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-ß-D-glucoside-dirhamnoside, genistein-7-O-ß-D-glucosyl-6″-O-malonate, formononetin-7-O-ß-D-glucoside-6″-O-malonate, and afrormosin and the phenolic acid chlorogenic acid.

Isolation and characterisation of procyanidins from Rumex obtusifolius.

An acetone:water (7:3) extract obtained from the leaves of Rumex obtusifolius was fractionated into procyanidin oligomer and polymer fractions using a linear gradient and a simple step method on Sephadex LH-20. The chemical characteristics of the procyanidin fractions were studied by 13C-NMR spectroscopy, acid-catalysed degradation in the presence of benzyl mercaptan, matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) MS and electrospray ionisation (ESI) MS. The 13C-NMR showed that the polymer fraction consisted predominantly of procyanidin polymers, some with galloyl groups attached. The thiolysis reaction products indicated a mean degree of polymerisation (DP) of 4.3 for the step method, and a range of 2.3-8.2 mean DP for the gradient fractionation, with epicatechin as the most abundant flavan-3-ol extension unit, while the terminal units consisted of equal proportions of catechin, epicatechin and epicatechin gallate. Singly charged ions observed in MALDI-TOF/MS showed a range of oligomeric procyanidins and their polygalloyl derivatives. These species (in the range DP 2-7) were also observed by ESI/MS but the spectra were more complex due to overlapping multiply charged ions. Isolation of oligomers from the Sephadex LH-20 fraction by chromatography on polyamide and C18 yielded B1, B2, B3 and B7 dimers, an A-type trimer and a B2 3,3'-O-digallate.

At-line near-infrared spectroscopy for prediction of the solid fat content of milk fat from New Zealand butter.

Near-infrared (NIR) spectroscopy calibrations that will allow prediction of the solid fat content (SFC) of milk fat extracted from butter by one measurement during manufacture were developed. SFC is a measure of the amount of the solid fraction of fat crystallized at a temperature expressed as a percentage (w/w). At-line SFC determinations are currently performed by nuclear magnetic resonance (NMR) spectroscopy, which involves a 16 h delay period for tempering of the milk fat at 0 degrees C prior to the SFC measurements, from 0 to 35 degrees C in a series of 5 degrees C increments. The NIR spectra (400-2500 nm) were obtained using a sample holder maintained at 60 degrees C. Accurate predictions for the SFC (%) were developed by principal component analysis (PCA) and partial least-squares (PLS) regression models to relate the NIR spectra to the corresponding NMR values. The independent validation samples (N = 22) had a standard error of prediction (SEP) of 0.385-0.762% for SFC between 0 and 25 degrees C, with SFC reference values ranging between 70.42 and 8.96% with a standard deviation range of 3.36-1.47. The low bias (from -0.351 to -0.025), the slopes (0.935-1.077), and the excellent predictive ability (R2; 0.923-0.978) supported the validity of these calibrations.

Variation of proanthocyanidins in Lotus species.

The proanthocyanidin (PA) chemistry of 12 Lotus species of previously unknown PA content was examined in comparison with agricultural cultivars of L. pedunculatus, L. corniculatus, and L. tenuis and a "creeping" selection of L. corniculatus. Herbage harvested in winter 2000 and again in spring had extractable PA concentrations, estimations of which varied between 0.2 and 10.9% of dry matter. The four novel Lotus spp. with the highest concentrations were selected for further evaluation together with the agricultural accessions. PA concentrations in herbage were estimated for individual plants harvested in spring 2001 and bulk samples harvested in summer 2002-2003. PA oligomer and polymer fractions were separated by Sephadex LH-20 chromatography from aqueous acetone PA extracts of herbage. The chemical characteristics of the fractions were examined by acid catalyzed degradation with benzyl mercaptan, (13)C nuclear magnetic resonance spectroscopy, electrospray ionization (ESI), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). A wide variation was found in the chemical composition, mean degree of polymerization (mDP), and polydispersity of PAs from Lotus spp. Fractions from L. americanus, L. corniculatus "creeping selection," and L. pedunculatus consisted predominantly of prodelphinidin (PD) units, whereas PA from L. angustissimus and L. corniculatus consisted predominantly of procyanidin (PC) units. An approximately equal composition in terms of PC and PD units was found in L. parviflorus and L. suaveolens. In L. angustissimus, epicatechin is dominant in both extender and terminal units. In all Lotus PA fractions, the 2,3-cis isomers (epicatechin or epigallocatechin) predominated. Only trace amounts of PA were extracted from L. tenuis. The mDP of the PA fractions ranged from 8 to 97, with high mDP found only for L. pedunculatus and L. americanus. In the ESI-MS and MALDI-TOF-MS of the L. angustissimus PA fraction, ions for homo-PC oligomers were dominant, whereas ions for hetero-oligomers predominated in the other Lotus spp. Ions indicative of A-type linkages were observed in the MS of L. americanus. The results are discussed in terms of possible relationships between the concentration and composition of the PAs of Lotus spp. and ecological factors.

