Activation of Vegetable Oils by Reaction with Maleic Anhydride as a Renewable Source in Chemical Processes: New Experimental and Computational NMR Evidence.

Vegetable oils are bio-based and sustainable starting materials that can be used to develop chemicals for industrial processes. In this study, the functionalization of three vegetable oils (grape, hemp, and linseed) with maleic anhydride was carried out either by conventional heating or microwave activation to obtain products that, after further reactions, can enhance the water dispersion of oils for industrial applications. To identify the most abundant derivatives formed, trans-3-octene, methyl oleate, and ethyl linoleate were reacted as reference systems. A detailed NMR study, supported by computational evidence, allowed for the identification of the species formed in the reaction of trans-3-octene with maleic anhydride. The signals in the 1H NMR spectra of the alkenyl succinic anhydride (ASA) moieties bound to the organic chains were clearly identified. The reactions achieved by conventional heating were carried out for 5 h at 200 °C, resulting in similar or lower amounts of ASA units/g of oil with respect to the reactions performed by microwave activation, which, however, induced a higher viscosity of the samples.

NMR Quantification of Carbohydrates in Complex Mixtures. A Challenge on Honey.

The knowledge of carbohydrate composition is greatly important to determine the properties of natural matrices such as foodstuff and food ingredients. However, because of the structural similarity and the multiple isomeric forms of carbohydrates in solution, their analysis is often a complex task. Here we propose an NMR analytical procedure based on highly selective chemical shift filters followed by TOCSY, which allows us to acquire specific background-free signals for each sugar. The method was tested on raw honey samples dissolved in water with no other pretreatment. In total, 22 sugars typically found in honey were quantified: 4 monosaccharides (glucose, fructose, mannose, rhamnose), 11 disaccharides (sucrose, trehalose, turanose, maltose, maltulose, palatinose, melibiose and melezitose, isomaltose, gentiobiose nigerose, and kojibiose), and 7 trisaccharides (raffinose, isomaltotriose, erlose, melezitose, maltotriose, panose, and 1-kestose). Satisfactory results in terms of limit of quantification (0.03-0.4 g/100g honey), precision (% RSD: 0.99-4.03), trueness (bias % 0.4-4.2), and recovery (97-104%) were obtained. An accurate control of the instrumental temperature and of the sample pH endows an optimal chemical shift reproducibility, making the procedure amenable to automation and suitable to routine analysis. While validated on honey, which is one of the most complex natural matrices in terms of saccharides composition, this innovative approach can be easily transferred to other natural matrices.

Performance Assessment in Fingerprinting and Multi Component Quantitative NMR Analyses.

An interlaboratory comparison (ILC) was organized with the aim to set up quality control indicators suitable for multicomponent quantitative analysis by nuclear magnetic resonance (NMR) spectroscopy. A total of 36 NMR data sets (corresponding to 1260 NMR spectra) were produced by 30 participants using 34 NMR spectrometers. The calibration line method was chosen for the quantification of a five-component model mixture. Results show that quantitative NMR is a robust quantification tool and that 26 out of 36 data sets resulted in statistically equivalent calibration lines for all considered NMR signals. The performance of each laboratory was assessed by means of a new performance index (named Qp-score) which is related to the difference between the experimental and the consensus values of the slope of the calibration lines. Laboratories endowed with a Qp-score falling within the suitable acceptability range are qualified to produce NMR spectra that can be considered statistically equivalent in terms of relative intensities of the signals. In addition, the specific response of nuclei to the experimental excitation/relaxation conditions was addressed by means of the parameter named NR. NR is related to the difference between the theoretical and the consensus slopes of the calibration lines and is specific for each signal produced by a well-defined set of acquisition parameters.

NMR sugar-profile in genuine grape must.

