Levels Of Sinigrin Research Articles

Screening of seeds from wild Pennycress (Thlaspi arvense L.) populations for sinigrin levels using a blood glucose meter and test strips

Field pennycress, Thlaspi arvense L., is a new oil seed cover crop being commercialized as a source of biofuel in the United States. Along the path to commercialization, pennycress seed and meal will be marketed as animal feed. As a member of the Brassicacaeae family, pennycress seed is abundant in glucosinolates which are anti-nutritional factors that make the seed and meal less palatable to animals. More specifically, sinigrin has been identified as the only glucosinolate present in pennycress seed. As work continues to develop varieties low in glucosinolates, a rapid assay is needed to determine the sinigrin level in pennycress seed.The objective of this study was to develop and utilize a high throughput, rapid assay to screen pennycress seeds from wild populations for total sinigrin content. Pennycress seeds were ground in potassium phosphate buffer with endogenous myrosinase to catalyze the enzymatic hydrolysis of sinigrin to equimolar amounts of glucose. Glucose content was determined using a blood glucose meter and test strips. From the molar concentration of glucose, the molar concentration of sinigrin was known and the sinigrin level in seeds was calculated in µmol/g. The results were confirmed by HPLC/MS analysis.A rapid and inexpensive method was developed and used to screen seeds of 429 wild pennycress lines for sinigrin. Sinigrin levels in seed ranged from 25 to 85 μmol/g in the pennycress lines screened. Environmental factors also seemed to contribute to the levels of sinigrin in seeds found within the line ‘tt8-t/ARV1’ grown at ten different locations in the Midwest, USA.This rapid and inexpensive assay will allow breeders to efficiently screen lines and select for varieties that produce seeds with low sinigrin levels for improved feed value.

An origami paper-based electrochemical biosensing platform for quality control of agri-food waste in the valorization strategy

The increasing demand for food and the need for a sustainability vision in the agri-food sector have boosted novel approaches for food management, enhancing the valorization of wastes and by-products belonging to the food industry. Herein, we present a novel paper-based origami device to assess the amount of both glucosinolate and glucose in a food waste product belonging to Brassicaceae plants, to evaluate the quality value and the correct management of waste samples. The device has been designed as an origami paper-based platform constituted of two paper-based biosensors to work synergistically in a multiplexed detection. In detail, a monoenzymatic biosensor and a bienzymatic biosensor were configured for the detection of glucose and glucosinolates, respectively, using filter paper pads preloaded with glucose oxidase and/or myrosinase. To complete the paper-based platform, the enzyme-preloaded pads were combined with office paper-based electrodes modified with Carbon black/Prussian Blue nanoparticles for the measurement of enzymatic by-product at a low applied potential (i.e., 0 V versus Ag/AgCl). Overall, this paper-based platform measured glucose and glucosinolate (i.e., sinigrin) with a linear range up to 2.5 and 1.5 mM, and detection limits of 0.05 and 0.07 mM, respectively. The repeatability corresponded to an RSD% equal to 5% by testing 10 mM of glucose, and 10% by testing 1 mM of sinigrin. The accuracy of the developed multiplex device was evaluated by recovery studies at two different levels of sinigrin, i.e., 0.25 and 0.5 mM, obtaining recoveries values equal to (111 ± 3) % and (86 ± 1) %, respectively. The multiplex detection of both glucose and glucosinolate in Brassicaceae samples evaluates the quality values of the waste sample, ensuring the quality of the re-used food product waste by using an eco-designed analytical tool. The combination of paper-based devices for quality control of food waste with the re-use of these food products represents a sustainable approach that perfectly matches sustainable agrifood practices as well as the overall approach of the circular economy.Graphical abstract

Glucosinolate profile and Myrosinase gene expression are modulated upon Plasmodiophora brassicae infection in cabbage.

