PARTIE IV : Etude des interactions
ARTICLE 6 La gaine des larves infestantes des nématodes parasites est la cible des
BRUNET, S., REGOS, I., FEUCHT, W., TREUTTER, D., NEUM LLER, M.,
SANCHEZ, M.E. et HOSTE, H.
RESUME - ARTICLE 6
Introduction
L’utilisation de plantes riches en polyphénols est envisagée comme une
alternative à l’emploi des anthelminthiques (AHs) de synthèse pour la maîtrise du
parasitisme gastro-intestinal (GI). Néanmoins, le mode d’action de ces plantes sur les
nématodes GIs reste mal connu.
Des études fonctionnelles ont montré in vitro qu’un extrait de sainfoin
(Onobrychis viciifolia) affectait la mobilité, le dégainement et la pénétration dans les
muqueuses digestives des larves infestantes (L3s). Récemment, nous avons décrit des
modifications de l’ultra-structure des L3s après un contact avec un extrait de sainfoin.
Dans ces études, les effets de l’extrait de sainfoin ont été attribués aux tannins
condensés (TCs). Les TCs sont des métabolites secondaires, appartenant à la grande
famille des flavonoïdes, qui ont la propriété de se fixer aux protéines.
Afin de mieux comprendre les mécanismes d’action des TCs sur les L3s, nous
avons adapté aux L3s deux méthodes histochimiques, initialement développées pour
l’étude des tissus végétaux. Le réactif de Neu permet de détecter les flavones et les
flavonols par une fluorescence jaune-orange sous lumière UV. La détection des flavan-
3-ols, les monomères des TCs, est réalisée par l’emploi du DMACA, dont la réaction
avec les flavanols induit une coloration bleue.
L’objectif de cette étude concernait la localisation des flavonoïdes chez les L3s en
utilisant le réactif de Neu pour détecter les flavones et les flavonols, et le réactif
DMACA pour détecter les flavanols.
Dans cette étude, le modèle parasitaire était Haemonchus contortus. Néanmoins,
la spécificité des interactions de l’extrait de sainfoin avec les L3s a été évaluée par
comparaison avec des observations sur Trichostrongylus colubriformis.
Matériel et méthodes
Les L3s ont été incubées à 20°C toute la nuit soit dans du PBS (témoin négatif),
soit avec un extrait de sainfoin (60mg/ml), soit avec un extrait de sainfoin prétraité
avec du polyvinyl polypyrrolidone (PVPP) (ratio extrait:PVPP de 1:3), soit avec l’un
des flavan-3-ols (catéchol, épicatéchol, gallocatéchol ou épigallocatéchol) (500µg/ml).
Les flavones et flavonols ont été détectés en appliquant le réactif de Neu aux
L3s. La détection des flavan-3ols a été obtenue par immersion des L3s dans le réactif
au DMACA.
Résultats et discussion
Les observations ont permis la détection des flavonoïdes du sainfoin, par une
fluorescence orange, au niveau de la gaine des L3s d’H.contortus et de
T.colubriformis. Par ailleurs, aucune fluorescence orange n’a été révélée chez les L3s
incubées avec l’extrait de sainfoin prétraité avec du PVPP, suggérant que les
flavonoïdes fixés sur la gaine des L3s sont des flavonols.
La méthode avec le DMACA ne nous a pas permis de confirmer la présence des
flavanols sur les L3s incubées avec l’extrait de sainfoin, probablement du à une
concentration en flavan-3-ols trop faible. Néanmoins, l’incubation des L3s
d’H.contortus avec des flavan-3-ols purs a été associée à une détection positive sur la
gaine selon le flavan-3-ol impliqué, une coloration de la gaine étant observée avec
le gallocatéchol et l’épigallocatéchol mais pas avec le catéchol et l’épicatéchol.
Ainsi, la variabilité des effets des flavan-3-ols selon leur structure, décrite lors
d’études précédentes, serait due à des différences d’affinité des flavan-3-ols pour la
gaine des L3s.
