Chemical Structures of Plant Hydrolyzable ... - ACS Publications

Article pubs.acs.org/JAFC

Chemical Structures of Plant Hydrolyzable Tannins Reveal Their in Vitro Activity against Egg Hatching and Motility of Haemonchus contortus Nematodes M. T. Engström,*,† M. Karonen,† J. R. Ahern,† N. Baert,† B. Payré,‡ H. Hoste,§,∥ and J.-P. Salminen† †

Department of Chemistry, Laboratory of Organic Chemistry and Chemical Biology, University of Turku, FI-20014 Turku, Finland Centre de Microscopie Electronique Appliquée à la Biologie, Faculté de Médecine Toulouse Rangueil, Université de Toulouse, 133, route de Narbonne, 31062 Toulouse Cedex 4, France § UMR 1225, INRA/DGER, Ecole Nationale Vétérinaire Toulouse, 23 Chemin des Capelles, 31076 Toulouse Cedex, France ∥ ENVT, Université de Toulouse, Toulouse F-31076, France ‡

S Supporting Information *

ABSTRACT: The use of synthetic drugs against gastrointestinal nematodes of ruminants has led to a situation where resistance to anthelmintics is widespread, and there is an urgent need for alternative solutions for parasite control. One promising approach is to use polyphenol-rich bioactive plants in animal feeds as natural anthelmintics. In the present work, the in vitro activity of a series of 33 hydrolyzable tannins (HTs) and their hydrolysis product, gallic acid, against egg hatching and motility of L1 and L2 stage Haemonchus contortus larvae was studied. The effect of the selected compounds on egg and larval structure was further studied by scanning electron microscopy. The results indicated clear relationships between HT structure and anthelmintic activity. While HT size, overall flexibility, the types and numbers of functional groups, together with the linkage types between monomeric HTs affected the activity differently, the optimal structure was found with pentagalloylglucose. KEYWORDS: bioactive plant, polyphenol, tannin, hydrolyzable tannin, nematode, Haemonchus contortus, natural anthelmintic, SEM

■

polyphenols, especially tannins.12 However, it is still mostly unknown what kinds of tannin molecules are most efficient against nematodes and which structural features of tannins drive the observed activities. By better understanding the structure−activity relationships between plant tannins and their anthelmintic properties, it would be possible to select plant species with an optimal tannin composition to be used as ruminant feeds or feed additives for the control of the nematodes. Further, this knowledge could be used to breed new plant varieties with enhanced tannin composition. Most of the studies focusing on the effects of tannin-rich plants or purified plant fractions against the different parts of the nematode lifecycle have focused on proanthocyanidins (PAs, syn. condensed tannins).15−19 These studies have utilized either plant extracts or chemically characterized mixtures of PAs as individual PA compounds are very difficult to purify. The other group of terrestrial plant tannins, hydrolyzable tannins (HTs), has not been studied as extensively, although they are easier to purify than PAs, and individual HT compounds could be used for in vitro and in vivo activity tests.20−22 The few studies that have utilized HT-containing plants or fractions have witnessed anthelmintic activity.23,24 To provide unique data on the structure−activity relationships between individual tannins and their in vitro anthelmintic

INTRODUCTION Gastrointestinal parasitic nematodes are increasingly problematic for small ruminant production, causing numerous infectious diseases on their hosts. 1−3 The efficacy of commercially available chemical anthelmintics is decreasing due to the growing frequency of anthelmintic resistant strains. For example, routine preventative treatment of all sheep in a herd with a chemical anthelmintic provides a strong selective force where only worms that are resistant to its specific mechanism will survive and reproduce, and, as a result, parasite resistance to this drug grows over time.4 Furthermore, the anthelmintic resistance becomes an even larger problem when parasites develop “multiple anthelmintic resistance”, that is, resistance against different classes of anthelmintics.5,6 Unfortunately, the diminishing efficacy of anthelmintics drug is often followed by increasing dosage, which further increases the risk of drug residues in food products for the consumers as well as in the environment.7,8 As a consequence of these multiple elements, national restrictions and regulations on the use and dosage of anthelmintics have increased. All of these factors clearly indicate that there is an urgent need for alternative solutions to control gastrointestinal nematodes. One possible and promising approach is the utilization of bioactive plants as natural anthelmintics to at least partially replace the use of chemical drugs.9 Plants and plant extracts have been used for centuries as dewormers for both humans and livestock, and several studies have explored the anthelmintic effects of plant extracts.10−14 Most anti-nematode activities have been obtained with plant extracts rich in © XXXX American Chemical Society

Received: December 1, 2015 Revised: January 12, 2016 Accepted: January 12, 2016

A

DOI: 10.1021/acs.jafc.5b05691 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Figure 1. Structures of the monomeric hydrolyzable tannins studied. HHDP, hexahydroxydiphenoyl; DHHDP, dehydrohexahydroxydiphenoyl; NHTP, nonahydroxytriphenoyl.

