Subathira Sivakumaran

Effect of flavan-3-ols on in vitro egg hatching, larval development and viability of infective larvae of Trichostrongylus colubriformis.

The effects of flavan-3-ols (the monomer units of condensed tannins (CT)) and their galloyl derivatives on the viability of eggs, the development of first stage (L1) larvae, and the viability of the infective larvae of Trichostrongylus colubriformis were investigated under in vitro conditions. Each of the flavan-3-ol gallates showed some inhibition of egg hatching at 100 microg/ml, and 100% inhibition at 1000 microg/ml, with epigallocatechin gallate being the most effective in the egg hatch (EH) assay. In contrast, none of the flavan-3-ols were able to completely inhibit egg hatching. The flavan-3-ols and galloyl derivatives dose-dependently inhibited the development of infective larvae as assessed by the larval development (LD) assay. A larval migration inhibition (LMI) assay was used to assess the effect of flavan-3-ols and their galloyl derivatives on the motility of the infective third-stage (L3) larvae of T. colubriformis. In general, the flavan-3-ol gallates were more effective than the flavan-3-ols at immobilising the infective larvae as evidenced by their ability to inhibit more (P<0.05-0.01) larvae from passing through the LMI sieves. At 500 microg/ml, epigallocatechin gallate inhibited significantly more (P<0.1) larvae from passing through the sieves than did catechin gallate, epicatechin gallate, or gallocatechin gallate. Comparisons were made between the flavan-3-ols and their galloyl derivatives with the in vitro effects of CT extracts from several forage legumes, which have exhibited effects on parasites in vivo. The forage legumes tested at 200-500 microg/ml reduced the proportion of eggs that hatch, with comparable results to those obtained using the flavan-3-ols. The activities may be influenced by the prodelphinidin: procyanidin (PD:PC) ratios: CT extracts from Lotus pendunculatus and sainfoin have PD:PC ratios of 70:30 and 77:23, respectively, whereas the less active CT extract from Lotus corniculatus has a PD:PC ratio of 27:73. The active CT extracts from forage legumes have epigallocatechin as the dominant flavan-3-ol extender unit, and epigallocatechin is the most active flavan-3-ol in both the EH and LD assays.

Alkylation or Silylation for Analysis of Amino and Non-Amino Organic Acids by GC-MS?

Gas chromatography–mass spectrometry (GC-MS) is a widely used analytical technique in metabolomics. GC provides the highest resolution of any standard chromatographic separation method, and with modern instrumentation, retention times are very consistent between analyses. Electron impact ionization and fragmentation is generally reproducible between instruments and extensive libraries of spectra are available that enhance the identification of analytes. The major limitation is the restriction to volatile analytes, and hence the requirement to convert many metabolites to volatile derivatives through chemical derivatization. Here we compared the analytical performance of two derivatization techniques, silylation (TMS) and alkylation (MCF), used for the analysis of amino and non-amino organic acids as well as nucleotides in microbial-derived samples. The widely used TMS derivatization method showed poorer reproducibility and instability during chromatographic runs while the MCF derivatives presented better analytical performance. Therefore, alkylation (MCF) derivatization seems to be preferable for the analysis of polyfunctional amines, nucleotides and organic acids in microbial metabolomics studies.

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, 13C 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.