Au vue de ces observations, il est suggéré que les TCs affectent les fonctions
larvaires en interagissant directement avec la gaine des L3s.
THE SHEATH OF INFECTIVE NEMATODE LARVAE IS THE TARGET FOR
FLAVONOIDS FROM SAINFOIN (Onobrychis viciifolia)
BRUNET, S.
(1)(2), REGOS, I.
(3), FEUCHT, W.
(3), TREUTTER, D.
(3), NEUM LLER, M.
(3),
SANCHEZ, M.E.
(1)(2), HOSTE, H.
(1)(2)
(1) INRA, UMR 1225, F-31076 Toulouse, France.
(2) Université de Toulouse ; ENVT ; UMR 1225 ; F-31076 Toulouse, France.
(3) Center of Life and Food Science Weihenstephan, Technische Universität München,
Unit Fruit Science-Fruit Tree Physiology, Dürnast 2. D 85354 Freising, Germany.
SUMMARY
Polyphenol rich forages offer an alternative solution to chemical anthelmintics (AHs) to
control gastrointestinal nematodes (GINs). However the mechanisms of action of such
plants remain obscure in regard to the interactions between GINs and the bioactive
molecules. Previous in vitro studies have shown that sainfoin extracts induced functional
and ultra-structural changes in infective larvae (L3s). It has been suggested that the AH
effects of sainfoin might be due to secondary metabolites, particularly to flavonoids, such
as the condensed tannins (CTs).
We aimed at localizing the site of interaction of flavonoids from sainfoin extract and
single flavan-3-ols on Haemonchus contortus L3s through a histochemical method using
the Neu reagent to detect flavonoids, like flavones and flavonols, and the DMACA reagent
for detecting flavan-3-ols in treated L3s. The specificity of the interactions was evaluated
by comparing observations on H.contortus and a second GIN species, Trichostrongylus
By using the Neu reagent, flavonoids were detected on the sheath of L3s incubated with
sainfoin extract of both H.contortus and T.colubriformis. The addition of polyvinyl
polypyrrolidone (PVPP) to sainfoin extract alleviated flavonoid fluorescence in the sheath.
Unfortunately, the detection of CTs was not achieved in L3s treated with sainfoin extract
probably due to a less sensitivity of DMACA towards CT polymers. However, after
incubation with single GC and EGC, a positive DMACA staining occurred. This indicates
that the hydroxylation pattern of the flavonoid moiety may modify the interaction of
flavonoids with the L3 sheath.
KEY WORDS
Haemonchus contortus; Trichostrongylus colubriformis; Sainfoin (Onobrychis viciifolia);
Condensed Tannins; Flavonoids; Flavan-3-ols; Histochemistry; DMACA reagent; Neu
reagent.
INTRODUCTION
The use of tannin-rich plants represent an alternative solution to synthetic
anthelmintics (AHs) to control parasitic gastro-intestinal nematodes (GINs) in ruminants
(Kahn and Diaz-Hernandez, 2000; Min and Hart, 2002; Min et al., 2003; Waller and
Thamsborg, 2004; Hoste et al., 2006).
Sainfoin (Onobrychis viciifolia) is one of the tannin-rich legume forages whose AH
properties have been examined both in vivo and in vitro. Under in vivo conditions, the
consumption of sainfoin has been associated with changes either in the fertility or the
number of GINs in sheep (Heckendorn et al., 2006; Heckendorn et al., 2007) or in goats
(L3s) has also been described in sheep grazing sainfoin (Thamsborg et al., 2003). Many in
vitro results have confirmed the AH effects of sainfoin extracts on adult worms (Paolini et
al., 2004) or on L3s. In vitro larval migration (Paolini et al., 2004; Barrau et al., 2005), larval
exsheathment (Brunet et al., 2007) and penetration into the digestive mucosae (Brunet et
al., 2008) of Haemonchus contortus and Trichostrongylus colubriformis were significantly
reduced after contact with sainfoin extract. Recently, Brunet et al. (2008 submitted) have
shown that in vitro incubation with sainfoin extracts induced ultra-structural changes in L3s
of both H.contortus and T.colubriformis. The overall conclusions of these studies were that
some secondary metabolites, particularly condensed tannins (CTs), of sainfoin affect the
L3s by a direct mechanism of action. Nevertheless, Barrau et al. (2005) have also
described inhibitory effects of flavonol glycosides, which represent another class of
flavonoids, on L3 mobility.
