been increased. Thus, the results showed that fermentation with Saccharomyces cerevisiae or Saccharomyces boulardii has reduced the content of antinutritive factors and increased the protein content of the rapeseed meal, without major adverse effects on its overall nutritive value.

Effect of fermentation stages on glucosinolate profiles in kimchi: Quantification of 14 intact glucosinolates using ultra-performance liquid chromatography-tandem mass spectrometry.

We developed and validated an ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry-based analytical method to determine intact glucosinolates in kimchi and evaluate the effects of fermentation stages on glucosinolate profiles. The developed method yielded reliable data in the kimchi matrix in terms of selectivity, matrix effect (88%-105%), linearity (coefficients of determination ≥0.9991), sensitivity (limits of quantification ≤35nmol/L), accuracy (82%-101%), and precision (≤8%). The kimchi samples contained progoitrin, sinigrin, glucoraphanin, glucoraphenin, glucoalyssin, gluconapin, glucobrassicanapin, glucobrassicin, glucoberteroin, gluconasturtiin, 4-methoxyglucobrassicin, and neoglucobrassicin, of which 4-methoxyglucobrassicin, glucobrassicanapin, and gluconapin were the major compounds. Total glucosinolate content was decreased by 31%-97% and 91-100% in the moderate-fermented and over-fermented samples, respectively, compared with that in the non-fermented samples, revealing sudden glucosinolate degradation between the moderate- and over-fermentation stages. In summary, we report an efficient analytical method to estimate kimchi glucosinolate profiles, which could be a foundation for future studies.

Identification and quantification of intact glucosinolates at different vegetative growth periods in Chinese cabbage cultivars by UHPLC-Q-TOF-MS

The aims of this study were to investigate glucosinolate variations in Chinese cabbage cultivars at different growth periods. Glucosinolates in two types of Chinese cabbage (Xiayangbai and Zaoshu-5) at different growth periods (seeds, germination, seedling, and rosette period) were investigated. Thirteen glucosinolates were identified and quantified using UHPLC-Q-TOF-MS. Concentrations of the glucosinolates were significantly different between Xiayangbai and Zaoshu-5. The seed period generated the highest concentration of glucosinolates, and aliphatic glucosinolate predominated in seeds, seedling, and leaves of the rosette as well as during germination. However, the dominant glucosinolate in the roots was an aromatic glucosinolate (gluconasturtiin). In addition, glucoerucin was only found in the roots of rosettes. There were positive significant correlations with each other among gluconapin, glucobrassicanapin, glucoraphanin, glucoalyssin, and 4-hydroxyglucobrassicin. Our results released the metabolism pathways of glucosinolates in Chinese cabbage, which provided scientific evidence to develop functional foods with higher glucosinolate.

Determination of 18 Intact Glucosinolates in Brassicaceae Vegetables by UHPLC-MS/MS: Comparing Tissue Disruption Methods for Sample Preparation.

Glucosinolates (GSLs) are important precursor compounds with anticancer activities in Brassicaceae vegetables and are readily hydrolyzed by myrosinase. Given the diversity of these species, establishing an accurate and universal method to quantify intact GSLs in different plant tissues is necessary. Here, we compared and optimized three tissue disruption methods for sample preparation. After microwave treatment for 90 s, 13 GSLs in homogenized Chinese cabbage samples were recovered at 73–124%. However, a limitation of this method was that different tissues could not be processed under the same microwave conditions. Regarding universality, GSLs in Brassicaceae vegetables could be extracted from freeze-dried sample powder with 70% methanol (v/v) or frozen-fresh sample powder with 80% methanol (v/v). Moreover, heating extraction is necessary for GSLs extracted from frozen-fresh sample powder. Average recoveries of the two optimized methods were 74–119% with relative standard deviations ≤ 15%, with the limits of quantification 5.72–17.40 nmol/g dry weight and 0.80–1.43 nmol/g fresh weight, respectively. Notably, the method for analyzing intact GSLs was more efficient than that for desulfo-GSLs regarding operational complexity, detection speed and quantification accuracy. The developed method was applied to identify the characteristic GSLs in 15 Brassicaceae vegetables, providing a foundation for further research on GSLs.

Glucosinolates and Isothiocyantes in Processed Rapeseed Determined by HPLC-DAD-qTOF.