■

activity, the present study examined the in vitro activity of 33 purified HTs and their most common hydrolysis product, gallic acid, against the egg hatching and 12 HTs against motility of L1 and L2 stage larvae of Haemonchus contortus. The studied HTs were selected from a set of HT-producing plant species so that the purified compounds represented a wide variety of HT structures (Figures 1 and 2) from 10 different biosynthetic branches of the HT pathway (Figure 3). We hypothesized that the anthelmintic activities of HTs change parallel to the progression of their biosynthesis via, for example, galloylation reactions and oxidative coupling within and between the HT monomers. Should that be true, it would enable the future estimation of anthelmintic activities of different plant species or plant products directly from their specific HT composition or by knowing the biosynthetic branch utilized in their production. In addition, cryo-scanning electron microscopy (cryo-SEM) was utilized to study possible changes in the egg and larval structure due to the tested HTs to gain insight into the possible mechanisms of anthelmintic actions of HTs.

MATERIALS AND METHODS

Chemicals. Technical grade acetone for extraction was purchased from VWR (Haasrode, Belgium). Formic acid and LC−MS Chromasolv acetonitrile for the UHPLC-ESI-QqQ-MS, DMSO, and glutaraldehyde were obtained from Sigma-Aldrich (Seelze, Germany). Water was purified with a Millipore Synergy water purification system from Merck KGaA (Darmstadt, Germany). Sephadex LH-20 was obtained from GE Healthcare (Uppsala, Sweden). Phosphate buffered saline (PBS) was from Biomérieux (Marcy l’Etoile, France). Plant Material. The plant materials used for isolation of the studied HTs were collected during the summer of 2011 from southwestern Finland, including willowherb flowers (Epilobium angustifolium), silverweed leaves (Potentilla anserina), herb Bennet leaves (Geum urbanum), English oak acorns (Quercus robur), purple loosestrife leaves and flowers (Lythrum salicaria), meadowsweet flowers (Filipendula ulmaria), raspberry leaves (Rubus idaeus), wood cranesbill leaves (Geranium sylvaticum), rose leaves (Rosa rugosa), and burnet rose leaves (Rosa pimpinellifolia). Sea buckthorn (Hippophae rhamnoides) material was the same as used in Moilanen et al.,25 and white birch (Betula pubescens) material was the same as in Salminen et al.26 B


Article


Figure 2. Structures of the oligomeric hydrolyzable tannins studied and the different types of linkages between the monomers. m-DOG, valoneoyl group; m-GOG, dehydrodigalloylgroup; m-GOD, sanguisorboyl group; 2DOG, macrocyclic structure. Isolation and Purification of HTs. The extraction and the isolation of the HTs followed mainly the methods reported in Baert et al.,27 shortly described in the following. Extraction. The plant materials were collected directly into 1 L glass bottles and macerated in 80% aqueous acetone at 4 °C. After maceration, the extract was filtered, the acetone was evaporated, and the remaining aqueous solution was frozen and lyophilized. First Fractionation. The water phase of the crude extract was mixed to a slurry of Sephadex LH-20 (in 100% water) material and eluted

with water, methanol/water (1:1 v/v), methanol, acetone/water (4:1 v/v), and acetone in a Büchner funnel (⌀ = 240 mm) in vacuo with a filter paper. Column Chromatography. Sephadex LH-20 was loaded into a glass column (40 × 4.8 cm i.d., Kimble-Chase Kontes Chromaflex) and equilibrated with 1500 mL of ultrapure water at a flow rate of 5 mL min−1. The samples were dissolved in 15 mL of ultrapure water, filtered (0.2 μm, PTFE), and applied on top of the gel. The eluent profile depended on the HT to be isolated; the solvents used were C


