Phenolic syringyl end groups in 13C-enriched hardwoods detected and quantified by solid-state NMR.

While syringyl units are the most abundant monolignols in hardwood lignin, their phenolic (i.e. hydroxyl) end group concentration has not been measured. In two uniformly 13C-enriched young hardwoods, from beech and oak, the syringyl units were quantitatively investigated by advanced solid-state 13C NMR. Small signals of OH-terminated syringyl units were resolved in spectrally edited two-dimensional 13C-13C NMR spectra of the two hardwoods. Their distinct peak positions predicted based on literature data were validated via the abundant OH-terminated syringyl units in hydrolyzed 13C-beechwood. In a two-dimensional 13C-13C exchange spectrum with diagonal-ridge suppression, a well-resolved peak of phenolic syringyl units was observed at the characteristic C-H peak position of syringyl rings, without significant overlap from guaiacyl peaks. Accurate 13C chemical shifts of regular and end-group syringyl units were obtained. Through spectrally edited 2D NMR after 1H inversion recovery, phenols of condensed tannin complexed with arginine were carefully analyzed and shown to overlap minimally with signals from phenolic syringyl units. The local structure and resulting spin dynamics of ether (chain) and hydroxyl (end-group) syringyl units are nearly the same, enabling quantification by peak integration or deconvolution, which shows that phenolic syringyl end groups account for 2 ± 1 % of syringyl units in beechwood and 5 ± 2 % in oakwood. The observed low end-group concentration needs to be taken into account in realistic molecular models of hardwood lignin structure.

Recent Advancement in NMR Based Plant Metabolomics: Techniques, Tools, and Analytical Approaches.

Plant metabolomics, a rapidly advancing field within plant biology, is dedicated to comprehensively exploring the intricate array of small molecules in plant systems. This entails precisely gathering comprehensive chemical data, detecting numerous metabolites, and ensuring accurate molecular identification. Nuclear magnetic resonance (NMR) spectroscopy, with its detailed chemical insights, is crucial in obtaining metabolite profiles. Its widespread application spans various research disciplines, aiding in comprehending chemical reactions, kinetics, and molecule characterization. Biotechnological advancements have further expanded NMR's utility in metabolomics, particularly in identifying disease biomarkers across diverse fields such as agriculture, medicine, and pharmacology. This review covers the stages of NMR-based metabolomics, including historical aspects and limitations, with sample preparation, data acquisition, spectral processing, analysis, and their application parts.

Identifying ligands directly interacting with target protein in medicinal herbs by metabolomic analysis of T2-filtered HSQC spectra.

A protocol for efficiently identifying ligands directly interacting with a target protein in complex extracts of medicinal herbs was proposed by combining an adapted 2D perfect-echo Carr-Purcell-Meiboom-Gill heteronuclear single quantum correlation (PE-CPMG HSQC) spectrum with metabolomic analysis. PE-CPMG HSQC can suppress the signal interference from the target protein, allowing more accurate peak quantification than conventional HSQC. Inspired from untargeted metabolomics, regions of interest (ROIs) are constructed and quantified for the mixture or complex extract samples with and without a target protein, and then a binding index (BI) of each ROI is determined. ROIs or corresponding peaks significantly perturbed by the presence of the target protein (BI ≥1.5) are detected as differential features, and potential binding ligands identified from the differential features can be equated with bioactive markers associated with the 'treatment' of the target protein. Quantifying ROI can inclusively report the ligand bindings to a target protein in fast, intermediate and slow exchange regimes on nuclear magnetic resonance (NMR) time scale. The approach was successfully implemented and identified Angoroside C, Cinnamic acid and Harpagoside from the extract of Scrophularia ningpoensis Hemsl. as ligands binding to peroxisome proliferator-activated receptor γ. The proposed 2D NMR-based approach saves excess steps for sample processing and has a higher chance of detecting the weaker ligands in the complex extracts of medicinal herbs. We expect that this approach can be applied as an alternative to mining the potential ligands binding to a variety of target proteins from traditional Chinese medicines and herbal extracts.

