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.

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.