Probing the interaction of polyphenols with lipid bilayers by solid-state NMR spectroscopy

Polyphenols are bioactive natural products that appear to act against a wide range of pathologies. Mechanisms of activity have not been established, but recent studies have suggested that some polyphenols bind to membranes. This study examined the interaction between lipid bilayers and three structurally diverse polyphenols. It was hypothesized that features of the polyphenols such as polarity, molecular size, molecular geometry, and number and arrangement of phenol hydroxyl groups would determine the tendency to interact with the bilayer. The examined compounds included a mixed polyphenol, (-)-epigallocatechin gallate (EGCg); a proanthocyanidin trimer comprising catechin-(4→8)-catechin-(4→8)-catechin (cat?; and a hydrolyzable tannin, 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (PGG). These polyphenols were incorporated at different levels into 2H-labeled 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) multilamellar vesicles (MLVs). 31P and 2H solid-state NMR experiments were performed to determine the dynamics of the headgroup region and the hydrophobic acyl chain region of the lipid bilayer upon addition of polyphenols. The chemical shift anisotropy (CSA) width of the 31P NMR spectra decreased upon addition of polyphenols. Addition of PGG induces a dramatic reduction on the CSA width compared with the control lipid bilayer sample, whereas addition of cat? barely reduces the CSA width. The 2H quadupolar splitting of the lipids also decreased upon addition of polyphenols. At the same concentration, PGG substantially reduced the quadrupolar splitting, whereas cat? barely reduced it when compared with the control sample. From a calculation of the order parameters of the acyl chain region of the lipid bilayer, it was concluded that the hydrophobic part of the lipid bilayer was perturbed by PGG, whereas cat? did not cause large perturbations. The data suggest that the polarity of the polyphenols affects the interaction between tannins and membranes. The interactions may relate to the biological activities of polyphenols.     

Applications of high-resolution solid-state NMR spectroscopy in food science

The principal applications of high-resolution solid-state NMR spectroscopy, in the field of food science, are reviewed, after a short general introduction, mainly focusing on the potential of these investigations, which are, today, routine tools for resolving technological problems. Selected examples of the applications in the field of food science of high-resolution solid-state NMR spectroscopy both in (13)C and in (1)H NMR particularly illustrative of the results obtainable are reported in some detail.     

Characterization of lycopene nanoparticles combining solid-state and suspended-state NMR spectroscopy

The core-shell structure of lycopene micronizates can be verified by employing a combination of solid-state and suspended-state NMR spectroscopy. The type of molecular aggregation of carotenoid nanoparticles can be clearly determined from their characteristic fingerprint pattern in the solid-state NMR spectra.     

Use of NMR spectroscopy to study transformations of nitrogenous substances during incubation of peat

Abstract. Samples of peat were incubated with ¹⁵N-labelled ammonium sulphate, urea, wheat straw and glycine and divided into six size fractions of solid components and a water-soluble fraction. The fractions were analysed by NMR spectroscopy to study the formation of humic substances and rind how fertilizer nitrogen is immobilized in peaty soils. After six months' incubation about half of the ammonium sulphate nitrogen was still present as ammonium in the soluble fraction, the urea had been entirely metabolized to ammonium and various organic compounds, about half the straw had been decomposed to ammonium and amino acid or peptide materials, and most of the glycine had been transformed to ammonium, amide and aliphatic amine.     

Chemical structures of manure from conventional and phytase transgenic pigs investigated by advanced solid-state NMR spectroscopy

Nonpoint phosphorus (P) pollution from animal manure is becoming a serious global problem. The current solution for the swine industry includes the enzyme phytase as a component in oil meal and cereal grain-based swine diets. A long-term approach is the production of transgenic phytase pigs that express phytase in the salivary glands and secrete it in the saliva. This study provides a detailed comparison of chemical structures of manure from conventional pigs and transgenic pigs that express phytase under growing and finishing phases using new solid-state NMR techniques. Spectral editing techniques and quantitative NMR techniques were used to identify and quantify specific functional groups. Two-dimensional (1)H- (13)C heteronuclear correlation NMR was used to detect their connectivity. Manure from conventional and transgenic pigs had similar peptide, carbohydrate, and fatty acid components, while those from transgenic pigs contained more carbohydrates and fewer nonpolar alkyls. There was no consistent effect from diets with or without supplemental phosphate or growth stages.     