Clubroot is a devastating disease of Brassicaceae caused by the biotrophic protist Plasmodiophora brassicae. The progression of clubroot disease is modulated by the glucosinolate (GSL) profile of the host plant. GSL is hydrolysed by the enzyme myrosinase upon cell disruption and gives rise to metabolites like isothiocyanate, nitriles, thiocyanates, epithionitriles and oxazolidines. Some of these metabolites play important roles in the plant's defence mechanism. We identified 13 Myrosinase (Myro) and 28 Myrosinase-Binding Protein-like (MBP) genes from Brassica oleracea L. using a comparative genomics approach and characterised them through in silico analyses. We compared the expression patterns of these genes in a clubroot-susceptible line and a resistant line following inoculation with P. brassicae. Two BolMyro and 12 BolMBP genes were highly expressed in the susceptible line, whereas only one BolMyro and five BolMBP genes were highly expressed in the resistant line. Principal component analysis confirmed that specific GSL profiles and gene expression were modulated due to pathogen infection. Plants with higher levels of neoglucobrassicin, glucobrassicin and methooxyglucobrassicin produced disease symptoms and formed galls, whereas, plants with higher levels of sinigrin, hydroxyglucobrassicin and progoitrin produced less symptoms with almost no galls. Our results provide insights into the roles of Myro and MBP genes in GSL hydrolysis during P. brassicae infection, which will help for developing clubroot resistant cabbage lines.

Exposure of kale root to NaCl and Na2SeO3 increases isothiocyanate levels and Nrf2 signalling without reducing plant root growth

A plant factory is a closed cultivation system that provides a consistent and modified environment for plant growth. We speculated that treatment of kale (Brassica oleracea) grown in a plant factory with NaCl, Na2SeO3, or both would increase the bioactive phytochemical levels including glucosinolates (GLSs) and isothiocyanates (ITCs), the key molecules in cancer prevention. The kale was harvested and analysed after treatment with NaCl and Na2SeO3 alone or in combination for 1 or 2 weeks. Exposure to NaCl alone but not Na2SeO3 increased plant root growth. Levels of sinigrin were increased by a 2-week exposure to Na2SeO3 alone or in combination with NaCl, whereas no changes were observed in glucoraphanin and gluconasturtiin gluconasturtiin levels. Importantly, the ITC concentration was affected by 2-week treatment with both compounds. To evaluate the bioactivity of kale, HepG2 human hepatoma cells were treated with plant extract for 6 h. Only the extract of kale roots exposed to a combination NaCl and Na2SeO3 for 2 weeks showed an increased expression of nuclear factor erythroid 2-related factor (Nrf2), which regulates genes encoding antioxidant proteins. These data suggest that co-treatment with NaCl and Na2SeO3 increased the ITC content and chemopreventive effects of kale root.

Evaluation of Seasonal Variations in the Glucosinolate Content in Leaves and Roots of Four European Horseradish (Armoracia rusticana) Landraces

In comparison with other cruciferous vegetables, horseradish has rarely been the object of scientific research, and the knowledge about the composition, content and distribution of glucosinolates (GLS) in different organs of horseradish plants is limited. Therefore, the aim of this study was to evaluate changes in the GLS content in leaves and roots of four horseradish landraces during the growing season. The presence of 13 GLS was determined in the examined horseradish tissues, and glucoraphanin, glucoraphenin and napoleiferin were noted for the first time in the species. During the growing season, the content of individual GLS changed significantly. The rate and direction of these changes varied across the examined landraces and plant organs. In the leaves, between May and June, the content of sinigrin, the main GLS in all horseradish landraces, decreased in Bavarian (40%) and Hungarian (11%) horseradish, increased (22%) in Creamy horseradish, whereas in Danish horseradish, the difference was not significant. Despite the changes observed in the first two months, the highest content of sinigrin was noted in July in all horseradish landraces. During the growing season (August-October), the content of sinigrin fluctuated in the roots of Creamy and Danish landraces, reaching the highest level in October and September, respectively, whereas in the roots of Hungarian and Bavarian landraces, sinigrin concentrations continued to increase and peaked in October. Changes in the content of other, minor GLS during the growing season often differed from those noted in sinigrin levels.