Glucosinolates are well known as natural antimicrobials and anticarcinogenic agents. However, these compounds can lose their properties and transform into antinutrients, depending on processing conditions. In addition, the bitterness of some glucosinolate in rapeseed meal can affect the likability of the final product. Therefore, it is important to identify and determine each glucosinolate and its derived form, not just the total glucosinolate content, in order to evaluate the potential of the final rapeseed protein product. This study provides a comprehensive report of the types and quantities of glucosinolates and their derived forms (isothiocyanates) associated with different rapeseed processing conditions. Glucosinolates and isothiocyanates were determined by HPLC-DAD-qTOF. In our study, the enzymatic degradation of glucosinolates by myrosinase was the main factor affecting either glucosinolate or isothiocyanate content. Other factors such as pH seemed to influence the concentration and the presence of glucosinolates. In addition, process parameters, such as extraction time and separation technology, seemed to affect the amount and type of isothiocyanates in the final protein extracts. Overall, both determined intact glucosinolates and their derived forms of isothiocyanates can give different types of biological effects. More studies should be performed to evaluate the impact of glucosinolates and isothiocyanates on human health.

In vitro digestion using dynamic rat stomach-duodenum model as an alternative means to assess bioaccessibility of glucosinolates in dietary fiber powder from cabbage

Brassica vegetables are typically cooked or processed prior to consumption, resulting in inactivation of endogenous myrosinase; intact glucosinolates (GLSs) released during digestion are then hydrolyzed by gut bacteria. To understand release behavior of GLSs from vegetable matrix, two experimental systems, namely, dynamic in vitro rat stomach-duodenum (DIVRSD) model and conventional shaking-incubator in vitro digestion model, were comparatively utilized. Bioaccessible contents of selected GLSs, i.e., sinigrin, glucoraphanin and glucobrassicin, that could be released upon digestion of white cabbage powder, which was used as test material, were monitored. Approximately 92% and 82% of sinigrin and glucobrassicin in cabbage powder were released within 10 min during digestion in DIVRSD model. No degradation of these GLSs occurred during either simulated digestion process. Contractions mimicking gastric peristaltic waves in DIVRSD model facilitated release of GLSs, especially glucoraphanin; the content observed in DIVRSD model was around 4-fold higher than that in shaking-incubator model. DIVRSD model could well capture continuous digestion process and transport behavior of GLSs along the GI tract. After digestion in DIVRSD model for 180 min, a decrease in GLSs contents in the stomach (20–29%) and an increase in GLSs contents in the duodenum (63–80%) were clearly observed.

Controlling the quality of maca (Lepidium meyenii) dietary supplements: Development of compendial procedures for the determination of intact glucosinolates in maca root powder products

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.

Current analytical methods for determination of glucosinolates in vegetables and human tissues

Numerous epidemiological studies have indicated the potential effects of glucosinolates and their metabolites against cancer as well as other non-communicable diseases, such as cardiovascular disease and neurodegenerative disorders.However, information on the presence and quantity of glucosinolates in commonly consumed vegetables and in human fluids is sparse, largely because well-standardised methods for glucosinolate determination are not available, resulting in published data being inconsistent and conflicting. Thus, studies published since 2002 on the most recent developments of glucosinolate extraction and identification have been collected and reviewed with emphasis on determination of the intact glucosinolates by LC-MS and LC-MS/MS.This overview highlights the glucosinolate extraction methods used, the stability of glucosinolates during extraction, the availability of stable isotope labelled internal standards and the use of NMR for purity analysis, as well as the current analytical techniques that have been applied for glucosinolate analysis, e.g. liquid chromatography with mass spectrometric detection (LC-MS). It aims to interpret the findings with a focus on the development of a validated method, which will help to determine the glucosinolate content of vegetative plants and human tissues, and the identification and determination of selected glucosinolate metabolites.

