Metabolome profiling of cacao (Theobroma cacao L.) callus under drought stress conditions induced by polyethylene glycol (PEG) as osmoticant.

INTRODUCTION: The cacao tree (Theobroma cacao), a perennial crop that serves as a source of cacao beans, can suffer from drastic climate changes such as irregular rainfall and shorter rainy seasons. The search for hybrids which are capable of producing specific metabolites favoring adaptation in new climatic conditions is a challenge in cacao farming. OBJECTIVES: We aimed to (1) analyze the metabolic changes in calli of three cacao genotypes during water deficit induced by incubation with polyethylene glycol and (2) assess their response to water deficit stress with regard to somatic embryo differentiation. METHODS: Metabolic profiling was carried out using 1 H-NMR spectroscopy and multivariate data analysis was applied to crude extracts of calli grown in non-stress or water deficit stress conditions. RESULTS: Water deficit stress influences the capacity of calli to produce embryos. The SCA12 genotype exhibited the best conversion capacity under severe conditions and was considered as tolerant to drought, followed by the SCA6 genotype (mid-tolerant) and the MA12 genotype (sensitive). Fifty-four metabolites were identified in the three cacao genotypes and discriminant metabolites were identified. Metabolites involved in water stress tolerance such as fructose, trans-aconitic acid, leucine, and hydroxybenzene derivatives were observed in SCA12, the tolerant genotype. CONCLUSION: These results demonstrate the utility of 1 H-NMR metabolomics as an essential tool for the analysis of the drought tolerance characteristics of T. cacao.

Cytotoxicity and Identification of Antibacterial Compounds from Baillonella toxisperma Bark Using a LC-MS/MS and Molecular Networking Approach.

Baillonella toxisperma is a medicinal plant used in northern Gabon to treat microbial diseases. It is a plant well-known by local populations, but very few studies have focused on the molecules responsible for the antibacterial activities of B. toxisperma. This study proposes a dereplication strategy based on molecular networking generated from HPLC-ESI-Q/TOF data, allowing investigation of the molecules responsible for the antibacterial activity of B. toxisperma. From this strategy, eighteen compounds were putatively identified. All of these compounds belonged mainly to five families of natural compounds, including phenylpropanolamines, stilbenes, flavonoids, lignans and phenolic glycosides. The chemical study carried out from the bark of B. toxisperma allowed us to identify, for the first time, compounds such as resveratrol and derivatives, epicatechin, epigallocatechin and epigallocatechin gallate. In addition, antibacterial activity (diffusion method and microdilution) and cytotoxicity (Cell Counting Kit-8 (CCK-8 Assay)) in vitro were evaluated. The crude ethanolic extract, as well as the fractions of B. toxisperma, showed significant antibacterial activity. However, the ethanolic fractions F2 and F4 presented high antibacterial activity compared to the crude extract. Cytotoxicity studies on colon-cancer cells (Caco-2) and human keratinocyte cells (HaCaT) showed moderate cytotoxicity in both cell types. This study clearly shows the therapeutic potential of the ethanolic extract of the bark of B. toxisperma and provides information on the phytochemical composition and bioactive compounds of the plant.

Combined LC-MS/MS and Molecular Networking Approach Reveals Antioxidant and Antimicrobial Compounds from Erismadelphus exsul Bark.

Erismadelphus exsul Mildbr bark is widely used in Gabonese folk medicine. However, little is known about the active compounds associated with its biological activities. In the present study, phytochemical profiling of the ethanolic extract of Erismadelphus exsul was performed using a de-replication strategy by coupling HPLC-ESI-Q/TOF with a molecular network approach. Eight families of natural compounds were putatively identified, including cyclopeptide alkaloids, esterified amino acids, isoflavonoid- and flavonoid-type polyphenols, glycerophospholipids, steroids and their derivatives, and quinoline alkaloids. All these compounds were identified for the first time in this plant. The use of molecular networking obtained a detailed phytochemical overview of this species. Furthermore, antioxidant (2,2-diphenyl-1-picryl-hydrazylhydrate (DPPH) and ferric reducing capacity (FRAP)) and in vitro antimicrobial activities were assessed. The crude extract, as well as fractions obtained from Erismadelphus exsul, showed a better reactivity to FRAP than DPPH. The fractions were two to four times more antioxidant than ascorbic acid while reacting to FRAP, and there was two to nine times less antioxidant than this reference while reacting to DPPH. In addition, several fractions and the crude extract exhibited a significant anti-oomycete activity towards the Solanaceae phytopathogen Phytophthora infestans in vitro, and, at a lower extent, the antifungal activity against the wheat pathogen Zymoseptoria tritici had growth inhibition rates ranging from 0 to 100%, depending on the tested concentration. This study provides new insights into the phytochemical characterization and the bioactivities of ethanolic extract from Erismadelphus exsul bark.

