Investigations into the chemical analysis and bioactivity of plant proanthocyanidins to support sustainable livestock farming

Proanthocyanidins (PA) in some forage legumes have been linked to contradictory effects in animal health and nutrition. Ruminants fed with PA-containing plants do not suffer from bloat and can also reduce gastrointestinal parasite infections, improve protein use efficiency and reduce greenhouse gas emissions. Plants have a wide range of PA contents and compositions. Therefore, screening tools are required to determine the optimal contents and types to exploit PA bioactivities on farms. This research initially focused on the identification and isolation of PAs with contrasting characteristics from various plant species. These samples were then used to develop novel methods such as UPLC-ESI-MS/MS and 13C HSQC NMR for extractable PAs and 13C CPMAS NMR for PAs within plants, and to probe structure-activity relationships. These PAs were also subjected to complementary analytical methods, which demonstrated that depolymerisation techniques can provide quantitative information on PA contents and compositions and mass spectrometric techniques on molecular distributions. These analyses revealed an enormous range of molecular profiles. This diversity, however, led to good but not excellent correlations between the degradation methods. It also affected mass spectrometric and liquid-state NMR responses. In particular, there were some discrepancies between thiolysis-HPLC and UPLC-ESI-MS/MS results of sainfoin PA extracts. For solid-state NMR, the PAs from model plants proved too homogeneous for the analysis of the highly complex PAs in sainfoin plants. Nevertheless, this method could rank accessions on the basis of PA composition and discriminated between plant organs via signature spectra. Therefore, final decisions on which of these methods to use will depend on the research objectives and sample numbers. Finally, anti-parasitic assays discovered that the in vitro exsheathement inhibition of the abomasal parasite, Haemonchus contortus was dependent on the average molecular size of purified PA mixtures. In addition, collaborative studies showed that prodelphinidins or PA size also affected some anti-parasitic and ruminal fermentation results

Direct anthelmintic effects of condensed tannins from diverse plant sources against Ascaris suum

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

Anthelmintic activities against Haemonchus contortus or Trichostrongylus colubriformis from small ruminants are influenced by structural features of condensed tannins

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 based on 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 for H.contortus 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

Efficacy of condensed tannins against larval Hymenolepis diminuta (Cestoda) in vitro and in the intermediate host Tenebrio molitor (Coleoptera) in vivo

Natural anti-parasitic compounds in plants such as condensed tannins (CT) have anthelmintic properties against a range of gastrointestinal nematodes, but for other helminths such effects are unexplored. The aim of this study was to assess the effects of CT from three different plant extracts in a model system employing the rat tapeworm, Hymenolepis diminuta, in its intermediate host, Tenebrio molitor. An in vitro study examined infectivity of H. diminuta cysticercoids (excystation success) isolated from infected beetles exposed to different concentrations of CT extracts from pine bark (PB) (Pinus sps), hazelnut pericarp (HN) (Corylus avellana) or white clover flowers (WC) (Trifolium repens), in comparison with the anthelmintic drug praziquantel (positive control). In the in vitro study, praziquantel and CT from all three plant extracts had dose-dependent inhibitory effects on cysticercoid excystation. The HN extract was most effective at inhibiting excystation, followed by PB and WC. An in vivo study was carried out on infected beetles (measured as cysticercoid establishment) fed different doses of PB, HN and praziquantel. There was a highly significant inhibitory effect of HN on cysticercoid development (p = 0.0002). Overall, CT showed a promising anti-cestodal effect against the metacestode stage of H. diminuta

Carbon-13 cross-polarization magic-angle spinning nuclear magnetic resonance (CPMAS NMR) for measuring proanthocyanidin content and procyanidin to prodelphinidin ratio in sainfoin (Onobrychis viciifolia) tissues

A procedure based on 13C CPMAS NMR was developed to study procyanidins (PCs) and prodelphinidins (PDs) directly in milled sainfoin plant tissues. Blackcurrant and Tilia samples enabled reference spectra of purified proanthocyanidin (PA) fractions, crude extracts and milled plant tissues, with characteristic resonances at 155, 144 and 132 ppm. PC/PD ratios were estimated from the I132/I155 intensity ratio and differed by 2.5 to 5.9% compared to thiolysis data. Normalization to the 155 ppm signal intensity from reference spectra enabled analysis of PA contents with an error of ca 8 g PAs/100 g plant tissue. The procedure estimates the lignin contribution and allows for a correction of the PA content. In six sainfoin accessions, estimated PA contents, were 1.6- to 20.8-fold higher than the thiolysis and 1.4- to 2.6-fold higher than the HCl-butanol-acetone results. Method differences may reflect the presence of unextractable, possibly high molecular weight PAs in sainfoin

