Plant polygalacturonase structures specify enzyme dynamics and processivities to fine-tune cell wall pectins.

Polygalacturonases (PGs) fine-tune pectins to modulate cell wall chemistry and mechanics, impacting plant development. The large number of PGs encoded in plant genomes leads to questions on the diversity and specificity of distinct isozymes. Herein, we report the crystal structures of 2 Arabidopsis thaliana PGs, POLYGALACTURONASE LATERAL ROOT (PGLR), and ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE2 (ADPG2), which are coexpressed during root development. We first determined the amino acid variations and steric clashes that explain the absence of inhibition of the plant PGs by endogenous PG-inhibiting proteins (PGIPs). Although their beta helix folds are highly similar, PGLR and ADPG2 subsites in the substrate binding groove are occupied by divergent amino acids. By combining molecular dynamic simulations, analysis of enzyme kinetics, and hydrolysis products, we showed that these structural differences translated into distinct enzyme-substrate dynamics and enzyme processivities: ADPG2 showed greater substrate fluctuations with hydrolysis products, oligogalacturonides (OGs), with a degree of polymerization (DP) of ≤4, while the DP of OGs generated by PGLR was between 5 and 9. Using the Arabidopsis root as a developmental model, exogenous application of purified enzymes showed that the highly processive ADPG2 had major effects on both root cell elongation and cell adhesion. This work highlights the importance of PG processivity on pectin degradation regulating plant development.

NMR and LC-MS-based metabolomics to investigate the efficacy of a commercial bio stimulant for the treatment of wheat (Triticum aestivum).

INTRODUCTION: Bio stimulants are substances and/or microorganisms that are used to improve plant growth and crop yields by modulating physiological processes and metabolism of plants. While research has primarily focused on the broad effects of bio stimulants in crops, understanding their cellular and molecular influences in plants, using metabolomic analysis, could elucidate their effectiveness and offer possibilities for fine-tuning their application. One such bio stimulant containing galacturonic acid as elicitor is used in agriculture to improve wheat vigor and strengthen resistance to lodging. OBJECTIVE: However, whether a metabolic response is evolved by plants treated with this bio stimulant and the manner in which the latter might regulate plant metabolism have not been studied. METHOD: Therefore, the present study used 1H-NMR and LC-MS to assess changes in primary and secondary metabolites in the roots, stems, and leaves of wheat (Triticum aestivum) treated with the bio stimulant. Orthogonal partial least squares discriminant analysis effectively distinguished between treated and control samples, confirming a metabolic response to treatment in the roots, stems, and leaves of wheat. RESULTS: Fold-change analysis indicated that treatment with the bio stimulation solution appeared to increase the levels of hydroxycinnamic acid amides, lignin, and flavonoid metabolism in different plant parts, potentially promoting root growth, implantation, and developmental cell wall maturation and lignification. CONCLUSION: These results demonstrate how non-targeted metabolomic approaches can be utilized to investigate and monitor the effects of new agroecological solutions based on systemic responses.

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 1H-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 1H-NMR metabolomics as an essential tool for the analysis of the drought tolerance characteristics of T. cacao.

Consecutive action of two BAHD acyltransferases promotes tetracoumaroyl spermine accumulation in chicory.

Fully substituted phenolamide accumulation in the pollen coat of Eudicotyledons is a conserved evolutionary chemical trait. Interestingly, spermidine derivatives are replaced by spermine derivatives as the main phenolamide accumulated in the Asteraceae family. Here, we show that the full substitution of spermine in chicory (Cichorium intybus) requires the successive action of two enzymes, that is spermidine hydroxycinnamoyl transferase-like proteins 1 and 2 (CiSHT1 and CiSHT2), two members of the BAHD enzyme family. Deletion of these genes in chicory using CRISPR/Cas9 gene editing technology evidenced that CiSHT2 catalyzes the first N-acylation steps, whereas CiSHT1 fulfills the substitution to give rise to tetracoumaroyl spermine. Additional experiments using Nicotiana benthamiana confirmed these findings. Expression of CiSHT2 alone promoted partially substituted spermine accumulation, and coexpression of CiSHT2 and CiSHT1 promoted synthesis and accumulation of the fully substituted spermine. Structural characterization of the main product of CiSHT2 using nuclear magnetic resonance revealed that CiSHT2 preferentially catalyzed N-acylation of secondary amines to form N5,N10-dicoumaroyl spermine, whereas CiSHT1 used this substrate to synthesize tetracoumaroyl spermine. We showed that spermine availability may be a key determinant toward preferential accumulation of spermine derivatives over spermidine derivatives in chicory. Our results reveal a subfunctionalization among the spermidine hydroxycinnamoyl transferase that was accompanied by a modification of free polyamine metabolism that has resulted in the accumulation of this new phenolamide in chicory and most probably in all Asteraceae. Finally, genetically engineered yeast (Saccharomyces cerevisiae) was shown to be a promising host platform to produce these compounds.