Oviposition preference of aphidophagous hoverflies toward oviposition site quality: The presence of intra- and interspecific competitor, glucosinolate content, and prey species

The selection of oviposition sites by aphidophagous insects is complex because of tritrophic interactions among host plant, aphid, and natural enemies. Several factors are known to affect the choice of oviposition site by aphidophagous hoverflies. The decisions of ovipositing females about where to lay their eggs are even more important in insects whose newly hatched offspring have limited dispersal ability, such as most aphidophages. In this study, we focused on the oviposition responses of two generalist aphidophagous syrphids (Eupeodes luniger and Episyrphus balteatus) toward host plant–aphid combinations differing in sinigrin content and the presence of intra- and interspecific competitors. The oviposition preference of Eupeodes luniger was significantly influenced in a reverse manner by the presence of intraguild competitors and also by the sinigrin content of host plants (P<0.0001 for both factors). Likewise, the overall proportion of eggs laid by female Episyrphus balteatus was greater on aphids with relatively low levels of sinigrin. Our findings help in understanding the factors governing the oviposition responses of aphidophagous predators.

On the role of sinigrin (mustard oil) in a tritrophic context: plant–aphid–aphidophagous hoverfly

1. Plant secondary metabolites can govern prey–predator interactions by altering the diet breadth of predators and sometimes provide an ecological refuge to prey. Brassicaceae plants and their specialist pests can be used as a model system for understanding the role of chemically mediated effects restricting the diet breadth of natural enemies, and consequently the occurrence of enemy‐free space for the specialist pest.2. The objective of the present study was to test the performance of the generalist predatorEpisyrphus balteatusDe Geer(Diptera:Syrphidae) fed on the specialist herbivoreBrevicoryne brassicaeL.(Homoptera:Aphididae), reared on two different brassica species: black mustard (Brassica nigra), a wild species with high levels of sinigrin; and canola (Brassica napus), a cultivated species without sinigrin.3. The preference and performance of the predator and the performance of the prey were measured. Sinigrin was quantified by high‐performance liquid chromatography in both leaf samples and aphids reared on the two host plants.4. The cabbage aphid performed better on canola than on black mustard. The performance of the predator on this aphid when reared on canola was clearly better than when reared on black mustard. Females had a higher overall preference for cabbage aphids reared on canola than on black mustard.5. The ability of aphids reared on plants with high glucosinolate content to reduce the performance of their generalist predators indicates that the presence ofB.nigramay provide enemy‐free space for the cabbage aphid from its predator, a concept that has useful application in the context of biological control for agricultural systems.

Atmospheric H2S and SO2 as sulfur source for Brassica juncea and Brassica rapa: impact on the glucosinolate composition.

The impact of sulfate deprivation and atmospheric H2S and SO2 nutrition on the content and composition of glucosinolates was studied in Brassica juncea and B. rapa. Both species contained a number of aliphatic, aromatic and indolic glucosinolates. The total glucosinolate content was more than 5.5-fold higher in B. juncea than in B. rapa, which could solely be attributed to the presence of high levels of sinigrin, which was absent in the latter species. Sulfate deprivation resulted in a strong decrease in the content and an altered composition of the glucosinolates of both species. Despite the differences in patterns in foliarly uptake and metabolism, their exposure hardly affected the glucosinolate composition of the shoot, both at sulfate-sufficient and sulfate-deprived conditions. This indicated that the glucosinolate composition in the shoot was hardly affected by differences in sulfur source (viz., sulfate, sulfite and sulfide). Upon sulfate deprivation, where foliarly absorbed H2S and SO2 were the sole sulfur source for growth, the glucosinolate composition of roots differed from sulfate-sufficient B. juncea and B. rapa, notably the fraction of the indolic glucosinolates was lower than that observed in sulfur-sufficient roots.

Evaluation of Glucosinolates in Nordic Horseradish (Armoracia Rusticana)

Abstract Horseradish (Armoracia rusticana Gaertn., C.A. Mey &amp; Scherb.) has a long history as food and medicinal plant. Glucosinolates (GLS) or their breakdown products are responsible for the pungent taste and claimed medicinal effects. The dominate GLS in horseradish is sinigrin (&gt; 80%) followed by gluconasturtiin and glucobrassicin. A total of 168 Nordic accessions of horseradish were screened for the content of intact glucosinolates. Sinigrin levels varied between 10 and 45, gluconasturtiin between 1.3 and 7.4 and glucobrassicin between 0.1 and 2.6 μmol/g DM. Accessions with high levels of both sinigrin and gluconasturtiin were found. Horseradish accessions are kept as living plants in clonal archives in their respective countries. The task for plant gene banks is not only to conserve genetic resources for the future, but also to stimulate use of the collections for various products and breeding programmes. After further analyses to certify the screening results, selected accessions will form a base for breeding and increased use of horseradish as a condiment to food, natural preservative or in medical treatments

