In situ analysis and structural elucidation of sainfoin (Onobrychis viciifolia) tannins for high-throughput germplasm screening

A rapid thiolytic degradation and cleanup procedure was developed for analyzing tannins directly in chlorophyll-containing sainfoin ( Onobrychis viciifolia ) plants. The technique proved suitable for complex tannin mixtures containing catechin, epicatechin, gallocatechin, and epigallocatechin flavan-3-ol units. The reaction time was standardized at 60 min to minimize the loss of structural information as a result of epimerization and degradation of terminal flavan-3-ol units. The results were evaluated by separate analysis of extractable and unextractable tannins, which accounted for 63.6-113.7% of the in situ plant tannins. It is of note that 70% aqueous acetone extracted tannins with a lower mean degree of polymerization (mDP) than was found for tannins analyzed in situ. Extractable tannins had between 4 and 29 lower mDP values. The method was validated by comparing results from individual and mixed sample sets. The tannin composition of different sainfoin accessions covered a range of mDP values from 16 to 83, procyanidin/prodelphinidin (PC/PD) ratios from 19.2/80.8 to 45.6/54.4, and cis/trans ratios from 74.1/25.9 to 88.0/12.0. This is the first high-throughput screening method that is suitable for analyzing condensed tannin contents and structural composition directly in green plant tissue.     

Cytological characterisation of the underutilized forage crop Onobrychis viciifolia Scop. and other members of the Onobrychis genus

Sainfoin (Onobrychis viciifolia Scop.) is a perennial forage legume that possesses beneficial properties in the context of sustainable agriculture. A germplasm collection of O. viciifolia was assembled from many geographic regions, as well as a collection of Onobrychis species that might be crossed with O. viciifolia to improve its biological and agronomic properties. The objective of the present study was to obtain cytological information for a broad range of O. viciifolia varieties and related species, in order to confirm and extend previous findings. The study set was 143 accessions of O. viciifolia as well as 34 accessions of 17 other species from the Onobrychis genus. Three of the O. viciifolia accessions were diploid (2n = 2x = 14) and the remainder tetraploid (2n = 4x = 28). Chromosomes were <5 μm in length. Among the other Onobrychis species, roughly half were found to be diploid and the other half tetraploid, with two species, O. arenaria (Kit.) DC. and O. alba subsp. laconica (Orph. ex Boiss.) Hayek, including a mixture of diploid and tetraploid accessions. The basic number of chromosomes was seven, except for O. aequidentata (Sm.) d’Urv. and O. crista-galli Lam., where it was eight. The 2C value for O. viciifolia was estimated at 2.5 pg. These cytological data will be valuable as part of a pre-breeding programme aimed at the eventual production of improved O. viciifolia varieties for use in sustainable agriculture.     

Rapid quantification of proanthocyanidins (condensed tannins) with a continuous flow analyzer

Proanthocyanidins (condensed tannins) frequently need to be quantified in large numbers of samples in food, plant, and environmental studies. An automated colorimetric method to quantify proanthocyanidins with sulfuric acid (H(2)SO(4)) was therefore developed for use in a continuous flow analyzer. Assay conditions were optimized using 50% methanol extracts of paper birch, sugar maple, and quaking aspen leaves. Short extraction times and centrifugation of samples prevented proanthocyanidin degradation that otherwise occurred in 50% methanol extracts of aspen leaves. Extraction of birch and maple proanthocyanidins with 50% methanol was comparable to or better than that with 70% acetone. Proanthocyanidin levels in aspen were lower when extracted with aqueous methanol, but relative differences among samples were consistent with those found in aqueous acetone extracts. Results from the automated sulfuric acid assay were highly correlated with those of the conventional BuOH-HCl method for proanthocyanidins and, except for birch, with the Folin--Denis assay for total phenolics. This new technique significantly improves assay processing rate and repeatability compared to conventional colorimetric proanthocyanidin assays.     

葡萄果实和葡萄酒中缩合单宁的研究进展

缩合单宁是葡萄与葡萄酒中重要的组分之一,它影响着葡萄酒的色泽、风味、稳定性和贮藏寿命,与人的健康也有密切关系;该文介绍了缩合单宁的作用、结构及其与原花色素的关系,葡萄与葡萄酒中缩合单宁的存在形式及其在葡萄果实成熟和葡萄酒酿造过程中的变化和对葡萄酒口感的影响及缩合单宁的生物合成等方面的研究进展,提出了今后的研究方向.     