Extraction, Structural Characterization, and In Vivo Anti-Inflammatory Effect of Alginate from Cystoseira crinita (Desf.) Borry Harvested in the Bulgarian Black Sea.

The aim of this study was to identify the chemical composition and sequential structure of alginate isolated from C. crinita harvested in the Bulgarian Black Sea, as well as its effects in histamine-induced paw inflammation in rats. The serum levels of TNF-α, IL-1ß, IL-6, and IL-10 in rats with systemic inflammation, and the levels of TNF-α in a model of acute peritonitis in rats were also investigated. The structural characterization of the polysaccharide was obtained by FTIR, SEC-MALS, and 1H NMR. The extracted alginate had an M/G ratio of 1.018, a molecular weight of 7.31 × 104 g/mol, and a polydispersity index of 1.38. C. crinita alginate in doses of 25 and 100 mg/kg showed well-defined anti-inflammatory activity in the model of paw edema. A significant decrease in serum levels of IL-1ß was observed only in animals treated with C. crinita alginate in a dose of 25 mg/kg bw. The concentrations of TNF-α and IL-6 in serum were significantly reduced in rats treated with both doses of the polysaccharide, but no statistical significance was observed in the levels of the anti-inflammatory cytokine IL-10. A single dose of alginate did not significantly alter the levels of the pro-inflammatory cytokine TNF-α in the peritoneal fluid of rats with a model of peritonitis.

Antibacterial Activity and Untargeted Metabolomics Profiling of Acalypha arvensis Poepp.

The search for potent antimicrobial compounds is critical in the face of growing antibiotic resistance. This study explores Acalypha arvensis Poepp. (A. arvensis), a Caribbean plant traditionally used for disease treatment. The dried plant powder was subjected to successive extractions using different solvents: hexane (F1), dichloromethane (F2), methanol (F3), a 50:50 mixture of methanol and water (F4), and water (F5). Additionally, a parallel extraction was conducted using a 50:50 mixture of methanol and chloroform (F6). All the fractions were evaluated for their antimicrobial activity, and the F6 fraction was characterized using untargeted metabolomics using SPME-GC×GC-TOFMS. The extracts of A. arvensis F3, F4, and F5 showed antibacterial activity against Staphylococcus aureus ATCC 25923 (5 mg/mL), MRSA BA22038 (5 mg/mL), and Pseudomonas aeruginosa ATCC 27853 (10 mg/mL), and fraction F6 showed antibacterial activity against Staphylococcus aureus ATCC 29213 (2 mg/mL), Escherichia coli ATCC 25922 (20 mg/mL), Pseudomonas aeruginosa ATCC 27853 (10 mg/mL), Enterococcus faecalis ATCC 29212 (10 mg/mL), Staphylococcus aureus 024 (2 mg/mL), and Staphylococcus aureus 003 (2 mg/mL). Metabolomic analysis of F6 revealed 2861 peaks with 58 identified compounds through SPME and 3654 peaks with 29 identified compounds through derivatization. The compounds included methyl ester fatty acids, ethyl ester fatty acids, terpenes, ketones, sugars, amino acids, and fatty acids. This study represents the first exploration of A. arvensis metabolomics and its antimicrobial potential, providing valuable insights for plant classification, phytochemical research, and drug discovery.

Chemical extraction, characterization, and inspection of the antimicrobial and antibiofilm activities of shrimp chitosan against foodborne fungi and bacteria.

Nowadays, the exploitation of biopolymers in the industrial sector has become a trend. Chitosan is considered one of the most investigated biopolymers due to its abundance and antibacterial, antifungal, and antibiofilm activities. In this work, chitosan was chemically extracted from shrimp shells. Solutions of HCl 1 M, NaOH 4 M, and NaOH 15 M were used for the demineralization, deproteinization, and deacetylation process, respectively. The utilized methods of characterization (FTIR, 1 H NMR, 13 C NMR, and SEC-MALS) revealed that the obtained chitosan has a moderate degree of deacetylation and low molecular weight (DDA = 74% and Mw = 72.14 kDa). The microdilution method and inoculation of solid medium were carried out to assess the antibiofilm action of chitosan against Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus hirae, Escherichia coli, Rhizopus sp., and Aspergillus sp. which are known as foodborne microorganisms. Results showed that the produced chitosan at 1 g/L inhibits between 63.44 and 99.75% of the microbial biofilm depending on the tested strains. These promising results confirm the potential deployment of the obtained chitosan in the food industry as a replacement for synthetic antimicrobial agents.