Organic N forms of a subtropical Acrisol under no-till cropping systems as assessed by acid hydrolysis and solid-state NMR spectroscopy

This study was conducted to investigate the influence of land-use systems (grassland and cropland) and of long-term no-till cropping systems [bare soil, oat/maize (O/M), pigeon pea+maize (P+M)] on the composition of organic N forms in a subtropical Acrisol. Soil samples collected from the 0- to 2.5-cm layer in the study area (Eldorado do Sul RS, Brazil) were submitted to acid hydrolysis and cross-polarization magic angle spinning (CPMAS) ¹⁵N and ¹³C nuclear magnetic resonance (NMR) spectroscopies. The legume-based cropping system P+M contained the highest contents of non-hydrolysable C and N, hydrolysable C and N, amino acid N and hydrolysed unknown N. The relative proportion of non-hydrolysable N was higher in bare soil (30.0%) and decreased incrementally in other treatments based on the total C and N contents. The amino acid N corresponded to an average of 37.2% of total N, and was not affected by land use and no-till cropping systems. The non-hydrolysable residue contained lower O-alkyl and higher aromatic C concentrations, as revealed by CPMAS ¹³C NMR spectroscopy, and higher C:N ratio than the bulk soil. No differences in the bulk soil organic matter composition could be detected among treatments, according to CPMAS ¹³C and ¹⁵N NMR spectra. In the non-hydrolysable fraction, grassland showed a lower concentration of aromatic and a higher concentration of alkyl C than other treatments. From CPMAS ¹⁵N NMR spectra, it could be concluded that amide N from peptide structures are the main organic N constituent. Amide structures are possibly protected through encapsulation into hydrophobic sites of organic matter and through organomineral interaction.     

Similarities in chemical composition of soil organic matter across a millennia-old paddy soil chronosequence as revealed by advanced solid-state NMR spectroscopy

This study examined the chemical composition of soil organic matter (SOM) along a 2,000-year paddy soil chronosequence in eastern China by use of advanced solid-state nuclear magnetic resonance (NMR) spectroscopy as well as Fourier transform infrared spectroscopy (FTIR), aiming to identify changes in the chemical composition of SOM over a millennium timescale. The results showed that soil organic carbon concentration in the surface soil reached a steady state after 100 years of rice (Oryza sativa L.)–wheat (Triticum sp.) cropping on coastal tidal flats. The ¹³C NMR spectra and fractions of structural groups or components of the whole SOM samples differed little along the chronosequence, suggesting a similar chemical composition in SOM samples regardless of the duration of rice cultivation. The FTIR spectral pattern and relative intensities of some resolved functional groups or components of whole SOM were also similar along the soil chronosequence. The similarities in chemical composition of SOM can be attributed to the rice–wheat cropping system, in which SOM has undergone ongoing turnover under periodical fresh plant material input and wet–dry cropping alternation, leading to a similar chemical composition of bulk SOM.     

Effect of peat type and ph on breakdown of peat using fourier transform infrared spectroscopy

Abstract

Information on breakdown of peats as evidenced by shrinkage during cropping is generally lacking. The objective of this investigation was to study the breakdown of peat of various degrees of decomposition, effect of pH on breakdown and to relate the compositional changes during breakdown using Fourier Transform Infrared Spectroscopy (FTIR). Incubation studies were used in this investigation. Peat with a higher level of decomposition was less susceptible to breakdown. The pH had a major effect on breakdown with high pH leading to increased rate of breakdown. Lignin content of the peats was somewhat related to breakdown of the peats. The breakdown was also strongly correlated to the changes in the ratio of FTIR spectra of the start and to the end of the incubation particularly the 1600/1060 ratio. The 1600 spectra in mostly lignin 1060 spectra are mostly carbohydrate. There was relative enrichment of 1600 spectra in relation 1060 spectra. Other FTIR spectra ratio changes were also significantly correlated with breakdown. The FTIR technique has the potential to predict breakdown of peats.     

Metabolic characterization of Brassica rapa leaves by NMR spectroscopy

The Brassica has been intensively studied due to the nutritional and beneficial effects. However, many species, varieties, and cultivars of this genus and the resulting large metabolic variation have been obstacles for systematic research of the plant. In order to overcome the problems posed by the biological variation, the metabolomic analysis of various cultivars of Brassica rapa was performed by NMR spectroscopy combined with multivariate data analysis. Discriminating metabolites in different cultivars and development stages were elucidated by diverse 2D-NMR techniques after sorting out different significant signals using (1)H NMR measurements and principal component analysis. Among the elucidated metabolites, several organic and amino acids, carbohydrates, adenine, indole acetic acid (IAA), phenylpropanoids, flavonoids, and glucosinolates were found to be the metabolites contributing to the differentiation between cultivars and age of Brassica rapa. On the basis of these results, the distribution of plant metabolites among different cultivars and development stages of B. rapa is discussed.     