CTs (also called proanthocyanidins) are defined as polyphenols and belong to the
flavonoid family (Figure 1). CTs have the property to fix macromolecules, including the
proteins (Mueller-Harvey and Mc Allan, 1992; Bruneton, 1999). They present a wide range
of structural variations depending on the monomeric unit, i.e. the flavan-3-ols, and the
degree of polymerisation (Mueller-Harvey and Mc Allan, 1992; Bravo, 1998) (Figure 2). In
the legume forages, they are two main classes of CTs : the procyanindins (PCs), which
consist of catechin (C) and/or epicatechin (EC) units, and the prodelphinidins (PDs), which
consist gallocatechin (GC) and/or epigallocatechin (EGC) units (Marais et al., 2000).
Variations in the biochemical structure of flavan-3-ol have been shown to change their
biological activity (Waterman, 1999; Poncet-Legrand et al., 2006). Concerning the AH
effects, GC and EGC have been described as more potent inhibitors of larval functions
than C and EC (Molan et al., 2003; Brunet and Hoste, 2006; Brunet et al., 2008). A recent
suggested that the highest level of AH activity was associated with the consumption of
plants containing the highest PD/PC ratio, such as sainfoin (Hoste et al., 2006).
In order to understand the mechanisms of action of sainfoin on L3s, the current study
aimed at localizing compounds bound to L3s by using various histochemical methods,
which were originally developed for plant tissue analysis (Neu, 1956; Gutmann and
Feucht, 1994; Li et al., 1996; Laplaze et al., 1999). In the plant analysis, flavones and
flavonols were detected by characteristic yellow to orange fluorescence under UV light
after applying the Neu reagent (Figure 1)(Neu, 1956; Laplaze et al., 1999). Moreover, the
4-dimethylamino-cinnamaldehyde (DMACA) reagent was used for the selective detection
of flavan-3-ols, including CTs (Figure 2) (Treutter, 1989; Gutmann and Feucht, 1994; Li et
al., 1996; Polster et al., 2002; Feucht et al., 2007).
Using Haemonchus contortus as parasite model, the objectives were therefore, using
the Neu reagent, to localize flavonoids from sainfoin extracts on L3s. We also aimed at
detecting the interactions of the CTs from sainfoin extracts and the some individual flavan-
3-ols with L3s using the DMACA reagent. The specificity of the flavonoid detection
according to the GIN species was evaluated by comparing data acquired on H. contortus,
an abomasal species, with those acquired on T.colubriformis, an intestinal species.
7. MATERIALS AND METHODS
Chemicals and sainfoin extract
(+)Catechin (C), (-)epicatechin (EC), (-)gallocatechin (GC), (-)epigallocatechin (EGC),
polyvinyl polypyrrolidone (PVPP), 4-dimethylamino-cinnamaldehyde (DMACA reagent)
and 2-aminoethyl-diphenylborinate (Neu reagent) were purchased from Sigma, St Louis,
An acetone:water (7:3, v:v) extract was prepared from a sainfoin (Onobrychis viciifolia)
hay collected in the South-East of France in June 2005, as previously described by Brunet
et al. (2007; 2008). The condensed tannin content of the sainfoin hay was measured
according to the Butanol-HCl method and was estimated at 2.45 % of the dry matter
(Makkar, 2000).
Infective larvae
The third-stage larvae (L3s) were obtained from donor goats infected with pure strains
of either H. contortus or T. colubriformis. Larvae were stored at 4°C in water before use.
The same batches of 2-to-3-month old larvae were used in the experiments.