Inhibition of Listeria monocytogenes on bologna sausages by an antimicrobial film containing mustard extract or sinigrin

The ability of Listeria (L.) monocytogenes to convert glucosinolates into antimicrobial isothiocyanates was investigated. Mustard glucosinolates in pure (sinigrin) or extract forms (sinigrin, oriental; sinalbin, yellow mustard) were used in broth media and in a polyvinyl polyethylene glycol graft copolymer (PPG) packaging film with bologna to examine their value as antimicrobial precursors for the control of L. monocytogenes viability and extension of bologna shelf-life at 4°C. During broth tests with deodorized (myrosinase-inactivated) mustard extracts (10d at 20°C) or with purified sinigrin (21d at 20°C) L. monocytogenes was only inhibited when exogenous myrosinase was added. None the less, the organism was able to hydrolyze almost half the pure sinigrin by 21d in tests without added enzyme. Reductions in sinigrin levels were measured by reversed-phase liquid chromatography, and in the absence of L. monocytogenes or added myrosinase the glucosinolate was stable. When pure sinigrin, oriental or yellow mustard extracts were incorporated in PPG films containing 3, 5 and 6% (w/w) of the corresponding glucosinolate and used to package bologna inoculated with 4 log CFU/g L. monocytogenes, the pathogen became undetectable in bologna packed with the oriental mustard extract at 52d storage and remained undetectable at 70d. The yellow mustard extract was less inhibitory and the pure sinigrin was not antimicrobial. L. monocytogenes numbers reached >7 log CFU/g in the film and untreated controls at 17d storage. At 35d storage, samples packed with control film contained sufficient numbers of lactic acid bacteria (LAB) (>7 log CFU/g) to be considered spoiled, whereas treatments containing mustard or sinigrin remained <7 log CFU/g LAB for ≤70d. L. monocytogenes played a key role in exerting control over its own viability in bologna by hydrolysis of the glucosinolate in the oriental mustard film, but other antimicrobials in treatments may have contributed.

Competition, herbivory and genetics interact to determine the accumulation and fitness consequences of a defence metabolite

Summary 1 Plant defence theory predicts that chemical defences should be optimally deployed to protect against herbivore damage, but avoid production costs when herbivores are rare. However, many chemicals involved in herbivore defence are also involved in other interactions, such as competition. 2 We studied how the accumulation of sinigrin, the dominant glucosinolate defence metabolite of Brassica nigra, responded to field manipulations of herbivory and competition in groups artificially selected for high or low constitutive sinigrin levels. Additionally, we quantified the fitness costs and benefits of sinigrin production in these treatments. 3 The induction response to molluscan herbivores was independent of a genotype's constitutive sinigrin level. In contrast, genotypes selected for low constitutive sinigrin levels were significantly more inducible in response to plant competitors as compared to genotypes with high constitutive levels. 4 Accordingly, the fitness costs and benefits of sinigrin production agreed with optimal defence theory in the absence, but not in the presence, of competition. 5 Thus, predictions for the optimal investment to defensive traits may be misleading if they do not incorporate all of the costs and benefits of traits involved in multiple interactions.