Floral Trifolium proanthocyanidins: polyphenol formation and compositional diversity.

Recent studies on the biosynthesis of proanthocyandins have identified key genes and enzymes in the formation of 2,3-cis-flavan-3-ols (epiafzelechin, epicatechin, and epigallocatechin). However, the enzymes that catalyze the polymerization of monomer units remain unknown. Studies of proanthocyanidin synthesis have involved the reference plant, Arabidopsis thaliana, forage legumes, tobacco, and grape. For this paper the floral proanthocyanidins of 10 Trifolium species were studied to identify candidates with contrasting proanthocyanidin chemistry, for the identification of factors involved in polymerization. Proanthocyandins were present in the floral portions (3.1-12.2 g/100 g of dry matter) of Trifolium spp. Thiolyic cleavage of proanthocyanidin fractions liberated flavan-3-ol extension units dominated by cis stereochemistry. The terminal units' stereochemistry of T. michelianum was exclusively trans (catechin), that of T. hirtum mixed trans (catechin and gallocatechin), and that of T. vesiculosum equal proportions of trans and cis (catechin and epicatechin). Compositional dispersion for oligomers was determined by MALDI-TOF MS, which showed a range of ions up to approximately 2200 Da. The three Trifolium spp. highlighted, all annuals, may warrant investigation for insights into proanthocyanidins biosynthesis.

Variation in antimicrobial action of proanthocyanidins from Dorycnium rectum against rumen bacteria.

The proanthocyanidin polymer fractions of the leaves of the forage legume Dorycnium rectum were analysed by acid catalysis with benzyl mercaptan, NMR and ES-MS. The results showed that D. rectum differs from other temperate proanthocyanidin-containing forage legumes in that the range of polymers extends up to very high degrees of polymerisation. Three fractions were characterised as low, medium, and high molecular weight proanthocyanidin fractions with mean degree of polymerisations of 10.3, 41 and 127, respectively. Epigallocatechin was the most abundant extension unit and the terminating flavan-3-ols comprised largely catechin and gallocatechin units in equal proportions. Formation of thiolyated dimer products showed the interflavan-linkages of the lower molecular weight proanthocyanidins to be predominantly C4-->C8 with a small amount of C4-->C6. ES-MS spectra distinguished lower from higher polymeric proanthocyanidins from M2- to M8(2)-. The antibacterial activity of proanthocyanidin fractions against pure cultures of microbes selected from the ruminal population to represent fibre degrading, proteolytic and hyper ammonia producing bacteria in broth culture was evaluated. The activity of proanthocyanidin fractions against Clostridium aminophilum, Butyrivibrio fibrisolvens and Clostridium proteoclasticum was significantly dependent on their structure but not so against Ruminococcus albus and Peptostreptococcus anaerobius. The latter observation was unique in that they were sensitive to all proanthocyanidin fractions evaluated, even at the lowest concentration (100 microg/ml). The results suggest the effects of the extractable proanthocyanidins on rumen microbes should be considered when evaluating an alternative proanthocyanidin-containing forage source for ruminants, such as D. rectum.

Floral procyanidins of the forage legume red clover (Trifolium pratense L.).

The chemical characteristics of the purified procyanidin polymers of the flowers of the forage legume red clover (Trifolium pratense L.) were studied by (13)C NMR, acid-catalyzed degradation with benzyl mercaptan, and electrospray ionization mass spectrometry (ESI-MS). The (13)C NMR showed that the fraction consisted of predominantly procyanidin polymers. The thiolysis reaction products indicated a mean degree of polymerization (mDP) of 9.3 with epicatechin (81%) as the abundant flavan-3-ol extension unit and the terminating units dominated by catechin (95%). ESI-MS showed a range of oligomeric procyanidin ions (DP of 2-11). The white clover floral prodelphinidins consist of terminal units with nearly equal proportions of epigallocatechin (52%) and gallocatechin (48%) and extender units showing epigallocatechin (56%) and gallocatechin (39%). The dramatic difference in the stereochemistry of the terminal and extender units observed for the red clover floral procyanidins contrasts with the mixture of cis and trans stereochemistry observed for white clover floral prodelphinidins.

Ovine metabolism of diosgenin coated on cellulose in relation to hepatogenous photosensitization of ruminants.

Diosgenin (2.25 g) was coated onto cellulose and dosed daily to a lamb for 12 d by intraruminal intubation. Upon completion of dosing, samples of gut contents and internal organs were subjected to chemical analysis and histological examination. No clinical signs, changes in serum chemistry, or microscopic organ changes were observed. GC-MS analyses showed that in the rumen diosgenin was metabolised to free and, to a lesser extent, conjugated derivatives of tigogenin, smilagenin and epismilagenin. Reduction of diosgenin to smilagenin and tigogenin, and epimerization of smilagenin to epismilagenin, appeared to continue in the duodenum, ileum and jejunum. Deconjugation and the epimerization of smilagenin to epismilagenin appeared to continue in the cecum and colon. Epismilagenin was the dominant conjugated sapogenin constituent identified in a bile sample.