Millettia dubia De Wild. (Fabaceae): Structural analysis of the oleanane-type glycosides and stimulation of the sweet taste receptors TAS1R2/TAS1R3.

From the root barks of a Central African tree Millettia dubia De Wild. (Fabaceae), ten previously undescribed oleanane-type glycosides were isolated by various chromatographic protocols. Their structures were elucidated by spectroscopic methods, mainly 2D NMR experiments and mass spectrometry, as mono- and bidesmosidic glycosides of mesembryanthemoidigenic acid, hederagenin and oleanolic acid. The stimulation of the sweet taste receptor TAS1R2/TAS1R3 by these glycosides was evaluated, and structure/activity relationships were proposed. Two of them showed an agonist effect on TAS1R2/TAS1R3.

Spectral Assignment in the [3 + 2] Cycloadditions of Methyl (2E)-3-(Acridin-4-yl)-prop-2-enoate and 4-[(E)-2-Phenylethenyl]acridin with Unstable Nitrile N-Oxides.

The investigation of cycloaddition reactions involving acridine-based dipolarophiles revealed distinct regioselectivity patterns influenced mainly by the electronic factor. Specifically, the reactions of methyl-(2E)-3-(acridin-4-yl)-prop-2-enoate and 4-[(1E)-2-phenylethenyl]acridine with unstable benzonitrile N-oxides were studied. For methyl-(2E)-3-(acridin-4-yl)-prop-2-enoate, the formation of two regioisomers favoured the 5-(acridin-4-yl)-4,5-dihydro-1,2-oxazole-4-carboxylates, with remarkable exclusivity in the case of 4-methoxybenzonitrile oxide. Conversely, 4-[(1E)-2-phenylethenyl]acridine displayed reversed regioselectivity, favouring products 4-[3-(substituted phenyl)-5-phenyl-4,5-dihydro-1,2-oxazol-4-yl]acridine. Subsequent hydrolysis of isolated methyl 5-(acridin-4-yl)-3-phenyl-4,5-dihydro-1,2-oxazole-4-carboxylates resulted in the production of carboxylic acids, with nearly complete conversion. During NMR measurements of carboxylic acids in CDCl3, decarboxylation was observed, indicating the formation of a new prochiral carbon centre C-4, further confirmed by a noticeable colour change. Overall, this investigation provides valuable insights into regioselectivity in cycloaddition reactions and subsequent transformations, suggesting potential applications across diverse scientific domains.

Extraction of chitin-glucan complex from shiitake (Lentinula edodes) fruiting bodies using natural deep eutectic solvents and its prebiotic potential.

Chitin-glucan complex (CGC) is an emerging novel prebiotic with numerous physiological activities in amelioration of clinical manifestations. In the present work, natural deep eutectic solvent (NADES), ultrasonication, and submerged fermentation using probiotic microorganisms were deployed for the extraction of CGC from Shiitake fruiting bodies. CGC obtained through non-ultrasonication assisted fermentation employing Lactiplantibacillus plantarum exhibited maximum polysaccharide yield (27.86 ± 0.82 % w/w). However, based on antioxidant potential, NADES combination of urea: glycerol (1:1 M ratio) was selected for further characterization. The rheological behavior of CGC under optimized conditions showed shear thinning property in both 0.1 M NaCl and salt-free solution. FTIR, 1H-(1D), and 2D 1H1H Homonuclear NMR spectra displayed distinctive patterns associated with ß-glycosidic linkage and ß-d-glucopyranose sugar moiety. XRD profiles of CGC exhibited characteristic peaks at 2θ = 23°, 25°, and 28° with corresponding hkl values of (220), (101), and (130) lattice planes, respectively. Enhanced radical scavenging activities were noticed due to the triple helical structure and anionic nature of CGC. CGC exhibited potential prebiotic activity (prebiotic score 118-134 %) and short chain fatty acids liberation (maximum 9.99 ± 0.41 mM by Lactobacillus delbrueckii). Simulated static in-vitro digestion demonstrated that CGC withstands acidic environment of gastric phase, which indicated its suitability for use as a prebiotic in nutraceutical-enriched food products.