Influence of foliar Methyl-jasmonate biostimulation on exudation of glucosinolates and their effect on root pathogens of broccoli plants under salinity condition

The rhizosphere is the closest area of soil around plant roots. Root exudates are the main plant factor responsible for changes in microbial communities. As brassica plants have a defence system based on secondary metabolites such as glucosinolates (GLS) and isothiocyanates (ITC), the aim of this study was to determine the root exudation of these metabolites by broccoli plants and the influence of elicitors on this process. Also, the effect of these exudates on plant pathogens Fusarium oxysporum, Pseudomonas syringae and Sphingomonas suberifaciens. Broccoli plants were grown under controlled conditions. Biostimulation treatment with methyl-jasmonate (MeJA) with/without NaCl conditions were applied. The glucosinolates and isothiocyanates were measured in the leaves, roots and growth solution. Also, these compounds present in the exudates were applied to cultures of pathogens to check the effect on their growth. The results indicate that broccoli plants exude both GLS and ITC, and that foliar application of MeJA has a systematic positive effect, enhancing their biosynthesis and exudation by root cells, however, the biostimulation produced a less plant growth under salinity conditions. The intact GLS plus the ITC detected in the growth solution showed a strong effect against the pathogens F. oxysporum, P. syringae, S. suberifaciens, providing the first evidence of their suppression to be implemented under agronomical conditions.

Analytical Methods for Quantification and Identification of Intact Glucosinolates in Arabidopsis Roots Using LC-QqQ(LIT)-MS/MS.

Glucosinolates are biologically active secondary metabolites in Brassicaceae plants that play a critical role in positive and negative interactions between plants and root-associated microbial communities. The aim of this study was to develop a reversed-phase liquid chromatography method to quantify and identify intact glucosinolates in the root of Arabidopsis thaliana (Arabidopsis) grown in non-sterile natural soil by using liquid chromatography-hybrid triple quadruple-linear ion trap (LC-QqQ(LIT)) mass spectrometry. The Synergi Fusion C18-based column was found to be effective for sufficient retention and separation of nine intact glucosinolates without the need for time-consuming desulfation or ion-pairing steps. Method validation results showed satisfactory inter-day and intra-day precision for all glucosinolates except for 4-hydroxyglucobrassicin. Good inter-day and intra-day accuracy and recovery results were observed for glucoiberin, gluconapin, glucobrassicin, 4-methoxyglucobrassicin and neoglucobrassicin. However, for 4-hydroxyglucobrassicin, glucoraphanin and glucoerucin corrections to quantification results might be necessary since the recovery and accuracy results were not optimal. Matrix effects were shown to have a negligible effect on the ionization of all target analytes. The established liquid chromatography–tandem mass spectrometry (LC-MS/MS) method was applied to quantify target intact glucosinolates in Arabidopsis root crude extract of four different wild-type accessions where differences in terms of concentration and composition of intact glucosinolates were observed. Employment of sensitive and selective precursor ion survey scan of m/z 97 in combination with the information-dependent acquisition (IDA) of the enhanced product ion (EPI) dependent scan (Prec97-IDA-EPI) using LC-QqQ(LIT) provided high confidence in structural characterization of diverse intact glucosinolate profiles in complex Arabidopsis root crude extract.

Chemical profiling and separation of bioactive secondary metabolites in Maca (Lepidium peruvianum) by normal and reverse phase thin layer chromatography coupled to desorption electrospray ionization-mass spectrometry.

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.

Glucosinolates: Natural Occurrence, Biosynthesis, Accessibility, Isolation, Structures, and Biological Activities.

Glucosinolates (GSLs) are secondary plant metabolites abundantly found in plant order Brassicales. GSLs are constituted by an S-β-d-glucopyrano unit anomerically connected to O-sulfated (Z)-thiohydroximate moiety. The side-chain of the O-sulfate thiohydroximate moiety, which is derived from a different amino acid, contributes to the diversity of natural GSL, with more than 130 structures identified and validated to this day. Both the structural diversity of GSL and their biological implication in plants have been biochemically studied. Although chemical syntheses of GSL have been devised to give access to these secondary metabolites, direct extraction from biomass remains the conventional method to isolate natural GSL. While intact GSLs are biologically inactive, various products, including isothiocyanates, nitriles, epithionitriles, and cyanides obtained through their hydrolysis of GSLs, exhibit many different biological activities, among which several therapeutic benefits have been suggested. This article reviews natural occurrence, accessibility via chemical, synthetic biochemical pathways of GSL, and the current methodology of extraction, purification, and characterization. Structural information, including the most recent classification of GSL, and their stability and storage conditions will also be discussed. The biological perspective will also be explored to demonstrate the importance of these prominent metabolites.

