Characterization of condensed tannins from Lotus species by thiolytic degradation and electrospray mass spectrometry

The properties of condensed tannins (CT) from Lotus corniculatus and L. pedunculatus (also known as L. uliginosus) depend on their structure in terms of monomer units, their mean degree of polymerisation (mDP) and the linkage-type between flavan-3-ol units with a considerable range of structural variation. To ascertain the identity of the individual units that make up the CT, the polymer chains are subjected to strong acid-catalysed cleavage in the presence of phloroglucinol or benzyl mercaptan. Mass spectrometry (MS) can also be utilized to probe CT structural differences. MALDI-TOF MS has been used to study L. corniculatus cultivars. We have examined the chemical composition of Sephadex LH-20 CT fractions from both L. corniculatus and L. pedunculatus by strong acid-catalysed cleavage with benzyl mercaptan (thiolysis), and by high-performance liquid-chromatography (HPLC)-electrospray mass spectrometry (ES/MS) with detection in negative ion mode. The thiolysis study confirmed earlier findings that CT from the two species are composed of procyanidin (PC) and prodelphinidin (PD) units with the L. pedunculatus CTs (PC:PD; 19:81) having a higher proportion of prodelphinidin units than L corniculatus CTs (PC:PD; 84:16). However, using a gradient fractionation approach eluting first with water, L. pedunculatus CT was found to include a high molecular weight polymer (mDP 44), not found in L. corniculatus. In the gradient fractionation a series of oligomer fractions were also separated for L. pedunculatus CTs (mDP 1.7, 3.6, 6.3 and 12) and L. corniculatus CTs (mDP 1.9, 6.3 and 19). Ions from oligomeric species (DP 2-10) were detected in these fractions by LC/ES/MS. Fractions with an identical mDP of 6.3 by thiolysis for the two species have shown a different compositional dispersion (range of DP) of oligomeric CTs by ES/MS with a DP range of 2-7 for L. corniculatus and 4-6 for L pedunculatus. Chemical degradation and MS techniques have shown that dispersion of polymer size, for L. corniculatus and L. pedunculatus CTs, and not solely their concentration in the plant may influence biological activity. (C) 2004 Elsevier B.V. All rights reserved.