Integument-Specific Transcriptional Regulation in the Mid-Stage of Flax Seed Development Influences the Release of Mucilage and the Seed Oil Content.

Flax (Linum usitatissimum L.) seed oil, which accumulates in the embryo, and mucilage, which is synthesized in the seed coat, are of great economic importance for food, pharmaceutical as well as chemical industries. Theories on the link between oil and mucilage production in seeds consist in the spatio-temporal competition of both compounds for photosynthates during the very early stages of seed development. In this study, we demonstrate a positive relationship between seed oil production and seed coat mucilage extrusion in the agronomic model, flax. Three recombinant inbred lines were selected for low, medium and high mucilage and seed oil contents. Metabolite and transcript profiling (1H NMR and DNA oligo-microarrays) was performed on the seeds during seed development. These analyses showed main changes in the seed coat transcriptome during the mid-phase of seed development (25 Days Post-Anthesis), once the mucilage biosynthesis and modification processes are thought to be finished. These transcriptome changes comprised genes that are putatively involved in mucilage chemical modification and oil synthesis, as well as gibberellic acid (GA) metabolism. The results of this integrative biology approach suggest that transcriptional regulations of seed oil and fatty acid (FA) metabolism could occur in the seed coat during the mid-stage of seed development, once the seed coat carbon supplies have been used for mucilage biosynthesis and mechanochemical properties of the mucilage secretory cells.

NMR and LC-MS-Based Metabolomics to Study Osmotic Stress in Lignan-Deficient Flax.

Lignans, phenolic plant secondary metabolites, are derived from the phenylpropanoid biosynthetic pathway. Although, being investigated for their health benefits in terms of antioxidant, antitumor, anti-inflammatory and antiviral properties, the role of these molecules in plants remains incompletely elucidated; a potential role in stress response mechanisms has been, however, proposed. In this study, a non-targeted metabolomic analysis of the roots, stems, and leaves of wild-type and PLR1-RNAi transgenic flax, devoid of (+) secoisolariciresinol diglucoside ((+) SDG)-the main flaxseed lignan, was performed using 1H-NMR and LC-MS, in order to obtain further insight into the involvement of lignan in the response of plant to osmotic stress. Results showed that wild-type and lignan-deficient flax plants have different metabolic responses after being exposed to osmotic stress conditions, but they both showed the capacity to induce an adaptive response to osmotic stress. These findings suggest the indirect involvement of lignans in osmotic stress response.

Age-Dependent Metabolic Profiles Unravel the Metabolic Relationships Within and Between Flax Leaves (Linum usitatissimum).

Flax for oil seed is a crop of increasing popularity, but its cultivation needs technical improvement. Important agronomic traits such as productivity and resistance to stresses are to be regarded as the result of the combined responses of individual organs and their inter-communication. Ultimately, these responses directly reflect the metabolic profile at the cellular level. Above ground, the complexity of the plant phenotype is governed by leaves at different developmental stages, and their ability to synthesise and exchange metabolites. In this study, the metabolic profile of differently-developed leaves was used firstly to discriminate flax leaf developmental stages, and secondly to analyse the allocation of the metabolites within and between leaves. For this purpose, the concentration of 52 metabolites, both primary and specialized, was followed by gas chromatography (GC-) and liquid chromatography coupled to mass spectrometry (LC-MS) in alternate pairs of flax leaves. On the basis of their metabolic content, three populations of leaves in different growth stages could be distinguished. Primary and specialized metabolites showed characteristic distribution patterns, and compounds similarly evolving with leaf age could be grouped by the aid of the Kohonen self-organising map (SOM) algorithm. Ultimately, visualisation of the correlations between metabolites via hierarchical cluster analysis (HCA) allowed the assessment of the metabolic fluxes characterising different leaf developmental stages, and the investigation of the relationships between primary and specialized metabolites.