7.1 Larval incubation
For each experiment, 2,000 L3s were incubated overnight at 20°C in the experimental
medium or in phosphate buffer solution (PBS; 0.1M phosphate, 0.005M NaCl, pH7.2)
(negative controls). The incubations were run in duplicates. After incubation, L3s were
centrifuged and the supernatant was removed.
Experiment 1: L3s of H. contortus were incubated with solutions of sainfoin extract
dissolved in PBS at a concentration of 60 mg/ml.
Experiment 2: L3s of T. colubriformis were incubated with solutions of sainfoin extract
dissolved in PBS at a concentration of 60 mg/ml.
Experiment 3: A 60 mg/ml sainfoin extract was pre-incubated with polyvinyl pyrrolydone,
(PVPP), a strong binding agent of polyphenols, for 8 hours at 4°C (Ratio extract:PVPP of
1:3) in order to remove all tannins. After centrifugation, the supernatant was used to
incubate L3s. In this experiment, L3s of H. contortus were incubated with either a solution
of sainfoin extract dissolved in PBS at a concentration of 60 mg/ml, or the supernatant of
Experiment 4: L3s of H. contortus were incubated with one of the four flavan-3-ols (C,
EC, GC, or EGC) at a concentration of 500 µg/ml in PBS.
Histochemical methods
Flavones and flavonols from sainfoin extracts were detected by applying the Neu
reagent (Neu, 1956) to L3s incubated according to Experiments 1, 2 or 3. After incubation,
the L3s were immersed overnight in a Neu reagent [1% (w/v) 2-aminoethyl-
diphenylborinate in absolute methanol]. The observations were performed at different
magnifications using a LEICA microscope fitted with a mercury vapour lamp (452- nm
excitation filter and 340- to 380-nm barrier filter). Treatment with the Neu reagent gave an
orange staining, or a bright yellow tint under higher magnifications, in the presence of
flavones and flavonols (Figure 1) (Laplaze et al., 1999; Polster et al., 2002).
To visualize the flavan-3-ols (Experiments 1, 2 and 4) in L3s, the DMACA reagent [1%
(w/v) DMACA dissolved in a cold mixture (4°C) of methanol and 6N HCl (v/v)] was used (Li
et al., 1996). It was applied to L3s for 10 minutes at 20°C. Then, L3s were immediately
observed with a ZEISS light microscope at magnifications of X100 or X200. The reaction
of DMACA with flavan-3-ols yields a blue-coloured product (Figure 2) (Gutmann and
Feucht, 1994).
RESULTS
1. Localization of flavonoids in L3s using the Neu reagent
Under UV light, the body of the control and the L3s incubated with sainfoin extracts was
entirely blue-fluorescent after applying the Neu reagent for both GIN species (Figures 3
In comparison to the control L3s, the treated L3s showed orange deposits on the whole
sheath after UV excitation for H.contortus (Figure 3E) and T.colubriformis (Figure 4D). At
higher magnifications, the sheath of the treated L3s appeared entirely yellow coloured with
orange fluorescent inclusions. In contrast, the sheath of control L3s remained uncoloured
after Neu staining for both H.contortus (Figures 3C and 3D) and T.colubriformis (Figure
4B). The yellow-orange fluorescence was particularly visible on the sheath tail of treated
L3s for both nematode species (Figures 3H and 4D).
2. Consequence of the pre-treatment of sainfoin extract with PVPP
As shown previously, the L3s of H.contortus treated with sainfoin extracts showed a
yellow tint of the sheath with orange fluorescent grains after applying the Neu reagent
(Figure 5B) compared to the control L3s (Figure 5A). In contrast, no differences were
observed between the control L3s and the L3s incubated with PVPP-treated sainfoin
extract (Figure 5C).
3. Localization of flavan-3-ols in L3s using the DMACA reagent
The L3s incubated with the sainfoin extract did not show any blue staining after applying
the DMACA reagent, for both H.contortus and T.colubriformis (Data not shown).