Identifying Anti-NSCLC Bioactive Compounds in Scutellaria via 2D NMR-Based Metabolomic Analysis of Pharmacologically Classified Crude Extracts.

We presented a strategy utilizing 2D NMR-based metabolomic analysis of crude extracts, categorized by different pharmacological activities, to rapidly identify the primary bioactive components of TCM. It was applied to identify the potential bioactive components from Scutellaria crude extracts that exhibit anti-non-small cell lung cancer (anti-NSCLC) activity. Four Scutellaria species were chosen as the study subjects because of their close phylogenetic relationship, but their crude extracts exhibit significantly different anti-NSCLC activity. Cell proliferation assay was used to assess the anti-NSCLC activity of four species of Scutellaria. 1H-13C HSQC spectra were acquired for the chemical profiling of these crude extracts. Based on the pharmacological classification (PCA, OPLS-DA and univariate hypothesis test) were performed to identify the bioactive constituents in Scutellaria associated with the anti-NSCLC activity. As a result, three compounds, baicalein, wogonin and scutellarin were identified as bioactive compounds. The anti-NSCLC activity of the three potential active compounds were further confirmed via cell proliferation assay. The mechanism of the anti-NSCLC activity by these active constituents was further explored via flow cytometry and western blot analyses. This study demonstrated 2D NMR-based metabolomic analysis of pharmacologically classified crude extracts to be an efficient approach to the identification of active components of herbal medicine.

Tritordeum, a hybrid cereal with a highly tricin-enriched lignin.

The lignin from tritordeum straw, a hybrid cereal from crossbreeding of durum wheat and wild barley, was isolated and chemically characterized. Its composition and structure were studied by analytical pyrolysis (Py-GC/MS), nuclear magnetic resonance spectroscopy (NMR), Derivatization Followed by Reductive Cleavage (DFRC) method, and gel permeation chromatography (GPC). The data revealed an enrichment of guaiacyl (G) units (H:G:S of 3:61:36), which had a significant impact on the distribution of inter-unit linkages. The predominant linkages were the ß-O-4' alkyl-aryl ethers (78 % of all linkages), with substantial proportions of condensed linkages such as phenylcoumarans (11 %), resinols (4 %), spirodienones (4 %), and dibenzodioxocins (2 %). Moreover, DFRC revealed that tridordeum straw lignin was partly acylated at the γ-OH with both acetates and p-coumarates. Acetates were principally attached to G-units, whereas p-coumarates were predominantly attached to S-units. Furthermore, and more importantly, tritordeum lignin incorporates remarkable amounts of a valuable flavone, tricin, exceeding 30 g per kilogram of straw. Given the diverse industrial applications associated with this high-value molecule, tritordeum straw emerges as a promising and sustainable resource for its extraction.

The structural characteristic of acidic-degraded polysaccharides from seeds of Plantago ovata Forssk and its biological activity.

In this study, a response surface methodology (RSM) was used to determine the best combination for acid degradation parameters to reduce the viscosity of Plantago ovata Forssk seed polysaccharide (POFP). Then, the two major homogeneous polysaccharides (AH-POFP1 and AH-POFP3) were obtained by DEAE-650 M and Sephadex G-100 column chromatography. The apparent structure of the main fraction AH-POFP1 was characterized by SEM, TG and XRD, and the linkage of AH-POFP1 was determined by a combination of partial acidolysis, Smith's degradation, methylation analysis and 2D NMR analysis. Structural analysis showed that AH-POFP1 was mainly composed of xylose, with a molecular weight of 618.1 kDa, and had a backbone of 1 â†’ 4-linked Xylp, as well as branches of T-linked Xylp, 1 â†’ 4-linked Xylp attached to the O-2 position. The antioxidant activity assays showed that the both AH-POFP1 and AH-POFP3 possess strong scavenging radical ability. Moreover, AH-POFP1 inhibits the secretion of pro-inflammatory factors, and promotes the secretion of anti-inflammatory factors, thereby exerting anti-inflammatory effects. These findings may help to guide future applications of Plantago ovata Forssk in the fields of food, health care, and pharmacy.