Understanding the native Californian diet: Identification of condensed and hydrolyzable tannins in tanoak acorns (Lithocarpus densiflorus)

The tanoak (Lithocarpus densiflorus) acorn was a staple food in the Native American diet and is still used in traditional dishes. Acorns from the genus Quercus have been shown to contain a large range of hydrolyzable tannins. However, neither hydrolyzable nor condensed tannins have been characterized in tanoak acorns. The aim of this study was to identify the full range of hydrolyzable and condensed tannins in extracts of tanoak acorns using liquid chromatography/electrospray ionization-mass spectrometry/mass spectrometry. Condensed tannins were identified as B type oligomers of (epi)-catechin (procyanidins) with a degree of polymerization up to six. Oligomers up to and including tetramers were identified by UV spectra and MS detection whereas pentamers and hexamers were detected only by MS. The total concentration of condensed tannins was 464 mg/100 g acorn pericarp. The concentration of propocyanidin monomers, dimers, trimers, and tetramers in acorn pericarp (mg/100 g acorn pericarp) were 95 +/- 10.9, 148 +/- 35.0, 90 +/- 17.9, and 131 +/- 1.9, respectively. No procyanidins were found in the acorn cotyledon tissue. A total of 22 hydrolyzable tannins were identified in methanolic extracts of acorn cotyledon tissue. Gallic acid derivatives predominated and included galloylated esters of glucose, hexahydrodiphenoyl esters of glucose, and methylated gallates. Galloylated esters of glucose were present as isomers of galloyl glucose, digalloyl glucose, and trigalloyl glucose. Mass spectral fragmentation patterns indicate the presence of one gallic acid-galloyl glucose isomer and two gallic acid-digalloyl-glucose isomers. No isomers of tetragalloyl glucose and pentagalloyl glucose were identified. Ellagic acid and ellagic acid pentoside were also identified.     

Effect of low root temperature on symbiotic nitrogen fixation and competitive nodulation of Onobrychis viciifolia (sainfoin) by strains of arctic and temperate rhizobia

Summary Symbiotic effectiveness and competitive nodulation of the temperate forage legume sainfoin (Onobrychis viciifolia cv. Melrose) by two strains of arctic rhizobia (from Astragalus or Oxytropis sp.) and two strains of temperate rhizobia (from sainfoin) were evaluated at temperatures of 9, 12, or 15°C for roots and 20°C (day) and 15°C (night) for shoots. The inocula consisted of effective individual or paired arctic and temperate strains which were identified on the basis of differential growth. At 9°C, the arctic strains were generally more competitive than the temperate strains, whereas at the highest temperature tested, the converse was apparent. Symbiotic effectiveness (shoot dry weight, nitrogenase activity, and number of nodules) was similarly affected by root temperature, except that at 15°C the arctic strains were generally as effective as the temperate strains. The marked interaction between temperature and strain on competitiveness or on effectiveness indicates symbiotic adaptation of the arctic rhizobia to low temperatures. On the basis of these data we suggest that the use of selected, cold-adapted rhizobia in sainfoin inoculants may be beneficial in temperate regions where low soil temperatures occur early in the growing season.Contribution no. 413 and 1334 of the Agriculture Canada Sainte-Foy Research Station and Plant Research Centre, respectively     

Linking the B ring hydroxylation pattern of condensed tannins to C, N and P mineralization. A case study using four tannins

Condensed tannins (CTs) are a major component of litter inputs, but little is known about the effects of tannin structural variations on soil biological processes and organic matter development. Four different CTs extracted from balsam fir, western red cedar, kalmia and black spruce were added to Corsican pine litter and subsequently incubated for 16 weeks in order to investigate the effect of the B ring hydroxylation pattern on C, N and P transformations. While for C mineralization the chain length and stereochemistry of the CTs seemed to be a more important parameter, net N and P mineralization rates were clearly reduced compared with non-amended litter. With regard to the B ring hydroxylation, the prodelphinidin (PD) CTs having predominantly three hydroxy groups at the B ring (balsam fir and western red cedar) exhibited significantly lower mineralization rates than the procyanidin (PC) CTs having two OH groups (kalmia and black spruce). The same was true for net nitrification, but this process was only slightly affected by the CTs. Although based on only four CTs, this study indicates that B ring hydroxylation is an important variable determining net N and P mineralization rates. Our results support previous suggestions that PD tannins bind to or react more strongly with soil organic matter. Therefore, more than PC tannins, they reduce the availability of organic N for mineralization as well as their own detectability by standard methods for soil CT.     