Quantitative solid-state 13C NMR spectroscopy of organic matter fractions in lowland rice soils

Spin counting on solid‐state ¹³C cross‐polarization (CP) nuclear magnetic resonance (NMR) spectra of two humic fractions isolated from tropical lowland soils showed that only 32–81% of potential ¹³C NMR signal was detected. The observability of ¹³C NMR signal (C_obs) was higher in the mobile humic acid (MHA) than in the calcium humate (CaHA) fraction, and increased with increasing intensity of irrigated rice cropping. NMR observability appeared to be related to the nature of the organic carbon, with phenol‐ and methoxyl‐rich samples having the higher values of C_obs. The Bloch decay (BD) technique provided more quantitatively reliable ¹³C NMR spectra, as evidenced by values of C_obs in the range 91–100% for seven of the eight humic fractions studied. The BD spectra contained considerably more aryl and carbonyl signal, and less O–alkyl and alkyl signal, with the greatest differences between CP and BD spectra observed for the samples with low C_obs(CP). The causes of low CP observability were investigated using the spectral editing technique RESTORE ( RE storation of S pectra via T _CH and T O ne R ho (T_1ρH) E diting). Rapid T_1ρH relaxation was found to be primarily responsible for the under‐representation of carbonyl carbon, whereas inefficient cross‐polarization was primarily responsible for the under‐representation of aryl carbon in CP spectra. Proton NMR relaxation rates T_1H and T_1ρH were found to correlate with other NMR properties and also with cropping management. Non‐uniform rates of T_1H relaxation in two of the CaHA fractions enabled the generation of proton spin relaxation editing subspectra.     

Analysis of technical lignins by two- and three-dimensional NMR spectroscopy

Modern multidimensional NMR spectroscopic methods were applied to investigate the effects of kraft pulping and oxygen delignification on lignin side-chain structures. In addition to the two-dimensional HSQC measurements, the three-dimensional HSQC-TOCSY technique was utilized to elucidate the (1)H-(1)H and (1)H-(13)C correlations of individual spin systems and thus indicate a certain lignin side-chain structure. Unlike earlier, nonlabeled samples were used for 3D measurements. According to 2D and 3D NMR spectra, most of the structures identified in milled wood lignin (MWL) are still present in technical lignins after kraft pulping and oxygen delignification. Although the main reaction during kraft pulping is the cleavage of beta-O-4 linkages, these structures are still left in spent liquor lignin as well as in residual lignin. The amount of coniferyl alcohol and dihydroconiferyl alcohol end groups, as well as some unidentified saturated end groups, is higher in technical lignins than in MWL. Contrary to our earlier observations, no diphenylmethane structures were observed in any technical lignins. Vinyl aryl ether structures could not be detected in technical lignins either.     

A comprehensive approach for quantitative lignin characterization by NMR spectroscopy

A detailed approach for the quantification of different lignin structures in milled wood lignin (MWL) has been suggested using a combination of NMR techniques. 1H-13C heteronuclear multiple quantum coherence and quantitative 13C NMR of nonacetylated and acetylated spruce MWL have been found to have a synergetic effect, resulting in significant progress in the characterization of lignin moieties by NMR. About 80% of side chain moieties, such as different beta-O-4, dibenzodioxocin, phenylcoumaran, pinoresinol, and others, have been identified on the structural level. The presence of appreciable amounts of alpha-O-alkyl and gamma-O-alkyl ethers has been suggested. Although the quantification of various condensed moieties was less precise than for side chain structures, reliable information can be obtained. Comparison of the calculated results with known databases on spruce MWL structure shows that the suggested approach is rather informative and comparable with the information obtained from the combination of various wet chemistry methods. Discrepancies between the results obtained in this study and those previously published are discussed.     

Decomposition of plant tissue submerged in an extremely acidic mining lake sediment: phenolic CuO-oxidation products and solid-state C NMR spectroscopy

