Ascaris suum is one of the most prevalent nematode parasites in pigs and causes significant economic losses, and also serves as a good model for A. lumbricoides, the large roundworm of humans that is ubiquitous in developing countries and causes malnutrition, stunted growth and compromises immunity to other pathogens. New treatment options for Ascaris infections are urgently needed, to reduce reliance on the limited number of synthetic anthelmintic drugs. In areas where Ascaris infections are common, ethno-pharmacological practices such as treatment with natural plant extracts are still widely employed. However, scientific validation of these practices and identification of the active compounds are lacking, although observed effects are often ascribed to plant secondary metabolites such as tannins. Here, we extracted, purified and characterised a wide range of condensed tannins from diverse plant sources and investigated anthelmintic effects against A. suum in vitro. We show that condensed tannins can have potent, direct anthelmintic effects against A. suum, as evidenced by reduced migratory ability of newly hatched third-stage larvae and reduced motility and survival of fourth-stage larvae recovered from pigs. Transmission electron microscopy showed that CT caused significant damage to the cuticle and digestive tissues of the larvae. Furthermore, we provide evidence that the strength of the anthelmintic effect is related to the polymer size of the tannin molecule. Moreover, the identity of the monomeric structural units of tannin polymers may also have an influence as gallocatechin and epigallocatechin monomers exerted significant anthelmintic activity whereas catechin and epicatechin monomers did not. Therefore, our results clearly document direct anthelmintic effects of condensed tannins against Ascaris and encourage further in vivo investigation to determine optimal strategies for the use of these plant compounds for the prevention and/or treatment of ascariosis.

Condensed tannins (CTs) account for up to 20% of the dry matter in forage legumes used as ruminant feeds. Beneficial animal responses to CTs have included improved growth, milk and wool production, fertility, and reduced methane emissions and ammonia volatilization from dung or urine. Most important is the ability of such forages to combat the effects of gastrointestinal parasitic nematodes. Inconsistent animal responses to CTs were initially attributed to concentration in the diet, but recent research has highlighted the importance of their molecular structures, as well as concentration, and also the composition of the diet containing the CTs. The importance of CT structural traits cannot be underestimated. Interdisciplinary research is the key to unraveling the relationships between CT traits and bioactivities and will enable future on‐farm exploitation of these natural plant compounds. Research is also needed to provide plant breeders with guidelines and screening tools to optimize CT traits, in both the forage and the whole diet. In addition, improvements are needed in the competitiveness and agronomic traits of CT‐containing legumes and our understanding of options for their inclusion in ruminant diets. Farmers need varieties that are competitive in mixed swards and have predictable bioactivities. This review covers recent results from multidisciplinary research on sainfoin (Onobrychis Mill. spp.) and provides an overview of current developments with several other tanniniferous forages. Tannin chemistry is now being linked with agronomy, plant breeding, animal nutrition, and parasitology. The past decade has yielded considerable progress but also generated more questions—an enviable consequence of new knowledge!

This paper presents the development of a rapid method with ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for the qualitative and quantitative analyses of plant proanthocyanidins directly from crude plant extracts. The method utilizes a range of cone voltages to achieve the depolymerization step in the ion source of both smaller oligomers and larger polymers. The formed depolymerization products are further fragmented in the collision cell to enable their selective detection. This UPLC-MS/MS method is able to separately quantitate the terminal and extension units of the most common proanthocyanidin subclasses, that is, procyanidins and prodelphinidins. The resulting data enable (1) quantitation of the total proanthocyanidin content, (2) quantitation of total procyanidins and prodelphinidins including the procyanidin/prodelphinidin ratio, (3) estimation of the mean degree of polymerization for the oligomers and polymers, and (4) estimation of how the different procyanidin and prodelphinidin types are distributed along the chromatographic hump typically produced by large proanthocyanidins. All of this is achieved within the 10 min period of analysis, which makes the presented method a significant addition to the chemistry tools currently available for the qualitative and quantitative analyses of complex proanthocyanidin mixtures from plant extracts.

Plants containing condensed tannins (CTs) may hold promise as alternatives to synthetic anthelmintic (AH) drugs for controlling gastrointestinal nematodes (GINs). However, the structural features that contribute to the AH activities of CTs remain elusive. This study probed the relationships between CT structures and their AH activities. Eighteen plant resources were selected on the basis of their diverse CT structures. From each plant resource, two CT fractions were isolated and their in vitro AH activities were measured with the larval exsheathment inhibition assay, which was applied to Haemonchus contortus and Trichostrongylus colubriformis. Calculation of mean EC50 values indicated that H. contortus was more susceptible than T. colubriformis to the different fractions and that the F1 fractions were less efficient than the F2 ones, as indicated by the respective mean values for H. contortus, F1 = 136.9 ± 74.1 μg/mL and F2 = 108.1 ± 53.2 μg/mL, and for T. colubriformis, F1 = 233 ± 54.3 μg/mL and F2 = 166 ± 39.9 μg/mL. The results showed that the AH activity against H. contortus was associated with the monomeric subunits that give rise to prodelphinidins (P < 0.05) and with CT polymer size (P < 0.10). However, for T. colubriformis AH activity was correlated only with prodelphinidins (P < 0.05). These results suggest that CTs have different modes of action against different parasite species.

