Aina Ramsay

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 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.

Use of pelleted sericea lespedeza (Lespedeza cuneata) for natural control of coccidia and gastrointestinal nematodes in weaned goats.

Infection with Eimeria spp. (coccidia) can be devastating in goats, particularly for young, recently-weaned kids, resulting in diarrhea, dehydration, and even death. Feeding dried sericea lespedeza [SL; Lespedeza cuneata (Dum.-Cours.) G. Don.] to young goats has been reported to reduce the effects of internal parasites, including gastrointestinal nematodes (GIN) but there have been no reports of the effects of feeding this forage on Eimeria spp. in goats. Two confinement feeding experiments were completed on recently-weaned intact bucks (24 Kiko-cross, Exp. 1; 20 Spanish, Exp. 2) to determine effects of SL pellets on an established infection of GIN and coccidia. The bucks were assigned to 1 of 2 (Exp. 1) or 3 (Exp. 2) treatment groups based upon the number of Eimeria spp. oocysts per gram (OPG) of feces. In Exp. 1, the kids were fed 1 of 2 pelleted rations ad libitum; 90% SL leaf meal+10% of a liquid molasses/lignin binder mix and a commercial pellet with 12% crude protein (CP) and 24% acid detergent fiber (n=12/treatment group, 2 animals/pen). For Exp. 2, treatment groups were fed (1) 90% SL leaf meal pellets from leaves stored 3 years (n=7), (2) 90% SL pellets from leaf meal stored less than 6 months, (n=7), and the commercial pellets (n=6) ad libitum. For both trials, fecal and blood samples were taken from individual animals every 7 days for 28 days to determine OPG and GIN eggs per gram (EPG) and packed cell volume (PCV), respectively. In Exp. 2, feces were scored for consistency (1=solid pellets, 5=slurry) as an indicator of coccidiosis. In Exp. 1, EPG (P<0.001) and OPG (P<0.01) were reduced by 78.7% and 96.9%, respectively, 7 days after initiation of feeding in goats on the SL pellet diet compared with animals fed the control pellets. The OPG and EPG remained lower in treatment than control animals until the end of the trial. In Exp. 2, goats fed new and old SL leaf meal pellets had 66.2% and 79.2% lower (P<0.05) EPG and 92.2% and 91.2% lower (P<0.05) OPG, respectively, than control animals within 7 days, and these differences were maintained or increased throughout the trial. After 4 weeks of pellet feeding in Exp. 2, fecal scores were lower (P<0.01) in both SL-fed groups compared with control animals, indicating fewer signs of coccidiosis. There was no effect of diet on PCV values throughout either experiment. Dried, pelleted SL has excellent potential as a natural anti-coccidial feed for weaned goats.

Protein Precipitation Behavior of Condensed Tannins from Lotus pedunculatus and Trifolium repens with Different Mean Degrees of Polymerization

The precipitation of bovine serum albumin (BSA), lysozyme (LYS), and alfalfa leaf protein (ALF) by two large- and two medium-sized condensed tannin (CT) fractions of similar flavan-3-ol subunit composition is described. CT fractions isolated from white clover flowers and big trefoil leaves exhibited high-purity profiles by 1D/2D NMR and purities >90% (determined by thiolysis). At pH 6.5, large CTs with a mean degree of polymerization (mDP) of ∼18 exhibited similar protein precipitation behaviors and were significantly more effective than medium CTs (mDP ∼9). Medium CTs exhibited similar capacities to precipitate ALF or BSA, but showed small but significant differences in their capacity to precipitate LYS. All CTs precipitated ALF more effectively than BSA or LYS. Aggregation of CT-protein complexes likely aided precipitation of ALF and BSA, but not LYS. This study, one of the first to use CTs of confirmed high purity, demonstrates that the mDP of CTs influences protein precipitation efficacy.

Anthelmintic activity of trans -cinnamaldehyde and A- and B-type proanthocyanidins derived from cinnamon ( Cinnamomum verum )

Cinnamon (Cinnamomum verum) has been shown to have anti-inflammatory and antimicrobial properties, but effects on parasitic worms of the intestine have not been investigated. Here, extracts of cinnamon bark were shown to have potent in vitro anthelmintic properties against the swine nematode Ascaris suum. Analysis of the extract revealed high concentrations of proanthocyanidins (PAC) and trans-cinnamaldehyde (CA). The PAC were subjected to thiolysis and HPLC-MS analysis which demonstrated that they were exclusively procyanidins, had a mean degree of polymerization of 5.2 and 21% of their inter-flavan-3-ol links were A-type linkages. Purification of the PAC revealed that whilst they had activity against A. suum, most of the potency of the extract derived from CA. Trichuris suis and Oesophagostomum dentatum larvae were similarly susceptible to CA. To test whether CA could reduce A. suum infection in pigs in vivo, CA was administered daily in the diet or as a targeted, encapsulated dose. However, infection was not significantly reduced. It is proposed that the rapid absorption or metabolism of CA in vivo may prevent it from being present in sufficient concentrations in situ to exert efficacy. Therefore, further work should focus on whether formulation of CA can enhance its activity against internal parasites.

Condensed tannins in extracts from European medicinal plants and herbal products.