The wine market has always faced the problem of fraud, including the addition of exogenous sugar solutions to grape musts to increase the final alcohol content. Since in some countries the practice of chaptalization is prohibited (except by adding concentrated must) it is necessary to broaden the analytical techniques that allow the identification of this type of fraud. The aim of this study was to define an NMR-based sugar profile of genuine grape must to set concentration limits for each sugar as parameters of authenticity. Glucose, fructose, together with eleven minor sugars were quantified in 82 genuine Italian grape musts, developing an analytical procedure based on highly selective chemical shift filters followed by TOCSY. Alongside the characteristic myo- and scyllo-inositol, significant contents of mannose, galactose, and trehalose were also found. Otherwise, maltose, rhamnose, arabinose, sucrose and lactose are present in lower concentrations and show great concentration variability.

Automatic NMR-based protocol for assessment of honey authenticity.

1H NMR analysis of organic extracts of honey is a powerful technique to confirm its botanical origin, thanks to the presence of signals that are specific to each floral typology. Similarly, signals from bee metabolites provide an important tool to verify honey entomological origin. Here, we present a method for honey screening that does not require any detailed analysis of the NMR spectrum for the detection and quantification of such markers. Our approach is based on the measurement of two spectral parameters, named entomological factor (EF) and aromatic factor (AF), calculated by integration of well-defined regions of the NMR spectrum. The values of EF and AF can reveal direct or indirect dilution of honey with sugar syrups. This method was tested on honeys of different floral origins and could identify all adulterated samples previously recognized by official techniques. Notably, several samples found compliant by official methods were proven non-genuine by the proposed approach.

Extraction and mass spectrometry identification of a major peach allergen Pru p 1.

BACKGROUND: Peach allergy can be caused by the allergen Pru p 1. This occurs by cross-reactivity with the homologous birch pollen allergen Bet v 1. However, the direct identification of Pru p 1 as an immunoglobulin E (IgE)-binding protein extracted from peach fruit has never been reported. RESULTS: Phosphate-buffered saline (PBS) and phenol extractions were applied to solubilise the proteins from peach peel and pulp, and IgE immunoblotting with sera of individual peach-allergic patients was used to detect the potential allergens. Most of the patients showed binding to an 18 kDa band in IgE immunoblotting performed with the phenolic extracts of peach peel and pulp, but not when the PBS extracts were used. Mass spectrometry of the 18 kDa spot excised from a two-dimensional electrophoretic gel showed this protein to correspond to the peach allergen Pru p 1. CONCLUSION: Phenol extraction was necessary to detect by IgE immunoblotting a major peach allergen, which showed very low extractability with PBS, indicating the appropriateness of adopting different extraction procedures to identify plant allergens. The 18 kDa peach protein was definitively identified as the Bet v 1-homologous peach allergen Pru p 1.

Quantification of polyols in sugar-free foodstuffs by qNMR.

We present a qNMR method for the determination of low calories sweeteners (erythritol, mannitol, maltitol, sorbitol, isomalt and xylitol) in sugar-free foodstuff. The structural similarities of these compounds determine often a severe spectral overlap that hampers their quantification via conventional 1D and 2D NMR spectra. This problem is here overcome by exploiting the resolving capabilities of the CSSF-TOCSY experiment, allowing the quantification of all six polyols, with satisfactory results in terms of LoQ (2.8-7.4 mg/L for xylitol, mannitol, sorbitol, 15 mg/L for erythritol, 38 mg/L for maltitol and 91 mg/L for isomalt), precision (RSD% 0.40-4.03), trueness (bias% 0.15-4.81), and recovery (98-104%). Polyol's quantification in different sugar-free confectionary products was performed after a simple water extraction without any additional sample treatment. While these results demonstrate the robustness of the proposed method for polyols quantification in low calories foods, its applicability can be further extended to other food matrices or biofluids.

Grape Berry Responses to Sequential Flooding and Heatwave Events: A Physiological, Transcriptional, and Metabolic Overview.