Two new triterpenoids from the stems of Euphorbia royleana.

Two new triterpenoids, namely 24-methylene-5,24-dien-19(10→9)-abeo-8α,9ß,10α-eupha-3ß-ol (1) and 24-methyl-5,23-dien-19(10→9)-abeo-8α,9ß,10α-eupha-3ß-ol (2) were isolated from the stems of Euphorbia royleana, together with three known analogs. The structures of the new compounds were elucidated by extensive 1H NMR,13C NMR, HSQC, HMBC, 1H-1H COSY, ROESY and HR-MS spectroscopic analyses.

Capacity of Aqueous Solutions of the Ionic Liquid 1-Ethyl-3-methylimidazolium Acetate to Partially Depolymerize Lignin at Ambient Temperature and Pressure.

Lignin is a very attractive and abundant biopolymer with the potential to be a biorenewable source of a large number of value-added organic chemicals. The current state-of-the-art methods fail to provide efficient valorization of lignin in this regard without the involvement of harsh conditions and auxiliary substances that compromise the overall sustainability of the proposed processes. Making an original approach from the set of mildest temperature and pressure conditions, this work identifies and explores the capacity of an aqueous solution of the nonvolatile ionic liquid 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) to partially depolymerize technical lignin (Indulin AT) by means of a treatment consisting in the simple contact at ambient temperature and pressure. Among a considerable number of valuable phenolic molecules that were identified in the resulting fluid, vanillin (yield of about 3 g/kg) and guaiacol (yield of about 1 g/kg) were the monophenolic compounds obtained in a higher concentration. The properties of the post-treatment solids recovered remain similar to those of the original lignin, although with a relatively lower abundance of guaiacyl units (in agreement with the generation of guaiacyl-derived phenolic molecules, such as vanillin and guaiacol). The assistance of the treatment with UV irradiation in the presence of nanoparticle catalysts does not lead to an improvement in the yields of phenolic compounds.

A multiplex approach of MS, 1D-, and 2D-NMR metabolomics in plant ontogeny: A case study on Clusia minor L. organs (leaf, flower, fruit, and seed).

INTRODUCTION: The genus Clusia L. is mostly recognised for the production of prenylated benzophenones and tocotrienol derivatives. OBJECTIVES: The objective of this study was to map metabolome variation within Clusia minor organs at different developmental stages. MATERIAL AND METHODS: In total 15 organs/stages (leaf, flower, fruit, and seed) were analysed by UPLC-MS and 1H- and heteronuclear multiple-bond correlation (HMBC)-NMR-based metabolomics. RESULTS: This work led to the assignment of 46 metabolites, belonging to organic acids(1), sugars(2) phenolic acids(1), flavonoids(3) prenylated xanthones(1) benzophenones(4) and tocotrienols(2). Multivariate data analyses explained the variability and classification of samples, highlighting chemical markers that discriminate each organ/stage. Leaves were found to be rich in 5-hydroxy-8-methyltocotrienol (8.5 µg/mg f.w.), while flowers were abundant in the polyprenylated benzophenone nemorosone with maximum level detected in the fully mature flower bud (43 µg/mg f.w.). Nemorosone and 5-hydroxy tocotrienoloic acid were isolated from FL6 for full structural characterisation. This is the first report of the NMR assignments of 5-hydroxy tocotrienoloic acid, and its maximum level was detected in the mature fruit at 50 µg/mg f.w. Seeds as typical storage organ were rich in sugars and omega-6 fatty acids. CONCLUSION: To the best of our knowledge, this is the first report on a comparative 1D-/2D-NMR approach to assess compositional differences in ontogeny studies compared with LC-MS exemplified by Clusia organs. Results derived from this study provide better understanding of the stages at which maximal production of natural compounds occur and elucidate in which developmental stages the enzymes responsible for the production of such metabolites are preferentially expressed.