Development and characterisation of a Brassica carinata inbred line incorporating genes for low glucosinolate content from B. juncea

The presence of high levels of sinigrin in the seeds represents a serious constraint for the commercial utilisation of Ethiopian mustard (Brassica carinata A. Braun) meal. The objective of this research was the introgression of genes for low glucosinolate content from B. juncea into B. carinata. BC1F1 seed from crosses between double zero B. juncea line Heera and B. carinata line N2-142 was produced. Simultaneous selection for B. carinata phenotype and low glucosinolate content was conducted from BC1F2 to BC1F4 plant generations. Forty-three BC1F4 derived lines were selected and subject to a detailed phenotypic and molecular evaluation to identify lines with low glucosinolate content and genetic proximity to B. carinata. Sixteen phenotypic traits and 80 SSR markers were used. Eight BC1F4 derived lines were very close to N2-142 both at the phenotypic and molecular level. Three of them, with average glucosinolate contents from 52 to 61 micromoles g−1, compared to 35 micromoles g−1 for Heera and 86 micromoles g−1 for N2-142, were selected and evaluated in two additional environments, resulting in average glucosinolate contents from 43 to 56 micromoles g−1, compared to 29 micromoles g−1 for Heera and 84 micromoles g−1 for N2-142. The best line (BCH-1773), with a glucosinolate profile made up of sinigrin (>95%) and a chromosome number of 2n = 34, was further evaluated in two environments (field and pots in open-air conditions). Average glucosinolate contents over the four environments included in this research were 42, 31 and 74 micromoles g−1 for BCH-1773, Heera and N2-142, respectively. These are the lowest stable levels of glucosinolates reported so far in B. carinata.

Accumulation of Glucosinolates by the Cabbage Aphid Brevicoryne brassicae as a Defense Against Two Coccinellid Species

Brassica nigra plants, characterized by high levels of sinigrin, and artificial aphid diets to which sinigrin was selectively added were used to rear the crucifer specialist, Brevicoryne brassicae. Aphids were provided as a food source to two species of polyphagous ladybird, Adalia bipunctata and Coccinella septempunctata. First instar A. bipunctata were unable to survive when fed with B. brassicae reared on B. nigra or diets containing 0.2% sinigrin, but when fed with aphids reared on diets containing 0% sinigrin, survival rates were high. By contrast, first instar C. septempunctata were able to survive when fed with aphids reared on B. nigra or artificial diets containing up to 1% sinigrin. However, the presence of sinigrin in the aphid diet decreased larval growth and increased the time necessary for larvae to reach second instar for this species of ladybird. These results indicate that the presence of sinigrin in the diet of B. brassicae makes this aphid unsuitable as a food source for A. bipunctata but not for C. septempunctata, although for this ladybird species, there appear to be costs associated with feeding on aphids that contain this secondary metabolite.

Sinigrin (2-Propenyl Glucosinolate) Content and Myrosinase Activity in BrassicaVegetables

Cruciferous vegetables can contribute to a healthy human diet. This may be due to the presence of the phytochemical glucosinolate. The sinigrin (2-propenyl glucosinolate) content and the myrosinase activity were estimated in edible portions of five broccoli (B. oleraceaL. var. italicaPlenck), three white cabbage (B. oleraceavar. capitataL. f. alba1 DC), two each of Chinese cabbage [B. rapaL. subsp. pekinensis(Lour.) Olsson], and cauliflower (B. oleraceaL. var. botrytisL.), and one each of Brussels sprouts (B. oleraceaL. var. gemmiferaDC.), kohlrabi (B. oleraceaL. var. gongylodesL.), red cabbage [B. oleraceavar. capitataL. f. rubra(L.) Jhell], and Savoy cabbage (B. oleraceavar. sabaudaL.). Significant differences for sinigrin (2-propenyl glucosino-late) content and myrosinase activity were recorded between Brassicasubspecies. Broccoli (3.23 mg/100 g) and Brussels sprouts (2.10 mg/ 100 g) had higher sinigrin content as compared with other Brassica vegetables. The highest myrosinase specific activity was recorded in Brussels sprouts (0.343 units/mg protein), and broccoli (0.324 units/mg protein), and the lowest myrosinase activity was for Savoy cabbage (0.036 units/mg protein) and Chinese cabbage (0.041 units/mg protein). Levels of sinigrin and myrosinase activity are dependent on the subspecies. Significant intraspecific variability for sinigrin and myrosinase activity in some of the Brassicavegetables can be utilized for selecting genotypes for the genetic improvement of such vegetables for phytochemical content through conventional breeding.