Proanthocyanidins (PAs) in sainfoin (Onobrychis viciifolia Scop.) are of interest to ameliorate the sustainability of livestock production. However, sainfoin forage yield and PA concentrations, as well as their composition, require optimization. Individual plants of 27 sainfoin accessions from four continents were analyzed with LC-ESI-QqQ-MS/MS for PA concentrations and simple phenolic compounds. Large variability existed in PA concentrations (23.0–47.5 mg g–1 leaf dry matter (DM)), share of prodelphinidins (79–96%), and mean degree of polymerization (11–14) among, but also within, accessions. PAs were mainly located in leaves (26.8 mg g–1 DM), whereas stems had less PAs (7.8 mg g–1 DM). Overall, high-yielding plants had lower PA leaf concentrations (R2 = 0.16, P < 0.001) and fewer leaves (R2 = 0.66, P < 0.001). However, the results show that these two trade-offs between yield and bioactive PAs can be overcome.

BackgroundPlant-derived condensed tannins (CT) show promise as a complementary option to treat gastrointestinal helminth infections, thus reducing reliance on synthetic anthelmintic drugs. Most studies on the anthelmintic effects of CT have been conducted on parasites of ruminant livestock. Oesophagostomum dentatum is an economically important parasite of pigs, as well as serving as a useful laboratory model of helminth parasites due to the ability to culture it in vitro for long periods through several life-cycle stages. Here, we investigated the anthelmintic effects of CT on multiple life cycle stages of O. dentatum.MethodsExtracts and purified fractions were prepared from five plants containing CT and analysed by HPLC-MS. Anthelmintic activity was assessed at five different stages of the O. dentatum life cycle; the development of eggs to infective third-stage larvae (L3), the parasitic L3 stage, the moult from L3 to fourth-stage larvae (L4), the L4 stage and the adult stage.ResultsFree-living larvae of O. dentatum were highly susceptible to all five plant extracts. In contrast, only two of the five extracts had activity against L3, as evidenced by migration inhibition assays, whilst three of the five extracts inhibited the moulting of L3 to L4. All five extracts reduced the motility of L4, and the motility of adult worms exposed to a CT-rich extract derived from hazelnut skins was strongly inhibited, with electron microscopy demonstrating direct damage to the worm cuticle and hypodermis. Purified CT fractions retained anthelmintic activity, and depletion of CT from extracts by pre-incubation in polyvinylpolypyrrolidone removed anthelmintic effects, strongly suggesting CT as the active molecules.ConclusionsThese results suggest that CT may have promise as an alternative parasite control option for O. dentatum in pigs, particularly against adult stages. Moreover, our results demonstrate a varied susceptibility of different life-cycle stages of the same parasite to CT, which may offer an insight into the anthelmintic mechanisms of these commonly found plant compounds.

SUMMARYAn in vitro study was conducted to investigate the effects of condensed tannin (CT) structural properties, i.e. average polymer size (or mean degree of polymerization), percentage of cis flavan-3-ols and percentage of prodelphinidins in CT extracts on methane (CH4) production and fermentation characteristics. Condensed tannins were extracted from eight plants in order to obtain different CT types: blackcurrant leaves, goat willow leaves, goat willow twigs, pine bark, redcurrant leaves, sainfoin plants, weeping willow catkins and white clover flowers. They were analysed for CT content and CT composition by thiolytic degradation, followed by high performance liquid chromatography (HPLC) analysis. Grass silage was used as a control substrate. Condensed tannins were added to the substrate at a concentration of 40 g/kg, with or without polyethylene glycol (+ or −PEG 6000 treatment) to inactivate tannins, then incubated for 72 h in mixed buffered rumen fluid from three different lactating dairy cows per run. Total cumulative gas production (GP) was measured by an automated GP system. During the incubation, 12 gas samples (10 µl) were collected from each bottle headspace at 0, 2, 4, 6, 8, 12, 24, 30, 36, 48, 56 and 72 h of incubation and analysed for CH4. A modified Michaelis-Menten model was fitted to the CH4 concentration patterns and model estimates were used to calculate total cumulative CH4 production (GPCH4). Total cumulative GP and GPCH4 curves were fitted using biphasic and monophasic modified Michaelis-Menten models, respectively. Addition of PEG increased GP, GPCH4, and CH4 concentration compared with the −PEG treatment. All CT types reduced GPCH4 and CH4 concentration. All CT increased the half time of GP and GPCH4. Moreover, all CT decreased the maximum rate of fermentation for GPCH4 and rate of substrate degradation. The correlation between CT structure and GPCH4 and fermentation characteristics showed that the proportion of prodelphinidins within CT had the largest effect on fermentation characteristics, followed by average polymer size and percentage of cis flavan-3-ols.

Studies with a diverse array of 22 purified condensed tannin (CT) samples from nine plant species demonstrated that procyanidin/prodelphinidin (PC/PD) and cis/trans-flavan-3-ol ratios can be appraised by (1)H-(13)C HSQC NMR spectroscopy. The method was developed from samples containing 44-∼100% CT, PC/PD ratios ranging from 0/100 to 99/1, and cis/trans ratios ranging from 58/42 to 95/5 as determined by thiolysis with benzyl mercaptan. Integration of cross-peak contours of H/C-6' signals from PC and of H/C-2',6' signals from PD yielded nuclei-adjusted estimates that were highly correlated with PC/PD ratios obtained by thiolysis (R(2) = 0.99). cis/trans-Flavan-3-ol ratios, obtained by integration of the respective H/C-4 cross-peak contours, were also related to determinations made by thiolysis (R(2) = 0.89). Overall, (1)H-(13)C HSQC NMR spectroscopy appears to be a viable alternative to thiolysis for estimating PC/PD and cis/trans ratios of CT if precautions are taken to avoid integration of cross-peak contours of contaminants.