Article


samples were filtered with a syringe filter (4 mm, 0.2 μm PTFE, Thermo Fisher Scientific Inc., Waltham, U.S.) prior to the UPLC−MS analyses. The NMR experiments were performed for selected HTs with a Bruker Avance 500 spectrometer operating at 500.13 MHz for 1H and 125.77 MHz for 13C. Spectra were recorded at 25 °C using methanold4 as a solvent. The measurements included 1H and 13C NMR spectra, in addition, to several 2D spectra such as COSY, 1H−13C HSQC, and 1 H−13C HMBC. The structures of individual HTs are presented in Figures 1 and 2. The stereochemistries of castalagin and vescalagin were recently reinvestigated by computational methods, and the nonahydroxytriphenoyl group (NHTP) was found to exist in (S,R) configuration.28 Therefore, it is feasible that the NHTP group of vescavaloninic and castavaloninic acids is also in (S,R) configuration. The structures of tellimagrandin II, roshenin C, and salicarinin A were confirmed by NMR spectroscopy by comparing to the spectra from previous analyses. One of the compounds, the α-anomer of hippophaenin B, had not been previously reported. This novel compound was nominated as hippophaenin C (NMR data will be reported in a separate publication). The studied 33 HTs and their hydrolysis product with information on the original plant material, the purity by UPLC at 280 nm, and the ESI−MS identification are presented in the Supporting Information (S1 Appendix). Egg Hatch Assay. The method was based on a modification of the egg hatch assay that was previously used to assess anthelmintic resistance.29 Parasite eggs were freshly obtained from the feces of a donor sheep experimentally infected with H. contortus. A water suspension of feces was filtered through a mesh (150 μm pore size) and transferred into 15 mL centrifuge tubes. The suspension was centrifuged (Heraeus Labofuge 400 R, 2500 rpm, 3 min, 20 °C), and the supernatant was replaced with tap water. This was repeated three times. After the third centrifugation, the supernatant was removed, replaced with saturated sugar solution, and centrifuged. The eggs (located on the top of the sugar solution) were collected into a 15 mL falcon tube. The tube was filled with phosphate buffered saline (PBS; 0.1 M phosphate, 0.05 M NaCl, pH = 7.2), and after mixing, the egg solution was centrifuged, the supernatant removed, and the tube filled again with PBS solution to get rid of the sugar solution residues. After the washing was repeated four times, the egg solution was diluted with PBS to a final concentration of 1000 eggs mL−1. 100 μL of egg solution was pipetted into a 300 μL well of a 96 well plate together with 100 μL of tested HT solutions. After mixing, samples were incubated for 48 h at 26 °C (Incucell - V 111, MMM Medcenter Einrichtungen GmbH, Gräfelfing, Germany). Thereafter, a drop of Lugol solution (1 g of iodine + 2 g of potassium iodide in 50 mL of water) was added into each well to kill the larvae and to facilitate microscopic examination (Axio Scope.A1, Carl Zeiss Microscopy, LLC, U.S.). The number of larvae, eggs with larvae inside, and eggs with embryo inside were counted per well. Tested HTs were dissolved in DMSO/PBS (4:96, v/v). Six different tannin concentrations were tested: 2.0, 1.5, 1.0, 0.5, 0.25, and 0.125 mM. Four replicates for each concentration were run. In addition, a positive control (thiabendazole at a concentration of 50 μg mL−1 in DMSO/PBS (10:90, v/v)) and a negative control (DMSO/PBS (4:96, v/v)) were included in the assay. The negative control was run for each batch of egg solution to take into account possible differences in the egg quality between different runs/days. For controls, five replicates were run. The egg hatching percentage of the controls varied from 84 ± 2% to 95 ± 2% between the different daily batches of eggs. Thus, the 0% egg hatching inhibition level had to be calculated for each batch separately to be able to control the observed variation. The percentage of inhibition was determined as follows:

Figure 3. A simplified scheme of the current view of the hydrolyzable tannin (HT) biosynthetic pathway, showing HT subclasses studied in the present work and their biosynthetic connections. GG, galloylglucose; ET, ellagitannin; HHDP, hexahydroxydiphenoyl. It is not yet fully known if the dehydroETs derive only from pentagalloyl glucose or also from HHDP esters. See the bond types of the oligomeric ETs in Figure 2. ultrapure water, aqueous methanol, and aqueous acetone. Obtained fractions were analyzed by UPLC-DAD-MS, concentrated to the water phases, and lyophilized. Selected Sephadex fractions were further purified by preparative and semipreparative high pressure liquid chromatography (HPLC). Preparative and Semipreparative HPLC. The HPLC-DAD system consisted of a Waters Delta 600 liquid chromatograph, a Waters 600 Controller, a Waters 2998 Photodiode Array Detector, and of a Waters Fraction Collector III. The column (approximately 327 mm × 33 mm) was manually filled with LiChroprep RP-18 (40−63 μm) material (Merck KGaA, Darmstadt, Germany). A binary solvent system with methanol (A)/1% aqueous formic acid (B) at a constant flow rate of 8 mL min−1 was used, and the elution protocol depended on the composition of the fractions; the typical gradient was as follows: 0−5 min, 100% B; 5−180 min, 0−40% A in B; 180−220 min, 40−60% A in B; 220−240 min, 60−80% A in B. The final purification of HTs was performed by semipreparative HPLC with the same HPLC-DAD system described above. The column was a Gemini C18 column (150 × 21.2 mm, 10 μm, Phenomenex), and the eluents were acetonitrile (A) and 0.1% aqueous formic acid (B) at a constant flow rate of 8 mL min−1. Different gradients were used for different HTs; for example, a typical gradient for acyclic ETs was as follows: 0−5 min, 2% A in B; 5−51 min, 2−32% A in B; 51−55 min, 32−70% A in B. All steps in the preparative and semipreparative purifications were followed by UPLC-DAD-MS. Fractions with pure HTs were pooled, concentrated to the water phase, and lyophilized. Structural Characterization of Hydrolyzable Tannins. Sample analysis was carried out with an Acquity UPLC system (Waters Corp., Milford, MA) coupled with a Xevo TQ triple quadrupole mass spectrometer (Waters Corporation, Milford, MA). The UPLC system consisted of a sample manager, a binary solvent manager, a column, and a diode array detector. The column used was a 100 mm × 2.1 mm i.d., 1.7 μm, Acquity UPLC BEH Phenyl column (Waters Corp., Wexford, Ireland). The flow rate of the eluent was 0.5 mL min−1. The elution profile used two solvents, acetonitrile (A) and 0.1% aqueous formic acid (B): 0−0.5 min, 0.1% A in B; 0.5−5.0 min, 0.1−30% A in B (linear gradient); 5.0−5.1 min, 30−90% A in B (linear gradient); 5.1−8.5 min, column wash and stabilization. UV and MS data were collected from 0 to 6 min. Negative ionization mode was used for MS analyses. ESI conditions were: capillary voltage 2.4 kV, desolvation temperature 650 °C, source temperature 150 °C, desolvation and cone gas (N2) 1000 and 100 L/h, respectively, and collision gas of argon. All

inhibition (%) =

(

1 5

∑i = 1 Di

(

1 5

5 Ci ∑i = 1 Di

B− B×

5

Ci

A− B×

) × 100% )