NMR, HPLC-ESI-MS, and MALDI-TOF MS analysis of condensed tannins from Delonix regia (Bojer ex Hook.) Raf. and their bioactivities

The structures of the condensed tannins isolated from leaf, fruit, and stem bark of Delonix regia (Bojer ex Hook.) Raf. have been investigated with (13)C nuclear magnetic resonance ((13)C NMR) and high performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS) coupled with thiolysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses. The results showed that these condensed tannins from D. regia possessed structural heterogeneity in monomer units and degree of polymerization. Propelargonidin (PP) and procyanidin (PC) were found in the leaf, fruit, and stem bark of D. regia, while prodelphinidin (PD) was found only in the leaves. The polymer chain lengths of condensed tannins from leaf and fruit organs were detected to be trimers to hexadecamers but from trimers to tridecamers for stem bark. B-type linkages were present in all these compounds. Condensed tannins from different parts of D. regia can be explored as tyrosinase inhibitors and food antioxidants because of their potent antityrosinase and antioxidant activities. The inhibitor concentration leading to 50% enzyme activity (IC(50)) was estimated to be 38 ± 1, 73 ± 2, and 54 ± 1.5 μg/mL for the condensed tannins of leaf, fruit, and stem bark. Condensed tannins extracted from stem bark exhibited the highest antioxidant activity; the DPPH scavenging activity (IC(50)) and the FRAP values were 90 ± 2 μg/mL and 5.42 ± 0.09 mmol AAE/g, respectively.     

Identification and quantification of polybrominated hexahydroxanthene derivatives and other halogenated natural products in commercial fish and other marine samples

During routine analysis of commercial fish on halogenated pollutants, an unknown tribromo component (TriBHD) was initially detected as an abundant peak in sample extracts from the Mediterranean Sea. The molecular formula was established to be C16H19Br3O by gas chromatography with electron ionization high-resolution mass spectrometry (GC/EI-HRMS). GC/EI-MS data were virtually identical with a polybrominated hexahydroxanthene derivative (PBHD) previously isolated from an Australian sponge species known to occur in the Mediterranean Sea as well. A tetrabromo isomer (TetraBHD) was also found in the fish samples. The concentrations of TriBHD and other halogenated compounds in commercial fish (sea bass, gilt head bream, anchovy, sardine, and salmon) were estimated with GC/electron capture detection (ECD). Using the ECD response of trans-nonachlor, the concentration of TriBHD reached up to 90 ng/g lipid weight and accounted for up to >90% of the concentration of p,p'-DDE, which was the most abundant peak in the most samples investigated. On the basis of the GC/ECD response, TetraBHD amounted for approximately 1/7 of TriBHD in all fish samples investigated. The sample with the highest content was a green-lipped mussel from New Zealand (236 ng/g lipid weight). The halogenated natural products TBA, Q1, and MHC-1 were also present in most of the samples. We assume that the bulk of the residues in fish from aquaculture may originate from algae and sponges living in proximity of the fish farms. Detection of TriBHD and TetraBHD in blubber of a monk seal (Monachus monachus) suggests that both HNPs may reach the top predators of food webs and thus also humans.     