An untargeted liquid chromatography-mass spectrometry-based workflow for the structural characterization of plant polyesters.

Cell wall localized heterogeneous polyesters are widespread in land plants. The composition of these polyesters, such as cutin, suberin, or more plant-specific forms such as the flax seed coat lignan macromolecule, can be determined after total hydrolysis of the ester linkages. The main bottleneck in the structural characterization of these macromolecules, however, resides in the determination of the higher order monomer sequences. Partial hydrolysates of the polyesters release a complex mixture of fragments of different lengths, each present in low abundance and therefore are challenging to structurally characterize. Here, a method is presented by which liquid chromatography-mass spectrometry (LC-MS) profiles of such partial hydrolysates are searched for pairs of related fragments. LC-MS peaks that show a mass difference corresponding to the addition of one or more macromolecule monomers were connected in a network. Starting from the lowest molecular weight peaks in the network, the annotation of the connections as the addition of one or more polyester monomers allows the prediction of consecutive and increasingly complex adjacent peaks. Multi-stage MS (MSn) experiments further helped to reject, corroborate, and sometimes refine the structures predicted by the network. As a proof of concept, this procedure was applied to partial hydrolysates of the flax seed coat lignan macromolecule, and allowed to characterize 120 distinct oligo-esters, consisting of up to six monomers, and containing monomers and linkages for which incorporation in the lignan macromolecule had not been described before. These results showed the capacity of the approach to advance the structural elucidation of complex plant polyesters.

Phenolic Profiling of Flax Highlights Contrasting Patterns in Winter and Spring Varieties.

Flax (Linum usitatissimum) is a plant grown in temperate regions either for its fiber or for its seeds, which are rich in the essential fatty acid omega-3. It is also well known as a source of medicinal compounds. The chemical composition of its leaves is currently poorly described. In order to fill this gap, we have conducted a comprehensive analysis of flax leaf metabolome. The exploration of the metabolome allowed the characterization of compounds isolated for the first time in flax leaves. These molecules were isolated by preparative HPLC and then characterized by NMR, LC-MS and standard analysis. This work extended our picture of C-glycosyl-flavonoids and coniferyl alcohol derivatives accumulated in flax. The follow-up of the content of these different metabolites via UPLC-MS revealed significant accumulation differences in spring and winter flax leaves. In particular, two methylated C-glycosylflavonoids (swertisin and swertiajaponin) were the most abundant phenolic compounds in winter flax whereas they were not detected in spring flax. This result suggests that these 2 compounds are involved in cold stress tolerance in flax.

Cytological Approaches Combined With Chemical Analysis Reveals the Layered Nature of Flax Mucilage.

The external seed coat cell layer of certain species is specialized in the production and extrusion of a polysaccharide matrix called mucilage. Variations in the content of the released mucilage have been mainly associated with genetically regulated physiological modifications. Understanding the mucilage extrusion process in crop species is of importance to gain deeper insight into the complex cell wall biosynthesis and dynamics. In this study, we took advantage of the varying polysaccharide composition and the size of the flax mucilage secretory cells (MSCs) to study mucilage composition and extrusion in this species of agricultural interest. We demonstrate herein that flax MSCs are structured in four superimposed layers and that rhamnogalacturonans I (RG I) are firstly synthesized, in the upper face, preceding arabinoxylan and glucan synthesis in MSC lower layers. Our results also reveal that the flax mucilage release originates from inside MSC, between the upper and deeper layers, the latter collaborating to trigger polysaccharide expansion, radial cell wall breaking and mucilage extrusion in a peeling fashion. Here, we provide evidence that the layer organization and polysaccharide composition of the MSCs regulate the mucilage release efficiency like a peeling mechanism. Finally, we propose that flax MSCs may represent an excellent model for further investigations of mucilage biosynthesis and its release.

Processing of NMR and MS metabolomics data using chemometrics methods: a global tool for fungi biotransformation reactions monitoring.