Proanthocyanidins inhibit Ascaris suum glutathione-S-transferase activity and increase susceptibility of larvae to levamisole and ivermectin in vitro

Proanthocyanidins (PAC) are a class of plant secondary metabolites commonly found in the diet that have shown potential to control gastrointestinal nematode infections. The anti-parasitic mechanism(s) of PAC remain obscure, however the protein-binding properties of PAC suggest that disturbance of key enzyme functions may be a potential mode of action. Glutathione-S-transferases (GSTs) are essential for parasite detoxification and have been investigated as drug and vaccine targets. Here, we show that purified PAC strongly inhibit the activity of both recombinant and native GSTs from the parasitic nematode Ascaris suum. As GSTs are involved in detoxifying xenobiotic substances within the parasite, we hypothesised that this inhibition may render parasites hyper-susceptible to anthelmintic drugs. Migration inhibition assays with A. suum larvae demonstrated that the potency of levamisole (LEV) and ivermectin (IVM) were significantly increased in the presence of PAC purified from pine bark (4.6-fold and 3.2-fold reduction in IC50 value for LEV and IVM, respectively). Synergy analysis revealed that the relationship between PAC and LEV appeared to be synergistic in nature, suggesting a specific enhancement of LEV activity, whilst the relationship between PAC and IVM was additive rather than synergistic, suggesting independent actions. Our results demonstrate that these common dietary compounds may increase the efficacy of synthetic anthelmintic drugs in vitro, and also suggest one possible mechanism for their well-known anti-parasitic activity

Impact of chemical structure of flavanol monomers and condensed tannins on in vitro anthelmintic activity against bovine nematodes

Plants containing condensed tannins (CT) may have potential to control gastrointestinal nematodes (GIN) of cattle. The aim was to investigate the anthelmintic activities of four flavan-3-ols, two galloyl derivatives and 14 purified CT fractions, and to define which structural features of CT determine the anti-parasitic effects against the main cattle nematodes. We used in vitro tests targeting L1 larvae (feeding inhibition assay) and adults (motility assay) of Ostertagia ostertagi and Cooperia oncophora. In the larval feeding inhibition assay, O. ostertagi L1 were significantly more susceptible to all CT fractions than C. oncophora L1. The mean degree of polymerization of CT (i.e. average size) was the most important structural parameter: large CT reduced larval feeding more than small CT. The flavan-3-ols of prodelphinidin (PD)-type tannins had a stronger negative influence on parasite activity than the stereochemistry, i.e. cis- vs trans-configurations, or the presence of a gallate group. In contrast, for C. oncophora high reductions in the motility of larvae and adult worms were strongly related with a higher percentage of PDs within the CT fractions while there was no effect of size. Overall, the size and the percentage of PDs within CT seemed to be the most important parameters that influence anti-parasitic activity

Structural features of condensed tannins affect in vitro ruminal methane production and fermentation characteristics

An in vitro study was conducted to investigate the effects of condensed tannins (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 production (CH4) and fermentation characteristics. CT were extracted from eight plants in order to obtain different CT types: black currant leaves, goat willow leaves, goat willow twigs, pine bark, red currant leaves, sainfoin plants, weeping willow catkins and white clover flowers. They were analysed for CT content and CT composition by thiolytic degradation, followed by 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, and 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 gas production 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 analyzed 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 gas production and GPCH4 curves were fitted using biphasic and monophasic modified Michaelis-Menten models, respectively. Addition of PEG increased GP, GPCH4, and CH4 concentration compared to 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 27 polymer size and percentage of cis-flavan-3-ols

Facile purification of milligram to gram quantities of condensed tannins according to mean degree of polymerization and flavan-3-ol subunit composition

Unambiguous investigation of condensed tannin (CT) structure-activity relationships in biological systems requires well-characterized, high-purity CTs. Sephadex LH-20 and Toyopearl HW-50F resins were compared for separating CTs from acetone/water extracts and column fractions analyzed for flavan-3-ol subunits, mean degree of polymerization (mDP) and purity. Toyopearl HW-50F generated fractions with higher mDP values and better separation of procyanidins (PC) and prodelphinidins (PD) but required a pre-purification step, more time for large scale purifications and gave poorer recoveries. Therefore, two gradient elution schemes were developed for CT purification on Sephadex LH-20 providing 146 - 2000 mg/fraction. Fractions were analyzed by thiolysis and NMR spectroscopy. In general, PC/PD ratios decreased and mDP increased during elution. 1H NMR spectroscopy served as a rapid screening tool to qualitatively determine CT enrichment and carbohydrate impurities present, guiding fractionation towards re-purification or 1H-13C HSQC NMR spectroscopy and thiolysis. These protocols provide options for preparing highly pure CT samples

Large variability of proanthocyanidin content and composition in sainfoin (onobrychis viciifolia)

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