(1)

where A is the number of unhatched eggs; B is the total number of eggs and larvae; C is the number of unhatched eggs in control; D is the D


Article


Table 1. Studied Hydrolyzable Tannins and Gallic Acid, Their Molecular Weights, and Average Inhibition Activities against the Egg Hatching of Haemonchus contortus number

compound

1 gallic acid simple galloylglucoses 2 monogalloylglucose 3 tetragalloylglucose 4 pentagalloylglucose gallotannins 5 hexagalloylglucose 6 heptagalloylglucose 7 octagalloylglucose monomeric HHDPb esters 8 tellimagrandin I 9 tellimagrandin II 10 pedunculagin 11 casuarictin 12 isostrictinin monomeric DHHDPb esters 13 geraniin 14 carpinusin C-glucosidic ETb monomers 15 vescalagin 16 castalagin 17 vescavaloninic acid 18 castavaloninic acid 19 stachyurin 20 casuarinin 21 hippophaenin B 22 hippophaenin C

MW

average activity %

170.1

4

332.3 788.6 940.7

2 51 82

1092.8 1244.9 1397.0

38 21 18

786.6 938.7 784.5 936.7 634.5

29 64 6 11 6

952.6 952.6

27 6

934.6 934.6 1102.7 1102.7 936.7 936.7 1104.8 1104.8

7 12 7 9 19 29 49 42

number

compound

macrocyclic m-DOGb oligomers 23 oenothein B 24 oenothein A 25 tetramer 26 pentamer 27 hexamer m-DOGb oligomers 28 rugosin E 29 rugosin D m-GOGb oligomers 30 agrimoniin 31 gemin A m-GODb oligomers 32 sanguiin H6 33 lambertianin C C-glucosidic oligomers 34 salicarinin A

MW

average activity %a

1569.1 2353.6 3138.2 3922.7 4707.2

2 6 6 7 5

1723.2 1875.3

29 46

1871.3 1873.3

15 24

1871.3 2805.9

41 17

1869.2

29

a

The average % unhatched eggs as compared to control at the tested concentration range (2.0, 1.5, 1.0, 0.5, 0.25, and 0.125 mM). bHHDP, hexahydroxydiphenoyl; DHHDP, dehydrohexahydroxydiphenoyl; ET, ellagitannin; m-DOG, valoneoyl group; m-GOG, dehydrodigalloyl group; mGOD, sanguisorboyl group. Statistical Analysis. EC30 and EC50 values were calculated by fitting dose response curves in SAS (SAS Institute inc., version 9.3). Curves were fitted individually for each compound using logistic regression in probit procedure. Curves were fitted without transforming the dose variable, except for compound 4, where the dose was log10 transformed to achieve a proper model fit. EC30 or EC50 values were not presented for samples where the maximum inhibition was below 30% or 50%, respectively. Fate of the Hydrolyzable Tannins during the Egg Hatch and Motility Incubations. To get insight into whether the studied compounds are somehow modified during the 48 h incubation of the EHA and motility tests, selected HTs were exposed to experimental conditions and subsequently analyzed by UPLC-MS/MS. Two model compounds, 4 for galloylglucoses and 20 for ellagitannins, were incubated in water/MeOH (98:2, v/v), in PBS/MeOH (98:2, v/v), and in the egg matrix solution. The egg matrix was obtained similarly to that in EHA, but the eggs were removed from the solution by centrifuging. Concentration of the HTs in the incubation solution was 0.5 mM, and the final volume was 2 mL. Samples were taken from the incubation mixture after 10, 20, 30, 40, 50, 60, 120, 180, and 360 min, and 24 and 48 h. Samples were diluted 20× (v/v) prior to analysis and filtered with 0.2 μm PTFE filters. The UPLC-MS/MS analysis was carried out with the methods reported in Engström et al.30

total number of eggs and larvae in control; and i refers to replicate (1− 5). The average egg hatch inhibition values (Table 1) used in compound comparisons and the equation for predicting the egg hatching inhibition activity were calculated as an average from all of the individual dose averages. Effects of Hydrolyzable Tannins on the Motility after Hatching of the L1 and L2 Stage Larvae. The motility after hatching test was carried out similarly to EHA except that approximately 150 eggs were applied per well and no Lugol solution was added prior to microscopic examination. After 48 h incubation, the number of motile and nonmotile L1 and L2 stage larvae were counted per well. As the motility percentage of the controls varied from 80 ± 6% to 97 ± 3% between the different batches, the 0% motility inhibition level was taken into account in the calculations similarly to that in the egg hatch assay. Cryo-Scanning Electron Microscopy. Cryo-SEM images were performed with a FEG FEI Quanta 250 microscope (FEI Company, Eindhoven, Holland). The larvae and eggs obtained from the in vitro incubation in control or HT solutions were fixed with 2% glutaraldehyde in Sorensen buffer (0.1 M, pH = 7.4). Sample was deposited on filter then stub fixed with Tissue-Tek/colloidal graphite mounting media. Sample was frozen in nitrogen slush at −220 °C. The frozen sample was transferred under vacuum to the cryo-chamber apparatus (Quorum PP3000T Cryo Transfer System) at −140 °C. The temperature was then increased to −95 °C and maintained at this temperature during 1 h for sublimation. It was then metalized with Pd (60 s, 10 mA) and introduced into the microscope chamber where it was maintained at −140 °C during the observation, operating at 5 kV accelerating voltage.