Grapevine cultivation, such as the whole horticulture, is currently challenged by several factors, among which the extreme weather events occurring under the climate change scenario are the most relevant. Within this context, the present study aims at characterizing at the berry level the physiological response of Vitis vinifera cv. Sauvignon Blanc to sequential stresses simulated under a semi-controlled environment: flooding at bud-break followed by multiple summer stress (drought plus heatwave) occurring at pre-vèraison. Transcriptomic and metabolomic assessments were performed through RNASeq and NMR, respectively. A comprehensive hormone profiling was also carried out. Results pointed out a different response to the heatwave in the two situations. Flooding caused a developmental advance, determining a different physiological background in the berry, thus affecting its response to the summer stress at both transcriptional levels, with the upregulation of genes involved in oxidative stress responses, and metabolic level, with the increase in osmoprotectants, such as proline and other amino acids. In conclusion, sequential stress, including a flooding event at bud-break followed by a summer heatwave, may impact phenological development and berry ripening, with possible consequences on berry and wine quality. A berry physiological model is presented that may support the development of sustainable vineyard management solutions to improve the water use efficiency and adaptation capacity of actual viticultural systems to future scenarios.

Structure elucidation of the dye Acid Red 131: complete (1)H, (13)C and (15)N NMR data assignment.

The chemistry of dyes and pigments is relevant to the textile industry, because of the importance to establish the best conditions for the finishing process and to understand the interactions among various compounds to yield the correct hue and nuances. For this reason, the molecular structure of a monoazo acid dye, C.I. Acid Red 131, was elucidated and characterized by homo- and hetero-nuclear NMR, MS, IR and UV spectroscopy techniques.

Floral origin modulates the content of a lipid marker in Apis mellifera honey.

The compound DAGE (DiAcyl Glyceryl Ether, 1-stearyl-2,3-dioleoyl glycerol), present in Apis mellifera honey, is a lipidic entomological marker secreted by the salivary glands of worker bees. Its content was determined by NMR, analyzing the organic extracts of a number of Italian honeys of different floral typology. We have found that the DAGE content is related to the botanical origin of honey. This dependence on floral typology was further confirmed by a linear correlation (R2 > 0.83) observed between the content of DAGE and the enzymatic activity of invertase and diastase in honey. Also these enzymes originate from bee salivary secretions and their concentrations in honey are known to depend on the floral source. DAGE content appears to be a sensitive parameter to some forms of honey manipulations, as indicated by the results of artificial bee-feeding experiments. This suggests its possible use as indicator of honey authenticity.

NMR Metabolite Profiles of the Bivalve Mollusc Mytilus galloprovincialis Before and After Immune Stimulation With Vibrio splendidus.

The hemolymph metabolome of Mytilus galloprovincialis injected with live Vibrio splendidus bacteria was analyzed by 1H-NMR spectrometry. Changes in spectral hemolymph profiles were already detected after mussel acclimation (3 days at 18 or 25 °C). A significant decrease of succinic acid was accompanied by an increase of most free amino acids, mytilitol, and, to a smaller degree, osmolytes. These metabolic changes are consistent with effective osmoregulation, and the restart of aerobic respiration after the functional anaerobiosis occurred during transport. The injection of Vibrio splendidus in mussels acclimated at 18°C caused a significant decrease of several amino acids, sugars, and unassigned chemical species, more pronounced at 24 than at 12 h postinjection. Correlation heatmaps indicated dynamic metabolic adjustments and the relevance of protein turnover in maintaining the homeostasis during the response to stressful stimuli. This study confirms NMR-based metabolomics as a feasible analytical approach complementary to other omics techniques in the investigation of the functional mussel responses to environmental challenges.

Hypericum triquetrifolium and H. neurocalycinum as Sources of Antioxidants and Multi-Target Bioactive Compounds: A Comprehensive Characterization Combining In Vitro Bioassays and Integrated NMR and LC-MS Characterization by Using a Multivariate Approach.