Medicinal plant materials are not usually analysed for condensed tannins (CT). Thirty commercially available European medicinal plants and herbal products were screened for CT and fourteen CT samples were analysed in detail. This is also the first comprehensive CT analysis of pine buds, walnut leaves, heather flowers and great water dock roots. Acetone/water extracts contained between 3.2 and 25.9 g CT/100g of extract, had CT with mean degrees of polymerisation of 2.9 to 13.3, procyanidin/prodelphinidin ratios of 1.6/98.4 to 100/0 and cis/trans flavan-3-ol ratios of 17.7/82.3 to 97.3/2.7. The majority of samples contained procyanidins, four contained A-type linkages (blackthorn flowers, heather flowers, bilberry leaves and cowberry leaves) and one sample also had galloylated procyanidins (great water dock roots).

Identification of structural features of condensed tannins that affect protein aggregation

A diverse panel of condensed tannins was used to resolve the confounding effects of size and subunit composition seen previously in tannin-protein interactions. Turbidimetry revealed that size in terms of mean degree of polymerisation (mDP) or average molecular weight (amw) was the most important tannin parameter. The smallest tannin with the relatively largest effect on protein aggregation had an mDP of ~7. The average size was significantly correlated with aggregation of bovine serum albumin, BSA (mDP: r = -0.916; amw: r = -0.925; p<0.01; df = 27), and gelatin (mDP: r = -0.961; amw: r = -0.981; p<0.01; df = 12). The procyanidin/prodelphinidin and cis-/trans-flavan-3-ol ratios gave no significant correlations. Tryptophan fluorescence quenching indicated that procyanidins and cis-flavan-3-ol units contributed most to the tannin interactions on the BSA surface and in the hydrophobic binding pocket (r = 0.677; p<0.05; df = 9 and r = 0.887; p<0.01; df = 9, respectively). Circular dichroism revealed that higher proportions of prodelphinidins decreased the apparent α-helix content (r = -0.941; p<0.01; df = 5) and increased the apparent β-sheet content (r = 0.916; p<0.05; df = 5) of BSA.

Galloylated proanthocyanidins from shea (Vitellaria paradoxa) meal have potent anthelmintic activity against Ascaris suum.

Proanthocyanidins (PA) from shea (Vitellaria paradoxa) meal were investigated by thiolytic degradation with benzyl mercaptan and the reaction products were analysed by high performance liquid chromatography-mass spectrometry. These PA were galloylated (≈40%), contained only B-type linkages and had a high proportion of prodelphinidins (>70%). The mean degree of polymerisation was 8 (i.e. average molecular size was 2384Da) and epigallocatechin gallate (EGCg) was the major flavan-3-ol subunit in PA. Shea meal also proved to be a potentially valuable source for extracting free flavan-3-ol-O-gallates, especially EGCg (575mg/kg meal), which is known for its health and anti-parasitic benefits. Proanthocyanidins were isolated and tested for bioactivity against Ascaris suum, which is an important parasite of pigs. Migration and motility tests revealed that these PA have potent activity against this parasitic nematode.

Co-operative suppression of inflammatory responses in human dendritic cells by plant proanthocyanidins and products from the parasitic nematode Trichuris suis

Interactions between dendritic cells (DCs) and environmental, dietary and pathogen antigens play a key role in immune homeostasis and regulation of inflammation. Dietary polyphenols such as proanthocyanidins (PAC) may reduce inflammation, and we therefore hypothesized that PAC may suppress lipopolysaccharide (LPS) ‐induced responses in human DCs and subsequent T helper type 1 (Th1) ‐type responses in naive T cells. Moreover, we proposed that, because DCs are likely to be exposed to multiple stimuli, the activity of PAC may synergise with other bioactive molecules that have anti‐inflammatory activity, e.g. soluble products from the helminth parasite Trichuris suis (TsSP). We show that PAC are endocytosed by monocyte‐derived DCs and selectively induce CD86 expression. Subsequently, PAC suppress the LPS‐induced secretion of interleukin‐6 (IL‐6) and IL‐12p70, while enhancing secretion of IL‐10. Incubation of DCs with PAC did not affect lymphocyte proliferation; however, subsequent interferon‐γ production was markedly suppressed, while IL‐4 production was unaffected. The activity of PAC was confined to oligomers (degree of polymerization ≥ 4). Co‐pulsing DCs with TsSP and PAC synergistically reduced secretion of tumour necrosis factor‐α, IL‐6 and IL‐12p70 while increasing IL‐10 secretion. Moreover, both TsSP and PAC alone induced Th2‐associated OX40L expression in DCs, and together synergized to up‐regulate OX40L. These data suggest that PAC induce an anti‐inflammatory phenotype in human DCs that selectively down‐regulates Th1 response in naive T cells, and that they also act cooperatively with TsSP. Our results indicate a novel interaction between dietary compounds and parasite products to influence immune function, and may suggest that combinations of PAC and TsSP can have therapeutic potential for inflammatory disorders.

Sodium Hydroxide Enhances Extractability and Analysis of Proanthocyanidins in Ensiled Sainfoin (Onobrychis viciifolia).

Little information exists on the effects of ensiling on condensed tannins or proanthocyanidins. The acetone-butanol-HCl assay is suitable for measuring proanthocyanidin contents in a wide range of samples, silages included, but provides limited information on proanthocyanidin composition, which is of interest for deciphering the relationships between tannins and their bioactivities in terms of animal nutrition or health. Degradation with benzyl mercaptan (thiolysis) provides information on proanthocyanidin composition, but proanthocyanidins in several sainfoin silages have proved resistant to thiolysis. We now report that a pretreatment step with sodium hydroxide prior to thiolysis was needed to enable their analysis. This alkaline treatment increased their extractability from ensiled sainfoin and facilitated especially the release of larger proanthocyanidins. Ensiling reduced assayable proanthocyanidins by 29%, but the composition of the remaining proanthocyanidins in silage resembled that of the fresh plants.