■

RESULTS Tannin Selection and Purification. Thirty-three HTs (Figures 1 and 2) were successfully purified (purities between 97.5% and 99.9%) from 11 plant species to achieve proper tannin diversity to be tested for their anthelmintic activities. The commercially available precursor for all HTs, gallic acid E


Article


into account and to rank the HTs by the most informative compound-specific activity value, we calculated an average inhibition value for every compound (Table 1). When the average inhibition values were compared against the chemical characteristics of the tested HT structures, several clear structure−activity patterns were revealed. One of the simplest parameters to be measured from HT structures is their molecular weight,31 and this showed that compounds with molecular weight below 700 or above 2000 Da had no or very little effect on the egg hatching of H. contortus. The most active compounds had molecular weights relatively close to that of 4 (940 Da), but this type of a match did not guarantee high activity, if the structures were biosynthetically distant from 4. For instance, 9 is the immediate biosynthetic product from 4, and it differs from 4 only by 2 Da; 9 was the second most active of the tested HTs. However, masses of 11, 15, and 16 differed from 4 by only 4 or 6 Da, but their activity was one-sixth of that of 4. Detailed structural reasons for these activity differences were revealed by comparing the activities of several compound pairs. The following pairs, 4 versus 9, 8 versus 10, and 9 versus 11, were otherwise similar in structures, but two galloyls of the former were oxidatively coupled to form an HHDP group of the latter. In each case, the activity decreased (82% → 64%, 29% → 6%, and 64% → 11%) due to the formation of the HHDP group from the two galloyls. A similar comparison could be made between 19 versus 15, 20 versus 16, 21 versus 17, and 22 versus 18 where galloyl and HHDP groups of the former HT were coupled to form a NHTP group of the latter. Also, in all of these examples the activity decreased (19% → 7%, 29% → 12%, 49% → 7%, and 42% → 9%) due to the additional oxidative coupling between the galloyl and HHDP groups. These results clearly indicated that the intramolecular oxidative coupling that results in the formation of HHDP and NHTP groups decreases the inhibition activity of HTs against egg hatching of H. contortus. However, the presence of these two groups cannot be considered as signs of inactive HTs, because they do contribute to the average activity. For instance, compounds 2, 12, and 11 each contain one galloyl group, but zero, one, or two HHDP groups, respectively. The average activity increased with the increasing numbers of HHDP groups (2% → 6% → 11%). This highlights the earlier note that HT size close to 4 is important for the activity, not only the numbers of galloyl groups. In plants, HTs can gain galloyl groups via galloylation reactions or lose them via catabolic processes. Comparisons within the galloylglucose series 2−7 indicated that additional galloyl groups increase the activity until all of the OHs of the glucose core are galloylated (as in 4). After that, further galloyl additions to 4 (as in its biosynthetic gallotannin-type successors 5, 6, and 7) did not increase the activity further, but rather decreased it. The importance of galloyl groups attached to the HT glucose core was further emphasized when comparing the following three compound pairs of ETs, that is, 9 versus 8, 11 versus 10, and 29 versus 28. In these cases, the former compounds with a galloyl group at the anomeric position were more active (64% → 29%, 11% → 6%, and 46% → 29%) than the latter compounds that had lost this specific galloyl presumably during a catabolic reaction. Thus, galloyls are important in enhancing the activity, especially if they are attached directly to the glucose core. Those ETs that possess a valoneoyl group in their structure (17, 18, 21, 22) may be produced by the catabolism of oligomeric ETs but also in an anabolic manner via the oxidative