Hypericum triquetrifolium and H. neurocalycinum were evaluated for their phytochemical content and in vitro bioactivity. NMR analyses were performed on the methanol extract of the aerial parts of H. triquetrifolium to establish the main classes of phytoconstituents. Then, LC-DAD-MSn analyses were performed in order to compare the composition of aerial parts and roots extracts of both Hypericum species, obtained using either methanol or water as solvents. Results, processed using multivariate data analysis, showed a significantly higher phenolic content of methanol extracts compared to water extracts, while minor qualitative differences were observed between the two. Distinctive flavonoid and PAC patterns were observed for H. triquetrifolium and H. neurocalycinum, and specific compounds were exclusively detected in one or the other species. Specifically, the phloroglucinols 7-epiclusianone, hyperfirin and hyperforin were present only in H. neurocalycinum, while hyperforin was detected only in H. triquetrifolium. Extracts were assayed using different in vitro tests to evaluate their antioxidant properties and their inhibitory activity against several enzymes, showing significant antioxidant and metal chelating activities. Furthermore, inhibitory properties against acetylcholinesterase, butyrylcholinesterase and tyrosinase were observed. Multivariate approaches were used to correlate biological data with the phytochemical composition of the different extracts. The results, showing positive correlations between specific chemical constituents and the measured bioactivities, represent preliminary data that could guide future studies aimed at isolating bioactive constituents from H. neurocalycinum and H. triquetrifolium for further pharmacological evaluations.

Total synthesis, characterization, and conformational analysis of the naturally occurring hexadecapeptide integramide A and a diastereomer.

Integramide A is a 16-amino acid peptide inhibitor of the enzyme HIV-1 integrase. We have recently reported that the absolute stereochemistries of the dipeptide sequence near the C terminus are L-Iva(14)-D-Iva(15). Herein, we describe the syntheses of the natural compound and its D-Iva(14)-L-Iva(15) diastereomer, and the results of their chromatographic/mass spectrometric analyses. We present the conformational analysis of the two compounds and some of their synthetic intermediates of different main-chain length in the crystal state (by X-ray diffraction) and in solvents of different polarities (using circular dichroism, FTIR absorption, and 2D NMR techniques). These data shed light on the mechanism of inhibition of HIV-1 integrase, which is an important target for anti-HIV therapy.

Role of the guanidine group in the N-terminal fragment of PTH(1-11).

A series of PTH hybrids containing a diamine [NH(2)(CH(2))(n)NH(2); n = 4, 5, 6] in the C-terminal position was synthesized based on the H-Aib-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har-NH(2) (Har = homoarginine) template. The compounds were pharmacologically characterized at PTH1R receptors for agonist activity.

Synthesis and structural studies of new analogues of PTH(1-11) containing Cα-tetra-substituted amino acids in position 8.

The N-terminal 1-34 fragment of parathyroid hormone (PTH) is fully active in vitro and in vivo and it can reproduce all biological responses characteristic of the native intact PTH. Recently, analogues of PTH(1-11) fragments with helicity-enhancing substitutions have been demonstrated to yield potent analogues of PTH(1-34). The work describes the synthesis, biological activity and structure of analogues of the best modified PTH sequence H-Aib-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har-NH2 (I). In particular, the effect of the Ala/Aib substitution at positions 1 and 3 as well as of the replacement of Nle in position 8 with D-Nle, L-(αMe)-Nle and D-(αMe)-Nle was studied. The resulting peptides were characterized structurally by CD spectroscopy, solution NMR and MD, and in vitro for activity with respect to the cognate receptor, parathyroid hormone receptor.

Configurational assignment of D- and L-isovalines in intact, natural, and synthetic peptides by 2D-NMR spectroscopy.

