Facile purification of milligram to gram quantities of condensed tannins according to mean degree of polymerization and flavan-3-ol subunit composition

Unambiguous investigation of condensed tannin (CT) structure-activity relationships in biological systems requires well-characterized, high-purity CTs. Sephadex LH-20 and Toyopearl HW-50F resins were compared for separating CTs from acetone/water extracts and column fractions analyzed for flavan-3-ol subunits, mean degree of polymerization (mDP) and purity. Toyopearl HW-50F generated fractions with higher mDP values and better separation of procyanidins (PC) and prodelphinidins (PD) but required a pre-purification step, more time for large scale purifications and gave poorer recoveries. Therefore, two gradient elution schemes were developed for CT purification on Sephadex LH-20 providing 146 - 2000 mg/fraction. Fractions were analyzed by thiolysis and NMR spectroscopy. In general, PC/PD ratios decreased and mDP increased during elution. 1H NMR spectroscopy served as a rapid screening tool to qualitatively determine CT enrichment and carbohydrate impurities present, guiding fractionation towards re-purification or 1H-13C HSQC NMR spectroscopy and thiolysis. These protocols provide options for preparing highly pure CT samples

Analysis of sainfoin (Onobrychis viciifolia) proanthocyanidins by complementary and newly developed techniques

Sainfoin proanthocyanidins (PAs) are complex mixtures of homo- and hetero- polymers consisting of B-type procyanidins and prodelphinidins. Direct analysis by thiolytic degradation revealed a wide range of PA contents and compositions within the HealthyHay germplasm collection (46 accessions). PA contents varied from 0.57% to 2.80% (g PAl100g freeze-dried samples). PAs polymer size ranged from 12 to 84 flavan-3-ol units in terms of their mean degree of polymerisation. Prodelphinidin/procyanidin ratios ranged from 53/47 to 95/5 and trans/cis ratios varied from 12/88 to 34/66. Purified PAs fractions from 4 selected accessions showed a positive correlation between polymer size and prodelphinidin content within each accession (R2 from 0.69 to 0.92). Careful selection of MALDI-TOF MS matrices and analytical conditions made it possible to detect PAs up to 12 subunits and also ion signals that could be assigned to A-type and rarely reported glycosylated A-type PAs. For detecting and confirming the polymer size of underivatised higher molecular weight PAs a new HPLC-GPC technique consisting in a single calibration curve for galloyl glucoses, ellagitannins and PAs was developed. Peak-average molecular weights of sainfoin PA fractions were overestimated by 42.0% at 2436 Dalton and underestimated by 13.9% at 8318 Dalton. Number-average molecular weights were overestimated by 30.0% and underestimated by 25.8%, respectively. Cluster analysis of the HealthyHay germplasm collection revealed that accessions clustered into two main clusters, Western Europe and Eastern Europe/Asia, and that accessions from Armenia, Canada and USA clustered into another group. This seems to be in agreement with the strong links between geographic origin and accession performance found in the HealthyHay sainfoin germplasm. This research made significant contributions to the fields of PA analysis and germplasm screening in terms of novel analytical techniques for determining the average molecular weight distribution, content, subunit composition and linkages, purification and fractionation of proanthocyanidins in sainfoin.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Simple solution for a complex problem: Proanthocyanidins, galloyl glucoses and ellagitannins fit on a single calibration curve in high performance-gel permeation chromatography

This study was undertaken to explore gel permeation chromatography (GPC) for estimating molecular weights of proanthocyanidin fractions isolated from sainfoin (Onobrychis viciifolia). The results were compared with data obtained by thiolytic degradation of the same fractions. Polystyrene, polyethylene glycol and polymethyl methacrylate standards were not suitable for estimating the molecular weights of underivatized proanthocyanidins. Therefore, a novel HPLC-GPC method was developed based on two serially connected PolarGel-L columns using DMF that contained 5% water, 1% acetic acid and 0.15 M LiBr at 0.7 ml/min and 50 degrees C. This yielded a single calibration curve for galloyl glucoses (trigalloyl glucose, pentagalloyl glucose), ellagitannins (pedunculagin, vescalagin, punicalagin, oenothein B, gemin A), proanthocyanidins (procyanidin B2, cinnamtannin B1), and several other polyphenols (catechin, epicatechin gallate, epicallocatechin gallate, amentoflavone). These GPC predicted molecular weights represented a considerable advance over previously reported HPLC-GPC methods for underivatized proanthocyanidins. (C) 2011 Elsevier B.V. All rights reserved

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

Structural diversity of sainfoin (Onobrychis viciifolia) tannins

Tannins are oligomeric and polymeric polyphenols that are produced by many plants. The study of their biological activities is of interest because they can generate valuable nutritional, veterinary and environmental effects in ruminant livestock production [1]. Isolated tannin fractions from sainfoin (Onobrychis viciifolia), which is potentially a very useful animal feed, were characterised by MALDI-TOF MS and thiolytic degradation with benzyl mercaptan. Condensed tannins were analysed and characterised in more than different 40 sainfoin varieties to provide guidelines for future plant breeding programmes. Several different techniques were used to study these complex tannin mixtures

Deciphering the complexity of sainfoin (Onobrychis viciifolia) proanthocyanidins by MALDI-TOF mass spectrometry with a judicious choice of isotope patterns and matrixes

Use of superdihydroxybenzoic acid as the matrix enabled the analysis of highly complex mixtures of proanthocyanidins from sainfoin (Onobrychis viciifolia) by MALDI-TOF mass spectrometry. Proanthocyanidins contained predominantly B-type homopolymers and heteropolymers up to 12- mers (3400 Da). Use of another matrix, 2,6-dihydroxyacetophenone, revealed the presence of A-type glycosylated dimers. In addition, we report here how a comparison of the isotopic adduct patterns, which resulted from Li and Na salts as MALDI matrix additives, could be used to confirm the presence of A-type linkages in complex proanthocyanidin mixtures. Preliminary evidence suggested the presence of A-type dimers in glycosylated prodelphinidins and in tetrameric procyanidins and prodelphinidins

Proanthocyanidin diversity in the EU 'HealthyHay' sainfoin (onobrychis viciifolia) germplasm collection

This study investigated 37 diverse sainfoin (Onobrychis viciifolia Scop.) accessions from the EU ‘HealthyHay’ germplasm collection for proanthocyanidin (PA) content and composition. Accessions displayed a wide range of differences: PA contents varied from 0.57 to 2.80 g/100 g sainfoin; the mean degree of polymerisation from 12 to 84; the proportion of prodelphinidin tannins from 53% to 95%, and the proportion of trans-flavanol units from 12% to 34%. A positive correlation was found between PA contents (thiolytic versus acid–butanol degradation; P < 0.001; R2 = 0.49). A negative correlation existed between PA content (thiolysis) and mDP (P < 0.05; R2 = −0.30), which suggested that accessions with high PA contents had smaller PA polymers. Cluster analysis revealed that European accessions clustered into two main groups: Western Europe and Eastern Europe/Asia. In addition, accessions from USA, Canada and Armenia tended to cluster together. Overall, there was broad agreement between tannin clusters and clusters that were based on morphological and agronomic characteristics