Synergistic inhibition of Haemonchus contortus exsheathment by flavonoid monomers and condensed tannins
- Under a Creative Commons license
Highlights
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- Tannins and flavonoids showed anti-parasitic effects against Haemonchus contortus.
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- This is the first report of their synergistic effects on larval exsheathment.
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- Flavonoids enhanced especially the anthelmintic activities of procyanidin tannins.
Abstract
This study investigated the separate and combined anthelmintic (AH) effects of different phenolic compounds, including condensed tannins and flavonoids, all of which are known to occur in willow leaves, a potentially valuable dry season feed. A range of contrasting model tannins, which span the whole range of willow tannins, were isolated from tilia flowers, goat willow leaves, black currant leaves and red currant leaves. All together, the tested compounds represented the major tannin types (procyanidins and prodelphinidins) and flavonoid types (flavonols, flavones and flavanones). The larval exsheathment inhibition assay (LEIA) was used to assess their in vitro effects onHaemonchus contortus third stage larvae. Arbutin, vanillic acid, and taxifolin proved to be ineffective whereas naringenin, quercetin and luteolin were highly effective at 250 μM concentrations. Procyanidin (PC) tannins tended to be less active than prodelphinidin tannins (PD). Experiments with combinations of tannins and quercetin or luteolin revealed for the first time the existence of synergistic AH effects between tannins and flavonoid monomers. They also provided evidence that synergistic effects appear to occur at slightly lower concentrations of PC than PD. This suggests that the AH activity of condensed tannins can be significantly enhanced by the addition of quercetin or luteolin. This information may prove useful for plant breeding or selection and for designing optimal feed mixtures.
Graphical abstract
Keywords
- Nematodes;
- Procyanidins;
- Prodelphinidins;
- Proanthocyanidins;
- Mean degree of polymerisation;
- Flavan-3-ols;
- Quercetin;
- Luteolin
1. Introduction
Infection of small ruminants with gastrointestinal nematodes (GINs) remains a serious pathological problem across the world affecting animal health, welfare and production, as there is now a critically high level of drug resistance (Jackson et al., 2012). These GINs are able to develop resistance to new synthetic (anthelmintic, AH) drugs within a few years (Waller, 2006) and, therefore, sustainable livestock farming can no longer rely on deworming with AH drugs. New approaches for the sustainable control of GIN seek to lower parasite numbers to a manageable level or to modify their biological development and life cycle, rather than to eliminate them completely. Medicinal plants have been used since antiquity to treat worm infections (Hrckova and Velebny, 2013) and anti-parasitic properties of plants against GIN have been linked to the presence of proteinases (Stepek et al., 2004) and other secondary plant metabolites. Plant phenolics, flavonoids and condensed tannins have received considerable attention over the last few years (Barrau et al., 2005, Hoste et al., 2006 and Hoste et al., 2012) as plants that produce these compounds grow in all regions of the world. Condensed tannins (CT; syn. proanthocyanidins) can have direct and indirect AH effects ( Min et al., 2003 and Hoste et al., 2012), as well as having the potential to enhance the host's innate immune response ( Tibe et al., 2012). The hypothesis for direct effects of polyphenolic compounds has been substantiated by several in vitro assays against different life-cycle stages ( Bahuaud et al., 2006 and Novobilský et al., 2011).
Condensed tannins are oligomers or polymers of flavan-3-ols and are classified into different subgroups. The two major CT types are procyanidins (PC), which have two OH-groups, and prodelphinidins (PD), which have three OH-groups in the B-ring (Fig. 1). The stereochemistry at the heterocyclic C-ring gives rise to either 2–3 cis- or 2–3 trans-flavan-3-ols and the average polymer length of CT mixtures is described in terms of mean degree of polymerisation (mDP). It is important to note that most condensed tannin-containing forages tend to contain complex mixtures of PC and PD homo- and hetero-polymers that are difficult to separate. However, a few plants specialise in the synthesis of either PCs or PDs and can thus serve as valuable sources for the different tannin types and as research tools for probing structure–activity relationships.
- Fig. 1.Structures of condensed tannins, flavonoids (flavan-3-ols, luteolin, quercetin, naringenin and taxifolin) and other polyphenols (arbutin and vanillic acid).