Contents of total protein, L-canavanine and condensed tannins of the one-flowered vetch (Vicia articulata Hornem.) collection of the Bank of Plant Germplasm of Cuenca (Spain)

A total of 125 accessions of one-flowered vetch (Vicia articulata Hornem.) mostly from the Iberian Peninsula have been analysed for total protein, L-canavanine and condensed tannins. It was observed a high variability in the composition: the protein content ranged from 18.20 to 30.07%, L-canavanine from 0.27 to 0.67%, and condensed tannins from 0.13 to 0.35%, which indicates a scarce domestication of the crop. The inclusion of the seeds into a diet to non-ruminants should be limited to marginal percentages because of the contents of L-canavanine and it suggests that a reduction of this compound by breeding should be encouraged. The protein and the L-canavanine contents showed a low and positive significant correlation (r = 0.55, P < 0.001), which complicates the selection of seeds having simultaneously low L-canavanine and high protein contents. On the other hand, no correlation between protein and condensed tannins or between L-canavanine and condensed tannins was observed. There was a poor relationship between the origin and the composition of the accessions and only the protein content was able to discriminate among the regions.     

Penicillium expansum: consistent production of patulin, chaetoglobosins, and other secondary metabolites in culture and their natural occurrence in fruit products

Penicillium expansum is known for its destructive rot and patulin production in apple juice. According to the literature, P. expansum can, among other compounds, produce citrinin, ochratoxin A, patulin, penitrem A, and rubratoxin B. In this study the qualitative production of metabolites was examined using TLC (260 isolates), HPLC (85 isolates), and MS (22 isolates). The results showed that none of the 260 isolates produced ochratoxin A, penitrem A, or rubratoxin B. However, chaetoglobosin A and communesin B were produced consistently by all 260 isolates. Patulin and roquefortine C were produced by 98% of the isolates. Expansolides A/B and citrinin were detected in 91 and 85% of the isolates, respectively. Chaetoglobosins and communesins were detected in naturally infected juices and potato pulp, whereas neither patulin nor citrinin was found. Because most P. expansum isolates produce patulin, citrinin, chaetoglobosins, communesins, roquefortine C, and expansolides A and B, foods contaminated with this fungus should ideally be examined for chaetoglobosin A as well as patulin.     

Changes in the structure and protein binding ability of condensed tannins during decomposition of fresh needles and leaves

Alterations of the chemical structure of condensed tannins (CT) during decomposition of Norway spruce (Picea abies) needles and white willow (Salix alba) leaves were investigated by gel permeation chromatography (GPC), ¹³C nuclear magnetic resonance (NMR) spectroscopy, and matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS). The effect of these alterations on their protein binding capacity was determined by radial diffusion assay on an agarose plate. For these studies an incubation experiment was performed with spruce needles and willow leaves. From the fresh foliage and its decomposed materials obtained after 4-days, and 1-, 2-, 4-, and 8-weeks of incubation, CT were extracted and analyzed.Dynamics of CT alterations during decomposition of the fresh foliage were different for the two plant species, although the amount of extractable CT for both decreased soon after incubation and only slight amounts of CT were extractable after 8 weeks of incubation. The decrease was faster for the willow leaves than for the spruce needles. Solution ¹³C NMR revealed alterations of CT isolated from both degrading plant materials, but only to a small extent. However, considerable changes in chemical composition and chain length of the CT were detected by MALDI-TOF MS. Changes in the chemical composition of CT are expected to decrease the protein binding capacity. Applying the radial diffusion assay, this assumption was confirmed for spruce CT, but not for willow CT. This may be explained by (1) higher reactivity of prodelphinidin (PD) than procyanidin (PC), the former is contained in spruce CT but not in willow CT and (2) the slower decomposition rate of spruce needles than willow leaves. Thus, CT in spruce needles may have suffered a greater extent of chemical alteration, which formed partially altered CT with less protein binding capacity. Further, since the decomposition rate of spruce needles was slower than that of willow leaves, the partially altered CT remained extractable for a longer incubation period compared with the willow leave's CT.     

Spinach (Spinacia oleracea) powder as a natural food-grade antioxidant in deep-fat-fried products

The addition of spinach (Spinacia oleracea) powder in flour dough as a natural antioxidant was investigated, and oxidation of frying oil and the lipid in fried products during frying was also studied. Flour dough with spinach powder was rolled into sheets of 0.1 cm thickness and then cut into squares to be fried. Each frying was performed in 160 degrees C soybean oil for 1 min, repeated every 20 min for 20 h. Fried samples were analyzed immediately or after being stored at 60 degrees C for 12 days under dark. The lipid content of fried dough was lower in samples with the addition of spinach powder. Spinach in the dough decreased accumulation of the polar compounds in soybean oil during frying but had little effect on the fried dough. It also reduced conjugated diene and aldehyde formation in the lipid of fried dough during storage. Improvement in lipid oxidative stability, presumably due to pigments in spinach, was more noticeable in the fried products during storage than in the frying oil.     