6aH-Benzo[α]fluorene: NMR evidence of the unexpected product of the reaction of butyryl chloride with 1,2-diphenylacetylene.

The reaction of butyryl chloride with ethynylbenzene in the presence of AlCl3 afforded a mixture of the Z/E-isomers of 1-chloro-2-phenylhex-1-en-3-one. 1,2-Diphenylethyne under these conditions gave a novel polycarbocycle core, 6aH-benzo[a]fluorene. The chemical structure of 11-chloro-5,6-diphenyl-6a-propyl-6aH-benzo[a]fluorene was established by means of IE-MS, 1 H, 13 C NMR, COSY, HSQC, HMBC, and 2D INADEQUATE technique.

Type I Lipoteichoic Acid (LTA) Purification by Hydrophobic Interaction Chromatography and Structural Analysis by 2D Nuclear Magnetic Resonance (NMR) Spectroscopy.

Type I lipoteichoic acid (LTA) is a glycerol phosphate polymer found in the cell envelope of diverse Gram-positive bacteria. The glycerol phosphate backbone is often further decorated with D-alanine and/or sugar residues. Here, we provide details of a 1-butanol extraction and purification method of type I LTA by hydrophobic interaction chromatography. The protocol has been adapted from methods originally described by Fischer et al. (Eur J Biochem 133:523-530, 1983) and further optimized by Morath et al. (J Exp Med 193:393-397, 2001). We also present information on a 2D nuclear magnetic resonance (NMR) analysis method to gain chemical and structural information of the purified LTA material.

Determination of De Novo Suberin-Lignin Ferulate Deposition in Xylem Tissue Upon Vascular Pathogen Attack.

Plant vascular pathogens use different ways to reach the xylem vessels and cause devastating diseases in plants. Resistant and tolerant plants have evolved various defense mechanisms against vascular pathogens. Inducible physico-chemical structures, such as the formation of tyloses and wall reinforcements with phenolic polymers, are very effective barriers that confine the pathogen and prevent colonization. Here, we use a combination of classical histochemistry along with bright-field and fluorescence microscopy and two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy to visualize and characterize wall reinforcements containing phenolic wall polymers, namely, lignin, ferulates, and suberin, which occur in different xylem vasculature in response to pathogen attack.

Marsogenin: a new pregnane ester from Marsdenia tenacissima.

A new pregnane, 12ß-O-acetyl-20(S)-O-N-methylanthranilyl-3ß, 8ß, 14ß, 17α-tetrol pregn-5-ene (12ß-O-acetyl-20(S)-O-N-methylanthranilyl-sarcostin) have been isolated from Marsdenia tenacissima (family: Asclepediaceae). The structure of new pregnane was elucidated by using spectroscopic techniques,1H,13C NMR, HMBC, HSQC, COSY and TOCSY and ESI-MS Mass spectrometry.

Investigating Betrixaban Maleate drug degradation profiles, isolation and characterization of unknown degradation products by mass-triggered preparative HPLC, HRMS, and NMR.

Betrixaban Maleate, a novel oral, once-daily factor Xa inhibitor drug substance, was subjected to stress testing under a wide range of degradation conditions, including acidic hydrolysis, alkaline hydrolysis, oxidative, thermal, and photolytic, to determine its inherent stability. The drug was biodegradable in acidic and alkaline environments, and three new degradation products were identified. Two degraded products are formed in an acidic environment, while the third is in alkaline conditions. The three degradants were identified using UPLC-ESI/MS and isolated using mass-triggered preparative HPLC, and their structures were unambiguously elucidated using HRMS and 2D NMR techniques. Based on spectral and chromatographic data, it was firmly proven that these distinct degradation products were the betrixaban chemical's hydrolysis components. The formation of the degradants has been hypothesized through several possible mechanisms.