Intact Glucosinolate Determination in Brassica Vegetables Using Anion Exchange Cleanup and HILIC (Hydrophilic Interaction Liquid Chromatography) HPLC Chromatography

In this work an HPLC/hydrophilic interaction liquid chromatography (HILIC) system for intact glucosinolates was optimized using a weak anion exchange resin to remove carboxylic phenolics that absorbed at the same wavelength (229 nm) as glucosinolates, by selectively protonating contaminating phenolics with pKas from 2.98 to 4 while leaving the second OH of the sulfate of the thioglycosidic linkage (pKa = 1.92) ionized. Glucosinolates bound to the anion exchange resin, while the contaminating phenolics washed through. The extraction was performed in 2-ml snap top centrifuge tubes with 50 to 80 mg of fresh tissue in 0.3 ml of extractant. The corresponding spin tubes contained 20–22 mg of anion exchange resin and were loaded with 100 μl of extractant. A 48-well pressure plate was used to load, wash, and finally elute the glucosinolates without a centrifuge into autosampler vials. Seventeen glucosinolates found in common vegetables were separated in a 16-min isocratic run vs 50 min for the C18 gradient used in the desulfoglucosinolate method. The recovery of a spiked sample of phenethyl glucosinolate was greater than 90%, and the limit of detection (LOD) and limit of quantitation (LOQ) were 6.85 and 20.78 nmol respectively. The time from sample weighing to the end of the first chromatographic run was approximately 2 h.

A cell suspension based uptake method to study high affinity glucosinolate transporters

BackgroundGlucosinolates are an important class of secondary metabolites characteristic to the order Brassicales. They are known to play a major role in plant defense and from the human perspective, can be anticarcinogenic or antinutritive. GTRs are plasma-membrane localized high affinity glucosinolate transporters, which are important components of the source (leaf) to sink (seed) translocation of intact glucosinolates in members of Brassicaceae family. GTRs are identified as major candidates for Brassica crop improvement, thus dictating a need for their functional characterization. However, currently there are limitations in availability of heterologous assay systems for functional characterization of plant secondary metabolite transporters. To date, the animal-based Xenopus oocyte system is the best established heterologous system for functional characterization of these transporters. Inherent biochemical and physiological attributes unique to the plant membranes necessitate the need for developing plant-based transporters assay systems as well.MethodsIn this study, Agrobacterium mediated transformation was used to develop GTR expressing cotton cell lines (CCL-1) for functional characterization of the Arabidopsis high affinity glucosinolate transporters, AtGTR1 and AtGTR2. Following sub-cellular localization of AtGTRs, we standardized the glucosinolate uptake assays using cell suspension cultures of AtGTR expressing CCL-1 its requirement of pH, salt, and time based glucosinolate uptake. Using the GTR expressing CCL-1, we subsequently performed kinetic analysis of AtGTR1 and AtGTR2 for different glucosinolate substrates, sinigrin, gluconapin and sinalbin.ResultsSeveral clones expressing each of AtGTR1 and AtGTR2 were obtained showing high level of GTR expression and were maintained through regular sub-culturing. Both AtGTR1 and AtGTR2 are predominantly plasma-localized proteins when overexpressed in CCL-1 cells. Uptake assays were standardized, suggesting that glucosinolate uptake of GTR expressing CCL-1 is robust within the physiological pH range 5–6, and at lower concentration of nitrate salts. GTR expressing CCL-1 cells show increasing glucosinolate accumulation in time course experiment. Kinetic studies over a wide glucosinolate concentrations (10–800 µM) revealed that our novel assay system displayed robust GTR-mediated uptake of different glucosinolates and unambiguously helps elucidate the saturable kinetics of GTRs. Our system confirms the high affinity of AtGTRs for both aliphatic and aromatic glucosinolates.ConclusionThe transporter assay system described in this study holds potential for studying sub-functionalization amongst GTR homologs present across Brassicaceae family. The fast growing CCL-1 cells, confer the benefits of an in vitro system for quick assays and is plant based thus enabling optimal expression without sequence modifications. The efficient functioning of the GTR transporters in the heterologous CCL-1 opens the possibility of using this plant cell suspension system for functional characterization of other metabolite transporters.