Measurement of condensed tannins and dry matter in red grape homogenates using near infrared spectroscopy and partial least squares

Samples (n = 620) of homogenized red grape berries were analyzed using a visible and near-infrared (NIR) spectrophotometer (400-2500 nm) in reflectance. The spectra and the analytical data were used to develop partial least-squares calibrations to predict dry matter (DM) content and condensed tannins (CT) concentrations. The coefficient of determination in cross-validation and the standard error of cross-validation were 0.92 and 0.83% w/w for DM and 0.86 and 0.46 mg/g epicatechin equivalents for CT, respectively. The standard error in prediction was 1.34% w/w for DM and 0.89 mg/g epicatechin equivalents for CT, respectively. By implementing a NIR spectroscopy method to measure DM and CT in red grape homogenates, we have developed an approach that is suited to large-scale compositional analysis in commercial wine production facilities, as it enables the analysis of large numbers of samples needed to stream batches of fruit. From an economical point of view, the calibration models could be achieved with relatively small data sets. Thus, NIR offers a suitable and efficient tool for the simultaneous measurement of DM and CT in addition to other important parameters in red grape homogenates such as total anthocyanins, total soluble solids, and pH, with minimal sample preparation and low cost.     

Allium chemistry: synthesis, natural occurrence, biological activity, and chemistry of Se-alk(en)ylselenocysteines and their gamma-glutamyl derivatives and oxidation products

Syntheses are reported for gamma-glutamyl Se-methylselenocysteine (Sa), selenolanthionine (16), Se-1-propenylselenocysteine (Gd), Se-2-methyl-2-propenyl-L-selenocysteine (6e), and Se-2-propynyl-L-selenocysteine (6f). Oxidation of 8a and Se-methylselenocysteine (Ga) gives methaneseleninic acid (24), characterized by X-ray crystallography, and dimethyl diselenide (25). Oxidation of Se-2-propenyl-L-selenocysteine (6c) gives allyl alcohol and 3-seleninoalanine (22). Compound 22 is also formed on oxidation of 16 and selenocystine (4). Oxidation of 6d gives 2-[(E,Z)-1-propenylseleno]propanal (36). These oxidations occur by way of selenoxides, detected by chromatographic and spectroscopic methods. The natural occurrence of many of the Se-alk(en)ylselenocysteines and their gamma-glutamyl derivatives and oxidation products is discussed. Three homologues of the potent cancer chemoprevention agents 6a and 6c, namely 6d-f, were evaluated for effects on cell growth, induction of apoptosis, and DNA-damaging activity using two murine mammary epithelial cell lines. Although each compound displays a unique profile of activity, none of these compounds (Gd-f) is likely to exceed the chemopreventive efficacy of selenocysteine Se-conjugates Ga and 6c.     

Condensed tannins and flavonoids from the forage legume sulla (Hedysarum coronarium)