Application of 2D NMR Spectroscopy in Combination with Chemometric Tools for Classification of Natural Lignins.

Lignin is considered a promising renewable source of valuable chemical compounds and a feedstock for the production of various materials. Its suitability for certain directions of processing is determined by the chemical structure of its macromolecules. Its formation depends on botanical origin, isolation procedure and other factors. Due to the complexity of the chemical composition, revealing the structural differences between lignins of various origins is a challenging task and requires the use of the most informative methods for obtaining and processing data. In the present study, a combination of two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy and multivariate analysis of heteronuclear single quantum coherence (HSQC) spectra is proposed. Principal component analysis and hierarchical cluster analysis techniques demonstrated the possibility to effectively classify lignins at the level of belonging to classes and families of plants, and in some cases individual species, with an error rate for data classification of 2.3%. The reverse transformation of loading plots into the corresponding HSQC loading spectra allowed for structural information to be obtained about the latent components of lignins and their structural fragments (biomarkers) responsible for certain differences. As a result of the analysis of 34 coniferous, deciduous, and herbaceous lignins, 10 groups of key substructures were established. In addition to syringyl, guaiacyl, and p-hydroxyphenyl monomeric units, they include various terminal substructures: dihydroconiferyl alcohol, balanopholin, cinnamic acids, and tricin. It was shown that, in some cases, the substructures formed during the partial destruction of biopolymer macromolecules also have a significant effect on the classification of lignins of various origins.

Preparation of glucuronides using liver microsomes and their characterization by 1D/2D NMR spectroscopy and mass spectrometry: Application to fentanyl metabolites.

A simple, low-cost method for preparing glucuronic acid-conjugated metabolites was developed using fentanyl, a potent synthetic opioid, as a model drug. Five glucuronic acid-conjugated metabolites of fentanyl were measured in the culture medium of fresh human hepatocytes incubated with fentanyl. These glucuronides were also formed by incubation of their corresponding substrates (e.g., 4'-hydroxy-fentanyl and ß-hydroxy-fentanyl) with uridine 5'-diphosphoglucuronic acid and human liver microsomes (HLM). Experiments using liver microsomes of several animals revealed that significant species differences exist in the glucuronide formation patterns; fentanyl glucuronide was only formed in HLM, and 4'-hydroxy-fentanyl glucuronide was formed much more in rat liver microsomes (RLM) than HLM and dog liver microsomes. Furthermore, surprisingly, HLM and RLM showed opposite substrate selectivity for the enantiomers of ß-hydroxy-fentanyl. Submilligram amounts of three of these metabolites, namely, 4'-hydroxy-fentanyl glucuronide and two glucuronides of ß-hydroxy-fentanyl, were prepared by using HLM or RLM. The products were readily purified with a reversed-phase/anion-exchange mixed-mode solid-phase extraction cartridge, and then, their chemical structures were confirmed by 1D/2D nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry data. In addition, the products were quantitated by quantitative NMR, and the yields were 3.6-69%.

A Modified 1H-NMR Quantification Method of Ephedrine Alkaloids in Ephedrae Herba Samples.

A previous 1H-NMR method allowed the quantification of ephedrine alkaloids; however, there were some disadvantages. The cyclized derivatives resulted from the impurities of diethyl ether were identified and benzene was selected as the better extraction solvent. The locations of ephedrine alkaloids were confirmed with 2D NMR. Therefore, a specific 1H-NMR method has been modified for the quantification of ephedrine alkaloids. Accordingly, twenty Ephedrae Herba samples could be classified into three classes: (I) E. sinica-like species; (II) E. intermedia-like species; (III) others (lower alkaloid contents). The results indicated that ephedrine and pseudoephedrine are the major alkaloids in Ephedra plants, but the concentrations vary greatly determined by the plant species and the collection locations.