(1), was accompanied by three simple galloyl glucoses (2−4) and three gallotannins (5−7). The competing biosynthetic branch with gallotannins from 4, that is, simple hexahydroxydiphenoyl (HHDP) esters, was presented by five ellagitannins (ETs, 8−12), each carrying the characteristic feature of ETs, the HHDP moiety. The HHDP group can be oxidized to form a dehydrohexahydroxydiphenoyl group (DHHDP), as in the so-called dehydroETs, 13 and 14. Their biosynthetic origin is not fully known, but they may derive directly from 4 or alternatively from the simple HHDP esters via more complex relocalizations of the galloyls and inversion of the glucose configuration. The C-glucosidic ETs (eight compounds, 15− 22) are considered as biosynthetic successors of the simple HHDP esters, and most of them contain a nonahydroxytriphenoyl (NHTP) group that cannot be found in other monomeric ET types. In addition, valoneoyl group with a free carboxylic acid group (COOH) was found in four of these monomers (17, 18, 21, and 22). The 12 oligomeric ETs were constructed of either simple HHDP esters or C-glucosidic monomers and were divided into five different oligomer types. Compounds 23−27 were macrocyclic DOG-oligomers, having the basic structure of two 8 bound to each other via two DOG-bonds (2DOG). Compounds 24−29 and 34 contained an intermolecular valoneoyl group, where a galloyl group of an ET monomer is bound to an HHDP group of another ET monomer via an ether bond (m-DOG; O-donating hydroxyl group is part of an HHDP group, and the acceptor is a galloyl group). Sanguisorboyl groups (m-GOD; O-donating hydroxyl group is part of a galloyl group, and the acceptor is a HHDP group) were found in 32 and 33. Dehydrodigalloyl groups, which are formed when galloyl groups of two monomeric ETs are attached to each other via C−O−C linkages (m-GOG), were found in 30 and 31. Egg Hatch Assay. The inhibition percentages for the 33 structurally different HTs and their hydrolysis product, gallic acid, against the egg hatching of H. contortus varied considerably between the compounds and tested concentrations as could be seen from the concentration versus inhibition plots (Supporting Information, Figures S1−S5). From the plots, it is evident that some of the compounds are quite ineffective anthelmintics (e.g., 1, 2, and 23), because their activity remained below the level of 20% inhibition at all tested concentrations. At the other end of the activity spectrum were compounds such as 4, 9, and 21 that showed over 50% inhibition levels already at the 0.5 mM range and at least 90% inhibition at the highest concentrations. Altogether there were 16 HTs that showed a minimum of 50% inhibition at least at one tested concentration. Interestingly, some of the remaining 17 tannins that did not reach the 50% inhibition level had relatively high activities at the low concentrations. For instance, 6, 7, and 33 were among the six best egg hatching inhibitors at the 0.125 mM concentration, but were outside the top 16 tannins with their maximal activities. Although EC50 values are often used to report compound-specific activities, these examples showed that the EC50 approach would give a false view of the overall activity related to the tested HTs. In addition, one cannot say if it is better for an HT to achieve a relatively good activity at low concentration or a very high activity at high concentration, because this totally depends on the concentration of the given tannin in a plant product. For the best anthelmintic HTs, both aspects are of course true as they are active across a wide concentration range. To take the whole concentration range F


Article

Journal of Agricultural and Food Chemistry coupling of the HHDP with an additional gallic acid.32 Addition of gallic acid to the 4,6-HHDP of NHTP-containing 15 and 16 to form 17 and 18, respectively, did not affect the inhibitory activities (7% → 7%, 12% → 9%). However, addition of gallic acid to the relatively more flexible structures of 19 and 20 (having HHDP and galloyl groups instead of NHTP group) to form 21 and 22, respectively, significantly increased the inhibitory effect (19% → 49% and 29% → 42%). The same selection of compounds enabled one also to compare the effect of the orientation of the hydroxyl group at the C-1 position on the anthelmintic activities of C-glycosidic ETs. In compound pair 19 versus 20, a significant difference in the activity was observed (19% → 29%), with the α-oriented hydroxyl group increasing the activity in comparison to the β-oriented hydroxyl group. In the other compound pairs, 15 versus 16, 17 versus 18, and 21 versus 22, the difference in activity was not significant (7% → 12%, 7% → 9%, and 49% → 42%). In the biosynthetic pathway, the formation of oligomeric ETs occurs via intermolecular oxidative coupling between the monomers. The effect of oligomerization was tested by three different series of ETs. The first series contained ETs 8 and 23−27. The results showed that only the monomer 8 had a significant inhibitory effect on the egg hatching process (29%), and its macrocyclic conjugates were rather ineffective in all tested concentrations (2−7%). The second series contained 15, 19, and their oligomerization product 34. Here, the average anthelmintic effect of the dimer (29%) was more active than either of the monomers (7% and 19%). The third comparison could be made between 8, 9, 28, and 29 where the dimerization of 8 and 9 to form 28 as well as the dimerization of two 9’s to form 29 decreased the inhibition of egg hatching (29% + 64% → 29% and 64% + 64% → 46%). Thus, it seemed that there is no common rule for how much ET oligomerization affects the activity. However, a detailed look at the results suggested that the macrocyclic structure present in 23−27 (2DOG, involving two DOG-linkages) decreased the activity the most, followed by the GOG- and DOG-linkages. On the contrary, the GODlinkage increased the activity of the oligomers. The compound-by-compound comparisons presented above enabled one to conclude what kind of an effect (positive or negative, large or small) a certain structural feature of HTs has on the egg hatching inhibition activity. For some of the average anthelmintic activity =

Figure 4. Correlation between the measured and the calculated average egg hatching inhibitory activities of the studied HTs against Haemonchus contortus. Red color indicates monomeric structures, and blue color represents oligomeric structures. Experimental values were calculated as the average % unhatched eggs as compared to control at the tested concentration range (2.0, 1.5, 1.0, 0.5, 0.25, and 0.125 mM).