Compared with the control L3s (Figures 6A and 6B), the L3s of H.contortus incubated
with individual flavan-3-ols showed a blue coloured sheath (Figure 6G and 6I) after
DMACA staining, which was particularly visible within the sheath tail (Figures 6H and 6J).
Differences in the sheath colour depended on the flavan-3-ols used to incubate the L3s. In
the case of C (Figures 6C and 6D) or EC (Figures 6E and 6F), no difference was observed
between the control L3s and the treated L3s. However, a deep blue staining of L3s was
DISCUSSION
The general objective of this study was to understand the mechanisms of action of a
flavonoid containing extract on GIN L3s and particularly, to localize the active constituents
of sainfoin extracts in L3s. Therefore, we have adapted the Neu and the DMACA staining
methods to nematode L3s. These histochemical methods were originally developed for
analysis of plant tissues (Neu, 1956; Gutmann and Feucht, 1994; Laplaze et al., 1999) and
more recently have also been applied to bovine tissues (Polster et al., 2002).
In our in vitro incubations, we used a higher concentration of sainfoin extract than in our
previous functional in vitro studies (Brunet et al., 2007; Brunet et al., 2008). The
concentration used in the present study was outside of physiological concentrations of
tannins measured in the gut of sheep fed with tanniniferous plants (Terrill et al., 1994).
However, the purpose of applying such an artificially high concentration was to facilitate
the detection of the flavonoids in L3s using the Neu and the DMACA reagents. It is well-
known that the histochemical detection depends on the concentration of the reactive
compounds in tissue, as demonstrated by Li et al. (1996) for the DMACA method.
The Neu reagent indicates that flavones and flavonols from sainfoin extracts were
mainly localized on the whole sheath of GIN L3s. Moreover, the observations performed
on H.contortus and T.colubriformis were similar suggesting that the interactions between
sainfoin flavonoids and L3s are not specific to the GIN species and not specific of a
particular region of the L3 sheath. This confirms previous results from in vitro studies on
the effects of sainfoin extracts on larval functions (Paolini et al., 2004; Brunet et al., 2007;
Brunet et al., 2008; Brunet, 2008 submitted). These observations suggest that sainfoin
active compounds interfere with L3 functions by interacting with the sheath as previously
Our study also aimed at distinguishing the role of flavonoids on the L3 functions. This
was done firstly by adding the polyphenols binding PVPP to the sainfoin extract and
secondly by staining with the DMACA reagent. It could be argued that the yellow-orange
fluorescence detected on the sheath of GIN L3s treated with sainfoin extracts were
flavonols because it disappeared when the sainfoin extracts were pre-treated with PVPP.
Despite the fact that the staining with DMACA failed in the L3s incubated with sainfoin
extracts, it is obvious that also flavanols, including the CTs, may bind to the sheath. This
assumption was confirmed by treating the L3s with monomeric flavan-3-ols.
The DMACA reaction is highly sensitive to monomeric flavan-3-ols, whereas CTs,
which are polymeric flavan-3-ols, are stained less intensely (Gutmann and Feucht, 1994;
Cadot and Miñana Castelló, 2006). Li et al. (1996) also reported that the DMACA reagent
is not very sensitive towards low CT concentrations. Nonetheless, we have demonstrated
that GC and EGC were localized on the sheath of H.contortus L3s, confirming that the
sheath is potentially the target of CTs.
In this study, the relationship between flavan-3-ol structure and their interaction with
the L3 sheath was investigated. Previous studies have shown that the flavan-3-ol structure
was a key factor in modulating the effects on larval functions (Molan et al., 2003; Molan et
al., 2004; Brunet and Hoste, 2006; Brunet et al., 2008). There is now considerable
evidence to suggest that PD monomers (GC and EGC) are more active than PC
monomers (C and EC). Our observations appear to confirm this structure/response
relationship since the sheath staining were observed with GC and EGC but not with C and
EC. Thus, the observed differences in efficacy of the various flavan-3-ols on L3s could be
explained by difference in their affinities for the L3 sheath.