INTRODUCTION: Biotransformation constitutes an important aspect of the drug discovery process, to mimic human metabolism of active principal ingredient but also to generate new chemical entities. Several microorganisms such as fungi are well adapted to transform drug, whether at the stage of screening or for large-scale production. OBJECTIVES: Due to the high chemical complexity of the biotransformation media, it seems attractive to develop new analytical strategies in order to guarantee an adequate monitoring and optimize the production of targeted metabolites or drug candidates. METHODS: The model designed for this purpose concerns the biotransformation of a potential histamine H3 antagonist (S38093) in order to produce phase I metabolites. MS, NMR and chemometrics tools were used to monitor biotransformation reactions. RESULTS: First, a screening of eleven filamentous fungi was carried out by UHPLC-UV-MS and principal component analysis to select the best candidates. Subsequently, MS (tR, m/z) and NMR (1H, JRES) fingerprints associated with Consensus OPLS-DA multiblock approach were used to better understand the bioreaction mechanisms in terms of nutrient consumption and hydroxylated metabolites production. Then an experimental design was set up to optimize the production conditions (pH, kinetic) of these target metabolites. CONCLUSION: This study demonstrates how NMR and MS acquisitions combined with chemometric methods offer an innovative analytical strategy to have a grasp of functionalization mechanisms, and identify metabolites and other compounds (amino acids, nutrients, etc.) in complex biotransformation mixtures.

A Deeper Investigation of Drug Degradation Mixtures Using a Combination of MS and NMR Data: Application to Indapamide.

A global approach that is based on a combination of mass spectrometry (MS) and nuclear magnetic resonance (NMR) data has been developed for a complete and rapid understanding of drug degradation mixtures. We proposed a workflow based on a sample preparation protocol that is compatible to MS and NMR, the selection of the most appropriate experiments for each technique, and the implementation of prediction software and multivariable analysis method for a better interpretation and correlation of MS and NMR spectra. We have demonstrated the efficient quantification of the remaining active pharmaceutical ingredient (API). The unambiguous characterization of degradation products (DPs) was reached while using the potential of ion mobility-mass spectrometry (IM-MS) for fragment ions filtering (HDMSE) and the implementation of two-dimensional (2D) NMR experiments with the non-uniform sampling (NUS) method. We have demonstrated the potential of quantitative NMR (qNMR) for the estimation of low level DPs. Finally, in order to simultaneously monitor multi-samples, the contribution of partial least squares (PLS) regression was evaluated. Our methodology was tested on three indapamide forced degradation conditions (acidic, basic, and oxidative) and it could be easily transposed in the drug development field to assist in the interpretation of complex mixtures (stability studies, impurities profiling, and biotransformation screening).

Optimizing 1D 1H-NMR profiling of plant samples for high throughput analysis: extract preparation, standardization, automation and spectra processing.

INTRODUCTION: Proton nuclear magnetic resonance spectroscopy (1H-NMR)-based metabolomic profiling has a range of applications in plant sciences. OBJECTIVES: The aim of the present work is to provide advice for minimizing uncontrolled variability in plant sample preparation before and during NMR metabolomic profiling, taking into account sample composition, including its specificity in terms of pH and paramagnetic ion concentrations, and NMR spectrometer performances. METHODS: An automation of spectrometer preparation routine standardization before NMR acquisition campaign was implemented and tested on three plant sample sets (extracts of durum wheat spikelet, Arabidopsis leaf and root, and flax leaf, root and stem). We performed 1H-NMR spectroscopy in three different sites on the wheat sample set utilizing instruments from two manufacturers with different probes and magnetic field strengths. The three collections of spectra were processed separately with the NMRProcFlow web tool using intelligent bucketing, and the resulting buckets were subjected to multivariate analysis. RESULTS: Comparability of large- (Arabidopsis) and medium-size (flax) datasets measured at 600 MHz and from the wheat sample set recorded at the three sites (400, 500 and 600 MHz) was exceptionally good in terms of spectral quality. The coefficient of variation of the full width at half maximum (FWHM) and the signal-to-noise ratio (S/N) of two selected peaks was comprised between 5 and 10% depending on the size of sample set and the spectrometer field. EDTA addition improved citrate and malate resonance patterns for wheat sample sets. A collection of 22 samples of wheat spikelet extracts was used as a proof of concept and showed that the data collected at the three sites on instruments of different field strengths and manufacturers yielded the same discrimination pattern of the biological groups. CONCLUSION: Standardization or automation of several steps from extract preparation to data reduction improves data quality for small to large collections of plant samples of different origins.

Flax phenolic compounds as inhibitors of lipid oxidation: Elucidation of their mechanisms of action.