structural features, such kinds of exact comparisons were not possible because in the compound-to-compound comparisons more than one structural feature was different (e.g., the effect of DHHDP group in 13 vs 14) or all corresponding monomers were not studied (e.g., the dimerization of two potentillin molecules to form 30 or oligomerization of potentillin with 8 or 11 to form 30−33). In those cases, the coefficient was taken from the structural feature that was closest to the evaluated one; DHHDP-group was treated as HHDP group, and the activity of potentillin was expected to be the same as for its isomer 11. On the basis of the comparisons, it was possible to create an equation for the estimation of the inhibitory activity on the egg hatching of H. contortus. Only those structural features that had a major effect on the activity were included in the equation, and coefficients reflecting the relative impact of each structural feature were adjusted in Microsoft Excel to produce activity estimates that would best correlate with the measured activities. The final equation was as follows:

30 × A − 40 × B + 10 × C − 130 × D − 50 × E + 20 × F 200 ×

where the letters present the numbers of following structural features in the HT: (A) galloyl group attached directly to the glucose core or gallic acid attached to the 4,6-HHDP forming valoneoyl group; (B) additional galloyl group attached to the pentagalloylglucose core; (C) HHDP-, DHHDP- or NHTPgroup; (D) 2DOG-linkage (i.e., the macrocyclic structure); (E) GOG- or DOG-linkage; (F) GOD-linkage; and (MW) molecular weight. The divider of the equation takes into account the relative distance to the optimal molecular weight of 4, that is, 940 Da. When applying the equation to the oligomers, the galloyl and HHDP groups involved in the different bond types (GOG, GOD, DOG, 2DOG) are counted also in “A” and “C”. Following this logic, the DOG group found in 28, for example, is counted as one galloyl, one HHDP group, and one DOG-linkage. The correlation between the calculated and measured average activities for the 33 HTs is presented in

1 + |940 − MW| 940

(2)

Figure 4. An examination of monomers and oligomers separately showed that the created equation better estimated the activities for the monomers (R-value 0.92) than for the oligomers (R-value 0.82). Motility after Egg Hatching. The effect of HTs on the motility of hatched L1 and L2 stage larvae of H. contortus was investigated by a selection of 12 structurally different HTs: 1, 2, 4, 6, 8, 9, 11, 13, 16, 19, 20, and 23. These compounds were select to represent the variety of results obtained from EHA. As in the EHA tests, the results varied between compounds (Supporting Information, Figure S6), but in contrast to EHA, inhibition was higher, allowing the estimation of EC30 and EC50 values (Table 2). In general, the most effective egg hatching inhibitors also markedly decreased motility, and the least effective egg hatching inhibitors were also less active in motility tests. However, compound 6, which reached only moderate G


Article


motility was evaluated by counting the percentage of moving and nonmoving larvae. However, in addition to this, it was noticed that for all studied compounds, the motility of the larvae decreased already at the low concentrations, progressively leading to total immobilization at high concentrations. Cryo-Scanning Electron Microscopy. The structural changes induced in L1 and L2 stage H. contortus larvae and egg after in vitro contact with two compounds, 4 and 20, were assessed using cryo-SEM. Figure 5 shows the changes found in the H. contortus larvae and egg after the exposure to either the control or the HT solutions. The main changes observed between treated and control (Figure 5A, D, G), 20 (Figure 5B, E, H), and 4 (Figure 5C, F, I) concerned the surface of the body (cuticle), the cephalic region, and the unhatched eggs. As compared to the control, small changes in the smoothness of the cuticle surface were observed (Figure 5A, B, C). However, no big structural alterations, such as longitudinal and transversal folds or thicker ridges in the cuticle, as previously reported with plant extracts,34 were observed in the present study with pure compounds. The most striking changes observed on L1 and L2 stage larvae were the aggregates located at the buccal capsule and the anterior amphidial channels (Figure 5D, E, F). For compound 4, these aggregates were more extensive than for 20, and they covered also parts of the surface of the other cephalic areas. As compared to control (Figure 5G), no significant changes were observed on the surface of the eggs incubated in solution with 20 (Figure 5H). Adversely, eggs incubated with 4

Table 2. EC50 and EC30 Motility Inhibition Values of the Studied HTs against Hatched H. contortus L1 and L2 Stage Larvae compound

EC30 (mM)a

1 2 4 6 8 9 11 13 16 19 20 23

n.c. 1.88 0.03 0.01 0.39 0.26 0.24 0.83 0.51 0.42 0.44 0.54

95% fiducial limits

EC50 (mM)

95% fiducial limits

n.c. 1.46, 0.02, 0.01, 0.30, 0.25, 0.23, 0.69, 0.40, 0.36, 0.39, 0.29,

2.98 0.04 0.02 0.47 0.28 025 0.96 0.60 0.47 0.49 0.73

0.06 0.03 0.58 0.31 0.33 1.20 0.64 0.65 0.72 1.37

0.06, 0.02, 0.50, 0.30, 0.31, 1.05, 0.55, 0.60, 0.68, 1.19,

0.07 0.05 0.67 0.33 0.34 1.40 0.76 0.70 0.77 1.61

a

n.c.: Data were not distribututed monotonically, so EC values were not calculated.

activity in EHA, efficiently inhibited the motility of H. contortus already at lower concentrations. The concentration−activity plot of 1 was distinctive: first the activity increased as concentration was increased, but after maximal inhibition (65%) was reached, the activity decreased as the concentration was further increased. Similar, nontypical dose−response with decreasing effectiveness at higher doses has been observed previously with some plant extracts.33 In the present work, the