In extremely acidic mining sediments of the Lusatian mining district, the alkalinisation process relies on organic C, which can serve as electron donor for microbially induced sulfate reduction. Plant material of the pioneer plant Juncus bulbosus is an important organic matter source in lake sediments. Therefore, decomposition of the plant tissue was assessed during the exposure of litterbags for 30 months in the 0-5 cm layer of waterlogged mining sediments, which have a pH between 2.5 and 3. The ash free dry weight (AFDW) and elemental content of the plant tissue were recorded several times during the exposure. Changes in chemical structure were analyzed by solid-state ¹³C cross polarization magic angle spinning nuclear magnetic resonance (CPMAS NMR) spectroscopy and the lignin component characterized by wet-chemical CuO oxidation. The AFDW accounted for about 34% of initial biomass after field exposure for 30 months. Mass loss of biomass occurred in two phases with decomposition rates varying between 30 and 430 mg AFDW d⁻¹. The mass loss increased considerably after 5-7 months when litterbags were invaded by fresh J. bulbosus plants. With respect to higher mass loss, ¹³C CPMAS NMR spectroscopy, showed slight changes of the bulk chemical composition after 11 months, indicating that microorganisms present in the sediments or in the rhizosphere degrade plant material as a whole, rather than selectively. During the second phase from about 11 months until the end of the exposure period, contribution of O-alkyl C most probably assignable to easily degradable polysaccharides decreased. In contrast, the contribution of alkyl, aromatic and carboxyl C increased. CuO oxidation showed that the lignin component of J. bulbosus is degraded oxidatively during field exposure. Our results indicate that the exposed plant material is decomposed in the sediment due to changes in sediment conditions that followed plant invasion of the litterbags. It is suggested that the rhizosphere of J. bulbosus by its influence on the redox potential, pH and the microbial component plays a crucial role in organic matter degradation in acidic mining sediments.     

Quantitative differences in evaluating soil humic substances by liquid- and solid-state C-NMR spectroscopy

We compared the quantitative responses of liquid-state (LS) and solid-state (CPMAS) ¹³C-NMR spectroscopy of four different soil humic substances. The intensities of signals for the alkyl carbons (0–40 ppm) were significantly larger in CPMAS than in LS spectra. This difference is in agreement with the pseudo-micellar model of the conformational nature of humic substances. By this view, the hydrophobic interactions holding together the heterogeneous molecules of humic micelles inhibit the molecular motions of the alkyl carbons, thereby enhancing the spin-lattice relaxation times and consequently lowering the sensitivity of liquid-state NMR. Conversely, regardless of their position in the humic conformation, a better estimation of the number of alkyl carbons can be obtained by CPMAS-NMR because of the cross-polarization of hydrogen nuclei in CH₂ and CH₃ groups. The intensity of the 40–110 ppm region is also slightly lower in LS than in CPMAS-NMR spectra, despite the hydrophilicity of the oxidized and peptidic carbons resonating in this chemical shift interval. Their molecular motion may also be reduced by either the formation of intra- and inter-molecular hydrogen bondings due to poorly acidic hydroxyl groups of saccharides, or the degree of conformational rigidity that a pseudo-micellar arrangement confers even to hydrophilic domains. The higher content of aromatic carbons (110–160 ppm) found in the LS spectra was attributed partly to the high degree of substitution of the aromatic ring that slows down cross-polarization in CPMAS experiments and partly to the relative overestimation of this region by LS-NMR due to a lack of signal in the aliphatic interval. The slightly lower content of carboxyl carbons estimated in CPMAS spectra as compared to LS spectra was also attributed to slow cross-polarization. This work shows that the combined use of both NMR techniques is profitable in conformational analysis of humic substances and of dissolved organic matter in general.     

Determination of phospholipids in olive oil by 31P NMR spectroscopy

A nondestructive analytical method based on NMR spectroscopy was developed for the determination of phospholipids in olive oil. The phospholipids extracted from virgin olive oil with a mixture of ethanol/water (2:1 v/v) were identified and quantified by high resolution (31)P NMR spectroscopy. The main phospholipids found in olive oil were phosphatidic acid, lyso-phosphatidic acid, and phosphatidylinositol. Validation of the (31)P NMR methodology for quantitative analysis of phospholipids in olive oil was performed. Sensitivity was satisfactory with detection limits of 0.25-1.24 mumol /mL. In addition, the composition of fatty acids in phospholipids model compounds and those in olive oil samples was estimated by employing one- and two-dimensional (1)H NMR. The results indicated that the fatty acid composition in phospholipids and triacylglycerols of olive oil was similar.     

Inositol phosphate profiling of fermented cowpeas by 1H NMR spectroscopy

The inositol phosphate content of naturally fermented cowpeas (Vigna sinensis var. Carilla) was studied using ion-pair HPLC and 1H NMR spectroscopy. The fermented flour was extracted with 0.5 M HCl, and the extract was purified and fractionated by ion-exchange chromatography. 1H NMR allowed for the identification of two monophosphates [Ins(1 or 3)P1 and Ins(4 or 6)P1], one inositol diphosphate [Ins(1,4)P2], three inositol triphosphates [Ins(1,2,6)P3, Ins(1,5,6)P3, and Ins(1,4,5)P3], one inositol tetraphosphate [Ins(1,3,4,5)P4], and one inositol pentaphosphate [Ins(1,2,3,5,6)P5]. Some of these isomers [Ins(1,4,5)P3 and Ins(1,3,4,5)P4] are considered to play important biological roles in intracellular signaling.     