Polyunsaturated fatty acids are particularly sensitive to the damages due to reactive oxygen species and lipid oxidation has been reported to be involved in the degradation of food as well as in the early stages of several diseases. Our objective was to study the mechanisms of action of flax (Linum usitatissimum) phenolic compounds to prevent membrane lipid oxidation. To do so, several biophysical techniques (oxidative stress, surface tension, fluorescence spectroscopy and HPLC) were used to investigate the ability of the compounds to prevent lipid oxidation and to interact with membranes. We evidenced a relationship between the structure and the antioxidant efficiency as aglycone compounds were significantly more efficient (p < 0.05) than glucoside compounds. In addition, our results revealed that aglycone lignans spontaneously penetrated the membrane contrary to aglycone hydroxycinnamic acids. To conclude, the comparison of the antioxidant efficiencies revealed that membrane inserted compounds better inhibited lipid oxidation than non-inserted compounds.

MuSeeQ, a novel supervised image analysis tool for the simultaneous phenotyping of the soluble mucilage and seed morphometric parameters.

BACKGROUND: The mucilage is a model to study the polysaccharide biosynthesis since it is produced in large amounts and composed of complex polymers. In addition, it is of great economic interest for its technical and nutritional value. A fast method for phenotyping the released mucilage and the seed morphometric parameters will be useful for fundamental, food, pharmaceutical and breeding researches. Current strategies to phenotype soluble mucilage are restricted to visual evaluations or are highly time-consuming. RESULTS: Here, we developed a high-throughput phenotyping method for the simultaneous measurement of the soluble mucilage content released on a gel and the seed morphometric parameters. Within this context, we combined a biochemical assay and an open-source computer-aided image analysis tool, MuSeeQ. The biochemical assay consists in sowing seeds on an agarose medium containing the dye toluidine blue O, which specifically stains the mucilage once it is released on the gel. The second part of MuSeeQ is a macro developed in ImageJ allowing to quickly extract and analyse 11 morphometric data of seeds and their respective released mucilages. As an example, MuSeeQ was applied on a flax recombinant inbred lines population (previously screened for fatty acids content.) and revealed significant correlations between the soluble mucilage shape and the concentration of some fatty acids, e.g. C16:0 and C18:2. Other fatty acids were also found to correlate with the seed shape parameters, e.g. C18:0 and C18:2. MuSeeQ was then showed to be used for the analysis of other myxospermous species, including Arabidopsis thaliana and Camelina sativa. CONCLUSIONS: MuSeeQ is a low-cost and user-friendly method which may be used by breeders and researchers for phenotyping simultaneously seeds of specific cultivars, natural variants or mutants and their respective soluble mucilage area released on a gel. The script of MuSeeQ and video tutorials are freely available at http://MuSeeQ.free.fr.

Insight into the Influence of Cultivar Type, Cultivation Year, and Site on the Lignans and Related Phenolic Profiles, and the Health-Promoting Antioxidant Potential of Flax (Linum usitatissimum L.) Seeds.

Flaxseeds are a functional food representing, by far, the richest natural grain source of lignans, and accumulate substantial amounts of other health beneficial phenolic compounds (i.e., flavonols, hydroxycinnamic acids). This specific accumulation pattern is related to their numerous beneficial effects on human health. However, to date, little data is available concerning the relative impact of genetic and geographic parameters on the phytochemical yield and composition. Here, the major influence of the cultivar over geographic parameters on the flaxseed phytochemical accumulation yield and composition is evidenced. The importance of genetic parameters on the lignan accumulation was further confirmed by gene expression analysis monitored by RT-qPCR. The corresponding antioxidant activity of these flaxseed extracts was evaluated, both in vitro, using ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC), and iron chelating assays, as well as in vivo, by monitoring the impact of UV-induced oxidative stress on the lipid membrane peroxidation of yeast cells. Our results, both the in vitro and in vivo studies, confirm that flaxseed extracts are an effective protector against oxidative stress. The results point out that secoisolariciresinol diglucoside, caffeic acid glucoside, and p-coumaric acid glucoside are the main contributors to the antioxidant capacity. Considering the health benefits of these compounds, the present study demonstrates that the flaxseed cultivar type could greatly influence the phytochemical intakes and, therefore, the associated biological activities. We recommend that this crucial parameter be considered in epidemiological studies dealing with flaxseeds.