We investigated, by means of 2D-NMR, the naturally occurring and chemically synthesized 16-mer integramides A and B, which belong to a group of bioactive, fungal peptides (peptaibiotics), that are characterized by an abundance of Aib as well as D- and L-Iva residues. The chemical shifts of the C(alpha)-alkyl groups in the Iva enantiomers depend on the alpha-C-atom configuration and on the helical screw sense of the peptides, the latter determined by CD. In the full-length, right-handed helical integramides, as well as in the partial sequences exploited for their total chemical syntheses, the gamma-Me H-atoms of the Et side chain of the D-Iva residues located near the C-terminus are significantly more shielded (delta<0.90 ppm) than those of the L-Iva residues (delta>0.95 ppm). The opposite behavior is observed for the left-handed, synthetic, intermediate Z-Aib-L-Hyp-L-Iva(14)-D-Iva(15)-O(t)Bu. Here, the gamma-Me H-atoms of L-Iva(14) are more shielded (0.838 ppm) than those of D-Iva(15) (0.905 ppm). The chemical-shift difference between the diastereotopic beta-CH(2) H-atoms of the Iva side chains in the right-handed helical peptides is much larger for D-Iva than for L-Iva. For D-Iva(14/15), the values range from 0.38 to 0.63 ppm, whereas, for D-Iva(1), the value is in the range of 0.26-0.31 ppm. In each case, the difference is always larger for the d-Iva than for the l-Iva residues (which is always

NMR carbohydrate profile in tracing acacia honey authenticity.

The sugar profile in honey can be used as a fingerprint to confirm the authenticity or reveal the adulteration of the product by sweetener addition. In this work, we have accurately determined the profile of 20 minor saccharides in a set of 46 European acacia honeys using a recently proposed NMR approach based on the CSSF-TOCSY experiment. Comparison of this reference profile with the sugar composition of several Chinese honey samples of the same declared botanical origin has revealed important differences. A detailed analysis of the saccharide profile of these Chinese honeys suggests product adulteration by overfeeding bee colonies with industrial sugars syrups during the main nectar flow period.

Limited genotypic and geographic variability of 16-O-methylated diterpene content in Coffea arabica green beans.

The acknowledged marker of Robusta coffee, 16-O-methylcafestol (16-OMC), can be quantified by NMR as a mixture with 16-O-methylkahweol (16-OMK), which accounts for approximately 10% of the mixture. In the present study, we detected and quantified 16-O-methylated diterpenes (16-OMD) in 248 samples of green Coffea arabica beans by NMR. We did not observe any differences between genotypes introgressed by chromosomal fragments of Robusta and non-introgressed genotypes. Environmental effects suggesting a possible protective role of 16-OMD for adaptation, as well as genotypic effects that support a high heritability of this trait were observed. Altogether, our data confirmed the presence of 16-OMD in green Arabica at a level approximately 1.5% that of a typical Robusta, endorsing the validity of 16-OMD as a marker for the presence of Robusta.

NMR quantification of trace components in complex matrices by band-selective excitation with adiabatic pulses.

The use of band-selective excitation with adiabatic pulses to rapidly obtain NMR spectra of trace components in the presence of strong signals is described, along with qualitative and quantitative examples from food matrices like olive oil and honey.

NMR assessment of European acacia honey origin and composition of EU-blend based on geographical floral markers.

A geographical discrimination of honey is an important prerequisite for quality and authenticity control. Here, we present a method based on an NMR-metabolomic analysis of organic extracts for a geographical discrimination of commercial European acacia honeys found on the Italian market. All 234 analysed samples show the characteristic 1H NMR profile of acacia (Robinia pseudoacacia) honey. However, a PLS2-DA model revealed variations among production zones allowing the successful geographical differentiation with a 100% of overall correct classification rate. Moreover, a PLS2 model is able to predict the proportions in binary blends of Italian and Eastern European acacia honeys. The geographical distinction and the possibility to characterize the content of blends derive from different markers content originating from minor nectar contributions of the acacia-accompanying flora.