Lucy P. Meagher

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.

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.

Antioxidant activity and polyphenol content of green tea flavan-3-ols and oligomeric proanthocyanidins

The antioxidant activity and total phenolics content (TPC) of freshly prepared green tea extract (GTE) as affected by time, temperature and stirring were determined using the ferric reducing antioxidant power (FRAP) and Folin–Ciocalteu assays, respectively. Acetone–water fractions of GTE containing flavan-3-ols and oligomeric proanthocyanidins were evaluated at concentrations between 25 and 500 µg/ml. Increasing the extraction time from 3 min to 10 min resulted in a significant increase in both the FRAP values and TPC. Increasing the extraction time from 10 min to 30 min was without any significant effects on both FRAP and TPC values. Moreover, the FRAP values were correlated with the TPC. GTE fractions had widely different FRAP values that were well correlated with the TPC of the fraction. It was concluded that brewing conditions such as extraction temperature, period of extraction, ratio of tea leaves to extracting water, and stirring are important factors for determining the FRAP values and TPC in GTE. These factors should be taken into consideration during preparation for nutritional benefits during usual consumption of this beverage. Elevated FRAP and TPC values corresponded to those GTE fractions with a higher amount of phenolic compounds, which have stronger antioxidant activities.

The effects of condensed tannins from Dorycnium rectum on skatole and indole ruminal biogenesis for grazing sheep

Skatole and indole are flavour compounds formed in the rumen, which are key factors in the pastoral flavour of meat and milk products. A selection of indolic compounds has been screened for their potential to form skatole and indole on in vitro fermentation, with rumen inocula collected from New Zealand pasture-fed sheep. Typically, 8% (P < 0.005) of added tryptophan was converted to indole and 54% (P < 0.001) was converted to skatole via indole acetic acid (IAA). The addition to rumen inocula of a condensed tannin (CT) fraction isolated from the forage legume, Dorycnium rectum, reduced the conversion of plant protein to skatole and indole by 75% and specifically inhibited the transformation of IAA to skatole by 85% (P < 0.001). The conversion of tryptophan to indole or IAA was not affected by CTs. The inhibitory effect of CTs on the conversion of tryptophan to skatole was prevented by the addition of polyethylene glycol (PEG) to the rumen inocula prior to the addition of CTs, but not by the addition of PEG subsequent to CTs, demonstrating that interactions between CTs and microbes involved in skatole biosynthesis are not reversible by PEG. This study is the first to demonstrate that isolated CTs have an inhibitory effect on the conversion of protein to skatole and indole by rumen microbes, and that CTs specifically inhibit the transformation of IAA to skatole by rumen microbes.

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