Structure elucidation of procyanidin oligomers by low-temperature 1H NMR spectroscopy

Procyanidin dimers and trimers, needed as reference compounds for biological studies, have been synthesized from various natural sources using a semisynthetic approach and purified by high-speed countercurrent chromatography (HSCCC). In the past, it has been difficult to elucidate the structure of these compounds, especially the determination of the interflavanoid bond. Here, the structure of two B-type procyanidin dimers, with (+)-catechin ((+)-C) in the upper unit, and eight C-type procyanidin trimers, with (-)-epicatechin ((-)-EC) in the upper unit, have been elucidated using low-temperature (1)H NMR spectroscopy, as well as circular dichroism (CD) spectroscopy. This is the first time NOE interactions have been used to characterize the interflavanoid linkage in underivatized procyanidin trimers. Complete analyses of procyanidin C1 (-)-EC-4β→8-(-)-EC-4β→8-(-)-EC, (-)-EC-4β→8-(-)-EC-4β→8-(+)-C, (-)-EC-4β→6-(-)-EC-4β→8-(-)-EC, (-)-EC-4β→6-(-)-EC-4β→8-(+)-C, (-)-EC-4β→8-(-)-EC-4β→6-(-)-EC, (-)-EC-4β→8-(-)-EC-4β→6-(+)-C, (-)-EC-4β→8-(+)-C-4α→8-(-)-EC, procyanidin C4 (-)-EC-4β→8-(+)-C-4α→8-(+)-C, and procyanidin dimers B6 (+)-C-4α→6-(+)-C and B8 (+)-C-4α→6-(-)-EC are presented.     

Soil organic matter in urban soils: Estimation of elemental carbon by thermal oxidation and characterization of organic matter by solid-state C nuclear magnetic resonance (NMR) spectroscopy

To reduce soil destruction by urban sprawl, land use planning has to promote the use of soils within cities. As soil functions are now protected by law in Germany, urban soil quality has to be evaluated before soil management. We studied contributions from elemental carbon (EC) and soil organic matter (SOM) quality in topsoil horizons at seven sites in Stuttgart, Germany, differing in impurities by technogenic substrates. The most disturbed site was found at a disused railway area while high-density areas, public parks and garden areas showed varying degrees of disturbance by anthropogenic activities. For most soils, compounds derived from plant litter dominated organic matter (OM) quality characterized by nuclear magnetic resonance (NMR) spectroscopy. Although high contents of EC (up to 70% of soil organic carbon) were indicated by thermal oxidation, this was not confirmed by aromatic C intensities in NMR spectra. Only for the highly aromatic railway soil were results for elemental carbon by thermal oxidation and NMR similar. As other technogenic substrates beside EC like plastics may also contribute in the long-term to OM in urban soils, new analytical techniques are therefore required. This knowledge will promote the evaluation of urban soil properties and their sustainable use.     

基于dbiPLS-SPA变量筛选的固态发酵湿度近红外光谱检测

为了提高基于近红外光谱技术的固态发酵关键过程参数——湿度快速检测的精度和稳定性,研究采用动态反向区间偏最小二乘（dbiPLS）法结合连续投影算法（SPA）进行最佳光谱子区间和特征组合变量的筛选,通过交互验证法确定偏最小二乘（PLS）模型的主成分因子数,并以预测均方根误差（RMSEP）和相关系数（Rp）作为模型的评价标准。试验结果显示,最佳dbiPLS-SPA模型筛选的组合变量个数为8,其RMSEP和Rp分别为1.1795%（质量分数）和0.9430。试验结果表明,dbiPLS-SPA是一个有效的波长组合变量筛选方法,可简化模型结构、增强模型精度和稳健性。     

Structural identification of two major anthocyanin components of boysenberry by NMR spectroscopy

The major anthocyanins of boysenberry fruit, a cross between Rubus loganbaccus and Rubus baileyanus Britt., were isolated by preparative high-performance liquid chromatography (HPLC). The structures of cyanidin-3-[2-(glucosyl)glucoside] (1) and cyanidin-3-[2-(glucosyl)-6-(rhamnosyl)glucoside] (2) were determined by NMR in 1% DCOOD/D(2)O. An unusually high chemical shift (delta 2.5) is reported for H-5' ' of cyanidin-3-[2-(glucosyl)glucoside].