The condensed tannin concentrations and composition and the characterization of the phenolic constituents in the leaves of the forage legume sulla (Hedysarum coronarium), a biennial forage legume found in temperate agricultural regions, were studied. The colorimetric butanol-HCl assay was used for the quantitation of the seasonal condensed tannin concentrations in the leaves of sulla. Fractionation of extracts on Sephadex LH-20 using step elution with aqueous methanol, followed with aqueous acetone or gradient elution with water, aqueous methanol, and aqueous acetone, gave condensed tannin and flavonoid fractions. The chemical characteristics of the purified condensed tannin fractions were studied by acid-catalyzed degradation with benzyl mercaptan and electrospray ionization mass spectrometry (ESI-MS). Thiolysis revealed that epigallocatechin was the major extender unit (15-75%) while gallocatechin was the major terminal unit (50-66%), thus indicating the extractable sulla condensed tannin fraction as the prodelphinidin type. Condensed tannin oligomers to polymers obtained from Sephadex LH-20 gradient fractions ranged between 2.9 and 46 mDP. The homo- and heterogeneous oligomer ions in condensed tannin gradient fractions detected by ESI-MS ranged from 2 to 10 DP and are consistent with the values obtained by thiolysis (2.9-6.9 DP). Lower molecular weight phenolics, including flavonoids and phenolic acids, were characterized by liquid chromatography atmospheric pressure chemical ionization mass spectrometry (LC-APCI/MS) and ESI/MS/MS on a linear ion trap. The flavonoids extracted with aqueous acetone and methanol from sulla leaves and identified included kaempferol, rutin, quercetin-7-O-α-L-rhamnosyl-3-O-glucosylrhamnoside, quercetin-3-O-α-L-rhamnosyl-7-O-glucoside, kaempferol-3-O-β-D-glucoside-dirhamnoside, genistein-7-O-β-D-glucosyl-6″-O-malonate, formononetin-7-O-β-D-glucoside-6″-O-malonate, and afrormosin and the phenolic acid chlorogenic acid.     

Evaluation of acibenzolar-S-methyl and other low-toxicity products for control of rose powdery mildew (Podosphaera pannosa) in greenhouses

Abstract

In an attempt to find alternative products to classical fungicides, several products with low toxicity were tested against powdery mildew of roses. These products included resistance inducers (Bion, BABA, and ROS), potassium salts (Resistim, monopotassium phosphate), and seed extracts. The best results were obtained with acibenzolar-S-methyl (Bion). The utilization of Bion as prophylactic treatment, watered at a concentration 0.1–0.2 mg/ml, together with good cultural practices can be enough to effectively control powdery mildew on roses. Treatments with Resistim reduced the disease incidence, but not always significantly compared to the controls. None of the other products had effect on powdery mildew.     

Phenolic antioxidants (hydrolyzable tannins, flavonols, and anthocyanins) identified by LC-ESI-MS and MALDI-QIT-TOF MS from Rosa chinensis flowers

Rosa chinensis (Yuejihua) is a well-known ornamental plant, and its flowers are commonly used in traditional Chinese medicine. Methanolic crude extracts of dried R. chinensis flowers were used for simultaneous determination of phenolic constituents by liquid chromatography-mass spectrometry (LC-MS) and matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (MALDI-QIT-TOF MS). A total of 36 known and unknown phenolics were identified as hydrolyzable tannins, flavonols, and anthocyanins, mainly including gallotannins (mono-, di-, or trigalloylglucopyranosides), ellagitannins, quercetin, quercetin/kaempferol mono- and diglycosides, and cyanidin/pelargonidin diglycosides. MALDI-QIT-TOF MS was applied not only to verify most phenolics isolated and identified by LC-MS but also to tentatively identify two ellagitannins (rugosins B and C) not isolated and unidentified by LC-MS. This study is the first to demonstrate the rapid and successful use of MALDI-QIT-TOF MS and LC-MS to directly and simultaneously identify phenolics in the crude extracts of R. chinensis flowers without any purification. The antioxidant activity of the crude extracts from R. chinensis flowers was also measured with three assay methods. The results showed that the phenolic antioxidants from R. chinensis flowers exhibited very strong radical scavenging effect and antioxidant power. High levels of flavonols and hydrolyzable tannins might be important bioactive principles in the dried R. chinensis flowers.     

Determination of hydrolyzable tannins (gallotannins and ellagitannins) after reaction with potassium iodate

A widely used method for analyzing hydrolyzable tannins afer reaction with KIO(3) has been modified to include a methanolysis step followed by oxidation with KIO(3). In the new method, hydrolyzable tannins (gallotannins and ellagitannins) are reacted at 85 degrees C for 20 h in methanol/sulfuric acid to quantitatively release methyl gallate. Dried plant samples can be methanolyzed under the same conditions to convert hydrolyzable tannins to methyl gallate. Oxidation of the methyl gallate by KIO(3) at pH 5.5, 30 degrees C, forms a chromophore with lambda(max) 525 nm, which is determined spectrophotometrically. The detection limit of the method is 1.5 microg of methyl gallate, and with plant samples, relative standard deviations of less than 3% were obtained.