Structural Characterization and In Vivo Anti-Inflammatory Activity of Fucoidan from Cystoseira crinita (Desf.) Borry.

The aim of this study was to evaluate the effects of fucoidan isolated from C. crinita on histamine-induced paw inflammation in rats, and on the serum levels of TNF-α, IL-1ß, IL-6, and IL-10 in rats during systemic inflammation response. The levels of TNF-α in a model of acute peritonitis in rats were also investigated. The isolated crude fucoidan was identified as a sulfated xylogalactofucan with high, medium, and low molecular weight fractions and a content of fucose of 39.74%, xylose of 20.75%, and galactose of 15.51%. Fucoidan from C. crinita showed better anti-inflammatory effects in the rat paw edema model, and this effect was present during all stages of the experiment. When compared to controls, a commercial fucoidan from F. vesiculosus, the results also displayed anti-inflammatory activity on the 60th, 90th, and 120th minute of the experiment. A significant decrease in serum levels of IL-1ß in rats treated with both doses of C. crinita fucoidan was observed in comparison to controls, whereas TNF-α concentrations were reduced only in the group treated with fucoidan from C. crinita at the dose of 25 mg/kg bw. In the model of carrageenan-induced peritonitis, we observed a tendency of decrease in the levels of the pro-inflammatory cytokine TNF-α in peritoneal fluid after a single dose of C. crinita fucoidan, but this did not reach the statistical significance margin. Single doses of C. crinita fucoidan did not alter serum levels of the anti-inflammatory cytokine IL-10 in animals with lipopolysaccharide-induced systemic inflammation.

Profiling of Essential Oils from the Leaves of Pistacia lentiscus Collected in the Algerian Region of Tizi-Ouzou: Evidence of Chemical Variations Associated with Climatic Contrasts between Littoral and Mountain Samples.

Leaves of Pistacia lentiscus were collected from two Algerian sites in the mountains and the littoral of the Tizi-Ouzou region. The harvest was conducted in four consecutive seasons on the same selected set of trees. Essential oils (EOs) were extracted by hydrodistillation; then, they were analyzed by gas chromatography coupled mass spectrometry (GC-MS). Forty-seven constituents could be detected and quantified, including α-pinene (2-13%), ß-caryophyllene (8-25%), ß-myrcene (0.3-19%), bornyl acetate (0.8-7%), δ-cadinene (3-8%), bisabolol (1-9%), ß-pinene (0.9-7%), caryophyllene oxide (4-9%), and α-cadinol (3-11%). Antioxidant (AOx) activities of the EOs were assessed by ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,2'-azino-bis (3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) assays. Significant differences in EO composition and AOx activities appeared dependent on the season and the site. Variations of AOx activities were significant for the FRAP and ABTS tests but not for DPPH. Characterization of the leaf fatty acyl (FA) profiles was performed by GC-MS. Variability appeared according to season and altitude. Polyunsaturated fatty acids levels were high (27-55%) at the coldest date and place. The levels of linolenic acyl in the leaves were significantly correlated with bisabolol levels in the EOs (Spearman's correlation coefficient: 0.818). Such results will be useful for the sustainable local valorization of wild P. lentiscus. These data also open new routes for further studies on terpenoid biosynthesis using correlation networks and fluxomic approaches.

Secondary Metabolism Rearrangements in Linum usitatissimum L. after Biostimulation of Roots with COS Oligosaccharides from Fungal Cell Wall.

In vitro culture of flax (Linum usitatissimum L.) was exposed to chitosan oligosaccharides (COS) in order to investigate the effects on the growth and secondary metabolites content in roots and shoots. COS are fragments of chitosan released from the fungal cell wall during plant-pathogen interactions. They can be perceived by the plant as pathogen-associated signals, mediating local and systemic innate immune responses. In the present study, we report a novel COS oligosaccharide fraction with a degree of polymerization (DP) range of 2-10, which was produced from fungal chitosan by a thermal degradation method and purified by an alcohol-precipitation process. COS was dissolved in hydroponic medium at two different concentrations (250 and 500 mg/L) and applied to the roots of growing flax seedlings. Our observations indicated that the growth of roots and shoots decreased markedly in COS-treated flax seedlings compared to the control. In addition, the results of a metabolomics analysis showed that COS treatment induced the accumulation of (neo)lignans locally at roots, flavones luteolin C-glycosides, and chlorogenic acid in systemic responses in the shoots of flax seedlings. These phenolic compounds have been previously reported to exhibit a strong antioxidant and antimicrobial activities. COS oligosaccharides, under the conditions applied in this study (high dose treatment with a much longer exposure time), can be used to indirectly trigger metabolic response modifications in planta, especially secondary metabolism, because during fungal pathogen attack, COS oligosaccharides are among the signals exchanged between the pathogen and host plant.