Whatever the GIN species, the L3 sheath is mainly composed of collagen-like
proteins with high contents of proline and hydroxyproline (Page, 2001). The interactions of
multiple hydrogen bondings between the phenolic groups of flavanol and the functional
groups of collagen, and hydrophobic interactions between the benzopyran rings of flavanol
and the hydrophobic residues of collagen are the major forces involved in these
interactions. The variation in affinity of flavan-3-ols for poly-proline peptides has also been
described (Poncet-Legrand et al., 2006). Based on their chemical structure, PD monomers
have 3 phenolic groups in B-ring whereas PC monomers have only two phenolic groups
(see Figure 2). A higher number of phenolic groups in GC/EGC and in the PDs might
favour increased hydrogen bondings with the sheath proteins. This might explain why
tanniniferous plants containing a high PD/PC ratio, such as sainfoin, have shown the
highest activity against GINs in ruminants (Marais et al., 2000; Min and Hart, 2002; Hoste
et al., 2006).
Overall, our study has shown for the first time that flavonoids, both flavonols and
flavanols, bind to the sheath of gastrointestinal nematodes. However, further research is
still required to identify the flavonoids from sainfoin involved in the interactions, to
understand the mechanisms of action of flavonoids on parasitic nematode and particularly,
to define which kinds of the sheath constituents interact with these phenolics.
ACKNOWLEDGEMENTS
The authors sincerely thank Dr. I. Mueller-Harvey from the University of Reading, United
Kingdom, for her pertinent advices to improve the manuscript.
This work received the financial support from the European Union as part of the Marie-
ABBREVIATIONS
AH: anthelmintic; C: (+)catechin; CT: condensed tannins; EC: (-)epicatechin; EGC:
(-)epigallocatechin; DMACA: 4-Dimethylamino-cinnamaldehyde; GC: (-)gallocatechin;
GINs: gastrointestinal nematodes; L3: third-stage larvae; PBS: phosphate buffer solution;
PCs: procyanidines; PDs: prodelphinidines; PVPP: polyvinyl polypyrrolidone; UV: ultra-
violet.
LEGENDS OF FIGURES
FIGURE 1 :
Illustration of the main classes of flavonoids.
FIGURE 2:
Chemical structure of flavan-3ols and condensed tannins.
FIGURE 3:
Images of Neu reagent stained infective larvae of Haemonchus contortus according to the
incubation either in phosphate buffer solution (negative control)(A, B, C and D) or in
sainfoin extract at 60mg/ml (E, F, G and H). Note the orange (or yellow) deposit (white
arrow) in sainfoin treated larvae.
Scales bars: 25µm in A and E; or 10 µm in B,C, D, F, G and H.
FIGURE 4 :
Images of Neu reagent stained infective larvae of Trichostrongylus colubriformis according
sainfoin extract at 60mg/ml (C and D). Note the orange (or yellow) deposit (white arrow) in
sainfoin treated larvae.
Scales bars: 25µm in A and C; or 10 µm in B and D.
FIGURE 5 :
Images of Neu reagent stained infective larvae of Haemonchus contortus according to the
incubation either in phosphate buffer solution (negative control)(A) or in sainfoin extract at
60mg/ml (B), or in sainfoin extract pre-treated with polyvinyl polypyrrolidone (PVPP)(C).
Note the orange-yellow deposit (white arrow) in sainfoin treated larvae.
Scales bars: 10µm in A, B and C.
FIGURE 6 :
Images of DMACA reagent stained infective larvae of Haemonchus contortus according to
the incubation either in phosphate buffer solution (negative control)(A and B) or with
catechin (C and D), or with epicatechin (E and F), or with gallocatechin (G and H), or with
epigallocatechin (I and J).
FIGURE 1
O
O
Flavone
O
O
OH
Flavonol
* These give a positive reaction with the Neu reagent.
FIGURE 2
O
OH
OH
R
OH
OH
O
H
O
OH
OH
R
OH
OH
O
H
O
OH
OH
R
OH
OH
O
H
O
OH
R3
OH
R1R2
OH