In Vivo Use of 1D and 2D 1H NMR to Examine the Glycosylation of Scopoletin in Duboisia myoporoides Cell Suspensions.

Cell suspensions initiated from Duboisia myoporoides-a shrub belonging to the Solanaceae family and being a rich source of tropane alkaloids-previously showed their ability to glycosylate scopoletin into scopolin, which represent coumarins showing health benefits. To investigate the time course of this glycosylation reaction, an in vivo NMR approach was developed using a perfusion system in an 8-mm NMR tube and 1H NMR with 1D and 2D (TOCSY and NOESY) experiments. The time course of metabolic changes could therefore be followed without any labeling.

Investigation of Linum flavum (L.) Hairy Root Cultures for the Production of Anticancer Aryltetralin Lignans.

Linum flavum hairy root lines were established from hypocotyl pieces using Agrobacterium rhizogenes strains LBA 9402 and ATCC 15834. Both strains were effective for transformation but induction of hairy root phenotype was more stable with strain ATCC 15834. Whereas similar accumulation patterns were observed in podophyllotoxin-related compounds (6-methoxy-podophyllotoxin, podophyllotoxin and deoxypodophyllotoxin), significant quantitative variations were noted between root lines. The influence of culture medium and various treatments (hormone, elicitation and precursor feeding) were evaluated. The highest accumulation was obtained in Gamborg B5 medium. Treatment with methyl jasmonate, and feeding using ferulic acid increased the accumulation of aryltetralin lignans. These results point to the use of hairy root culture lines of Linum flavum as potential sources for these valuable metabolites as an alternative, or as a complement to Podophyllum collected from wild stands.

Protein biosynthesis, a target of sorafenib, interferes with the unfolded protein response (UPR) and ferroptosis in hepatocellular carcinoma cells.

Sorafenib is the first line treatment for advanced hepatocellular carcinoma (HCC). We explored its impact on the proteostasis of cancer cells, i.e. the processes that regulate the synthesis, maturation and turn-over of cellular proteins. We observed that sorafenib inhibits the production of the tumour marker alpha-foetoprotein (AFP) in two different HCC cell lines, an effect that correlated with a radical inhibition of protein biosynthesis. This effect was observed at clinically relevant concentrations of sorafenib and was not related to the effect of sorafenib on the transport of amino acids across the plasma membrane or the induction of the unfolded protein response (UPR). Instead, we observed that sorafenib inhibits translation initiation and the mechanistic target of rapamycin (mTOR) signaling cascade, as shown by the analysis of phosphorylation levels of the protein 4EBP1 (eukaryotic translation initiation factor 4E binding protein 1). We explored the consequences of this inhibition in HCC cells. We observed that overall sorafenib is a weak inducer of the UPR that can paradoxically prevent the UPR induced by tunicamycin. We also found no direct synergistic anticancer effect between sorafenib and various strategies that inhibit the UPR. In agreement with the possibility that translation inhibition might be an adaptive stress response in HCC cells, we noted that it protects cancer cell from ferroptosis, a form of oxidative necrosis. Our findings point to the modulation of protein biosynthesis and mTOR signaling as being important, yet complex determinants of the response of HCC cells to sorafenib.

Use of qNMR for speciation of flaxseeds (Linum usitatissimum) and quantification of cyanogenic glycosides.

This report describes a routine method taking less than 20 min to quantify cyanogenic glycosides such as linustatin and neolinustatin from flaxseeds (Linum usitatissimum L.) using 1H nuclear magnetic resonance. After manual dehulling, a higher linustatin content was shown in the almond fraction, while neolinustatin and total cyanogenic glycoside contents were significantly higher in hulls. Linustatin and neolinustatin were quantified in seven cultivars grown in two locations in three different years. Linustatin, neolinustatin, and total cyanogenic glycosides ranged between 91 and 267 mg/100 g, 78-272 mg/100 g, and 198-513 mg/100 g dry weight flaxseeds, respectively. NMR revealed differences of up to 70% between samples with standard deviation variations lower than 6%. This study shows that NMR is a very suitable tool to perform flaxseed varietal selection for the cyanogenic glycoside content. Graphical abstract qNMR can be used to perform flaxseed varietal selection for the cyanogenic glycoside content.