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.

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.

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.

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.

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).

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.

A BAHD neofunctionalization promotes tetrahydroxycinnamoyl spermine accumulation in the pollen coat of the Asteraceae family.

In eudicotyledons, accumulation of trihydroxycinnamoyl spermidine that is restricted to the pollen wall constitutes an evolutionary conserved trait. However, the role of this compound, which is synthetized by the BAHD enzyme spermidine hydroxycinnamoyl transferase (SHT), is still a matter of debate. Here, we show that this particular phenolamide is replaced by tetrahydroxycinnamoyl spermine in the pollen coat of the Asteraceae. Phylogenetic analyses combined with quantitative RT-PCR experiments allowed the identification of two homologous genes from Cichorium intybus (chicory) putatively involved in its metabolism. In vitro biochemical characterization of the two enzymes, named CiSHT1 and CiSHT2, confirmed the capability of recombinant proteins to synthesize spermine as well as spermidine derivatives. The wild-type metabolic phenotype was partially restored in an Arabidopsis sht mutant expressing CiSHT2. Strikingly, the transgenic plants also accumulated spermine derivatives that were absent in the wild-type. Overexpression of CiSHT2 in chicory hairy roots led to the accumulation of spermine derivatives, confirming its in vivo function. Complementary sequence analyses revealed the presence of an amino acid motif typical of the SHTs among the BAHD enzyme family. Our results highlight a recent neofunctionalization among the SHTs that has promoted the emergence of new phenolamides in the Asteraceae, which could potentially have contributed to the evolutionary success of this family.

Selective Synthesis of 3-O-Palmitoyl-Silybin, a New-to-Nature Flavonolignan with Increased Protective Action against Oxidative Damages in Lipophilic Media.

A selective acylation protocol using cerium chloride (CeCl3) as catalyst was applied to functionalize silybinin (1), a natural antioxidant flavonolignan from milk thistle fruit, in order to increase its solubility in lipophilic media while retaining its strong antioxidant activity. The selective esterification of 1 at the position 3-OH with a palmitate acyl chain leading to the formation of the 3-O-palmitoyl-silybin (2) was confirmed by both mass spectroscopy (MS) and nuclear magnetic resonance (NMR) analyses. The antioxidant activity of 1 was at least retained and even increased with the CUPRAC assay designed to estimate the antioxidant activity of both hydrophilic and lipophilic compounds. Finally, the 3-O-palmitoylation of 1, resulting in the formation of 2, also increased its anti-lipoperoxidant activity (i.e., inhibition of conjugated diene production) in two different lipophilic media (bulk oil and o/w emulsion) subjected to accelerated storage test.

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.

Investigation of the Lignan Content in Extracts from Linum, Callitris and Juniperus Species in Relation to Their In Vitro Antiproliferative Activities.

Podophyllotoxin, a lignan still extracted from the rhizomes of Podophyllum hexandrum (Berberidaceae), is the starting molecule for the semisynthesis of widely used anticancer drugs such as etoposide. However, this source is threatened by the over-collection of P. hexandrum. Plants belonging to the Linaceae and Cupressaceae families could be attractive alternative sources with species that contain the lignan podophyllotoxin or its precursors and derivatives. Wild flax species, such as Linum flavum, as well as some Juniperus and Callitris species were investigated for their lignan content, and the in vitro antiproliferative capacity of their extracts was assayed on four tumor cell lines. Some of the lignans were detected by LC-HRMS for the first time in these extracts.In addition, lignans purified from these plants and compounds semisynthesized from commercially available podophyllotoxin were tested in terms of their in vitro antiproliferative activity. The genus Juniperus was the most promising given its in vitro antiproliferative effects, which were also observed with extracts from L. flavum and Callitris species.The in vitro antiproliferative effect of the plant extracts studied here appears to correlate well with the contents of the aryltetralin lignan podophyllotoxin and its glycoside as well as with deoxypodophyllotoxin and 6-methoxypodophyllotoxin. The strongest correlation between the lignan content of the extracts and the antiproliferative activity was observed for 6-methoxypodophyllotoxin. Regarding the possibility of producing large renewable amounts of 6-methoxypodophyllotoxin, this molecule could be of interest to produce new anticancer drugs and to bypass the resistance mechanisms against podophyllotoxin-derived drugs.