Since tannins and other flavonoids co-occur in plants, it is pertinent to investigate their combined potencies. To this end, we selected polyphenols, which are known to occur in willow (Salix spp, Salicaceae), as this species represents a potentially valuable nutraceutical forage ( Moore et al., 2003, Diaz Lira et al., 2008 and Mupeyo et al., 2011). Moreover, breeding programmes are currently focussing on increasing the Salix wood production as a renewable source of energy ( Karp et al., 2011) and, therefore, it is timely to explore, which phenolic compositions can contribute most to the AH properties of the leaf by-products. The phenolic composition of willow leaves has been studied already by several groups ( Jarrett and Williams, 1967, Pohjamo et al., 2003 and Enayat and Banerjee, 2009) and we recently reported that willow tannins cover a wide range of PC/PD ratios (from 15/85 to 98/2) and cis/trans flavan-3-ol ratios (from 2/98 to 81/19) (Falchero et al., 2011). We now describe the isolation of distinct PC and PD types from specialist CT plant sources in order to cover the full spectrum of Salix tannins and tested their AH effects in the presence of commercially available phenolic monomers that are known to be present in Salix leaves. This allowed us to test two hypotheses; the first stated that CT and other naturally occurring polyphenolic monomers exert synergistic inhibitory effects on the in vitro L3 exsheathment of Haemonchus contortus and the second that these AH interactions depend on CT types.
2. Materials and methods
2.1. Chemicals and plant materials
Arbutin, luteolin and taxifolin were purchased from Apin Chemicals Ltd (Abingdon, U.K.); vanillic acid and naringenin from Alfa Aesar (Blackpool, U.K.); gallocatechin and quercetin from Sigma–Aldrich (Gillingham, U.K.); phosphate buffered saline (PBS) from bioMerieux (Lyon, France), Milton solution, sodium hypochlorite 2% w/v and sodium chloride 16.5% w/v from Milton (Inibsa laboratorios, Barcelona, Spain). Goat willow (Salix caprea), black currant (Ribes nigrum) and red currant (Ribes rubrum) leaves were collected in July and August 2012. Tilia (Linden) flowers (Tilia × europaea) were purchased from Flos (Mokrsko, Poland). These plant samples were chosen as they allowed the testing of different tannin types, all of which are known to occur in willow.
2.2. Preparation of plant extracts and tannin fractions
Freeze-dried, powdered plant material (25 g of each species) was extracted with an acetone:water mixture (7:3, v/v; 300 ml) (Williams et al., 2014a) and filtered. The filtrate was extracted with dichloromethane (250 ml) and the organic phase discarded. The aqueous phase was rotary evaporated under vacuum at 40 °C to remove residual organic solvents and then lyophilised. The freeze-dried extract was re-dissolved in distilled water, filtered under vacuum to remove insoluble particles and applied to a Sephadex LH-20 column. Fraction 1 was eluted with acetone/water (3:7, v/v) and fraction 2 with acetone/water (1:1, v/v). Acetone was removed in a rotary evaporator and the aqueous residue freeze-dried. Tannins were quantified and characterized by thiolysis with benzylmercaptan (Gea et al., 2011). Tannin composition was determined in terms of the percentage of PCs and PDs, cis- and trans-flavan-3-ol subunits and mean degree of polymerisation (mDP), which were calculated according to Gea et al. (2011).
2.3. Bioassays
2.3.1. Haemonchus contortus strains
Third-stage larvae (L3) were obtained from sheep or goats infected with two monospecific strains of H. contortus that were susceptible to AH drugs ( Alonso-Díaz et al., 2008). The Juan strain was obtained from donor sheep, which were kept indoors and infected monospecifically with H. contortus, and the larvae had been maintained in the laboratory for four months. This strain was used initially to investigate the effects of four F1- and four F2-tannin fractions and of several phenolic compounds. The INRA strain was obtained from monospecifically infected goats and the larvae had been maintained in the laboratory for 1 month before use. This strain was used to repeat experiments with the F2 tannin fractions, two of the flavonoids (quercetin and luteolin) and also with the tannin-flavonoid mixtures. The facilities hosting the animals and the trial was performed according to French ethical and welfare rules (agreement number C 31 555 27 of 19 August 2010).