Flower vs. Leaf Feeding by Pieris brassicae: Glucosinolate-Rich Flower Tissues are Preferred and Sustain Higher Growth Rate

Interactions between butterflies and caterpillars in the genus Pieris and plants in the family Brassicaceae are among the best explored in the field of insect–plant biology. However, we report here for the first time that Pieris brassicae, commonly assumed to be a typical folivore, actually prefers to feed on flowers of three Brassica nigra genotypes rather than on their leaves. First- and second-instar caterpillars were observed to feed primarily on leaves, whereas late second and early third instars migrated via the small leaves of the flower branches to the flower buds and flowers. Once flower feeding began, no further leaf feeding was observed. We investigated growth rates of caterpillars having access exclusively to either leaves of flowering plants or flowers. In addition, we analyzed glucosinolate concentrations in leaves and flowers. Late-second- and early-third-instar P. brassicae caterpillars moved upward into the inflorescences of B. nigra and fed on buds and flowers until the end of the final (fifth) instar, after which they entered into the wandering stage, leaving the plant in search of a pupation site. Flower feeding sustained a significantly higher growth rate than leaf feeding. Flowers contained levels of glucosinolates up to five times higher than those of leaves. Five glucosinolates were identified: the aliphatic sinigrin, the aromatic phenyethylglucosinolate, and three indole glucosinolates: glucobrassicin, 4-methoxyglucobrassicin, and 4-hydroxyglucobrassicin. Tissue type and genotype were the most important factors affecting levels of identified glucosinolates. Sinigrin was by far the most abundant compound in all three genotypes. Sinigrin, 4-hydroxyglucobrassicin, and phenylethylglucosinolate were present at significantly higher levels in flowers than in leaves. In response to caterpillar feeding, sinigrin levels in both leaves and flowers were significantly higher than in undamaged plants, whereas 4-hydroxyglucobrassicin leaf levels were lower. Our results show that feeding on flower tissues, containing higher concentrations of glucosinolates, provides P. brassicae with a nutritional benefit in terms of higher growth rate. This preference appears to be in contrast to published negative effects of volatile glucosinolate breakdown products on the closely related Pieris rapae.

Determination of two intact glucosinolates in vegetables and Chinese herbs.

A reversed-phase HPLC method was developed for analyzing sinigrin and gluconasturtiin in six vegetable and two Chinese herb samples. A gradient program and mobile phases using methanol and 0.05% trifluoroacetic acid containing 20 mM ammonium acetate allowed sufficient retention and separation of the glucosinolates in the sample extracts. Quadrupole time-of-flight tandem mass spectrometry in negative ion electrospray ionization was used to analyze the fractions collected from the HPLC elution to confirm the identification of the glucosinolates. The levels of sinigrin and gluconasturtiin in the vegetables and Chinese herbs were determined by using an external calibration method. Concentrations of gluconasturtiin in the Chinese herbs were more than 15 times higher than those of sinigrin. Detection limits were 18 nmol g(-1) for sinigrin and 4 nmol g(-1) for gluconasturtiin when 50 g of fresh vegetable was analyzed.

Allelochemicals isolated from tissues of the invasive weed garlic mustard (Alliaria petiolata).

Garlic mustard (Alliaria petiolata) is a naturalized Eurasian species that has invaded woodlands and degraded habitats in the eastern United States and Canada. Several phytotoxic hydrolysis products of glucosinolates, principally allyl isothiocyanate (AITC) and benzyl isothiocyanate (BzITC), were isolated from dichloromethane extracts of garlic mustard tissues. AITC and BzITC were much more phytotoxic to wheat (Triticum aestivum) than their respective parent glucosinolates sinigrin and glucotropaeolin. However, garden cress (Lepidium sativum) growth was inhibited to a greater degree by glucotropaeolin than BzITC, possibly due to conversion to BzITC by endogenous myrosinase. Sinigrin and glucotropaeolin were not detected in leaf/stem tissues harvested at the initiation of flowering, but were present in leaves and stems harvested in the autumn. Sinigrin levels in roots were similar for both sampling dates, but autumn-harvested roots contained glucotropaeolin at levels over three times higher than spring-harvested roots. The dominance of garlic mustard in forest ecosystems may be attributable in part to release of these phytotoxins, especially from root tissues.