Figure 5. Comparison of the external structure of H. contortus midbody (A−C), cephalic region (D−F), and egg (G−I) incubated in PBS control (A, D, G), 20 (B, E, H), or 4 (C, F, I). The arrows in the cephalic region indicate the anterior amphidial channel. Images are from cryo-scanning electron microscopy. H


Article


Figure 6. UPLC-MS/MS fingerprints for galloyl derivatives and the proportion of intact pentagalloylglucose (PGG) and its degradation products monitored every hour when incubated in ultrapure water or egg matrix solution. (A) Initial PGG in H2O, (B) PGG after 48 h in H2O, (C) normalized peak areas of PGG and its degradation products in H2O, (D) initial PGG in egg matrix solution, (E) PGG after 48 h in egg matrix solution, and (F) normalized peak areas of PGG and its degradation products in egg matrix solution. 4GG = tetragalloylglucose, 3GG = trigalloylglucose, and 2GG = digalloylglucose.

change of color during the incubation to brownish/yellowish suggested that also oxidation occurred. Another difference between the two compounds was that 20 had completely disappeared from the PBS and egg matrix incubation solution after 24 h, while a quantifiable amount of 4 was present in both solutions still after 48 h.

were fully covered with chip-like layers of aggregates (Figure 5I). Fate of the Hydrolyzable Tannins during Incubation. In the present study, clear differences were observed in the stability of pentagalloylglucose (4) and casuarinin (20) when incubated in ultrapure water, PBS solution, or egg matrix solution. Both studied compounds remained intact during the 48 h incubation in water, but incubation in PBS and egg matrix solutions resulted in substantial degradation of both compounds. Figure 6 shows the fate of 4 during the 48 h incubation in water and in egg matrix solution. The UPLC-MS/MS analysis showed that for 4, all of the new peaks in the UV and MRM chromatogram were galloyl derivatives. Further, from full scan MS data, it was possible to identify tetra-, tri-, and digalloylglucoses, which are common hydrolysis products of 4. Similarly, the results with 20 indicated hydrolysis, but the

■

DISCUSSION All of the findings in the present study supported the general observation that regarding in vitro anthelmintic activity, the most optimal HT structures are those similar to pentagalloylglucose (4), and the further an HT is located from PGG in the biosynthetic pathway the lower is its in vitro anthelmintic activity. As previous studies have linked the protein precipitation capacity of HTs to their close location with PGG in the biosynthetic pathway of HTs,31,35−38 the in vitro I


Article


what kind of interaction takes place, and how strong the interaction is.42−44 Thus, in future in vitro activity studies, it would be essential to even more closely mimic the conditions of the different parts of the ruminant digestive track where HTs and the eggs and larvae are encountering each other. Altogether, from the HT point of view, the overall results in the present work indicated three different possibilities for the mode of action by which HTs disturb egg hatching and motility of the hatched larvae: (1) HTs with high PPC may bind to the eggs and larvae via noncovalent bonds. (2) HTs with high oxidative activity may be auto-oxidized, and the oxidation products bind covalently to the egg and larvae. (3) HTs undergo hydrolysis, and the hydrolysis products may interact with the eggs and larvae via noncovalent or covalent interactions. The mode of action behind the anthelmintic effect of tannins has been discussed, but is still largely unidentified.12,14,45 It has been suggested that the direct effect of plant tannins is based on their interactions with egg and larvae proteins vital for the development and biological functions of the larvae.11,46,47 In the present study, the amount of unhatched eggs with the embryo inside was constant in both control and HT solutions (results not shown), and only the amount of hatched versus unhatched eggs varied depending on the tested compound and test concentration. It was also observed that before the addition of the Lugol solution, most of the larvae were still moving inside the egg. Thus, it seems evident that the HTs did not penetrate inside the eggs and disturb the development of the embryo directly. More likely, the HTs bound to the surface of the egg shell, presumably via tannin−protein interactions, and either disturbed the proteins that evoke the actual hatching process as suggested in previous studies47 or, alternatively, the HT coat around the egg simply mechanistically disabled the penetration of the larvae through the egg shell. The HT coat could also disable vital functions such as oxygen exchange between the inside and outside of the egg, but this again would more likely disturb the development of the larvae inside the egg, which was not observed in the present study. The SEM results, when compound 4 was present in the incubation solution, support the theory of coat formation around the egg. However, for 29 no visible coat formation was observed, and this again supports the assumption above that different types of compounds may be effective via different modes of action. In regard to the hatched L1 and L2 stage larvae, the observed changes could suggest that the possible mode of action in inhibiting motility occurs via blocking the mouth and sensory neuron channels of the nematode. As a consequence, the food intake and the recognition of the environment would suffer and ultimately lead to the decease of the larvae. However, the motility test indicated reduction in motility already at low HT concentrations, which is in agreement with previous studies that have witnessed partial paralysis and interference with neurophysiology or neuromuscular coordination of the larvae48,49 that ultimately leads to the death of the nematode. After understanding the structural features behind the anthelmintic activities of HTs, it is important to consider the distribution of active versus inactive HTs in the plant kingdom. High anthelmintic activities of certain compounds would hardly be beneficial if these compounds were rarely found in plants suitable to be used as ruminant feed or if they were not present in high enough concentrations. In general, HTs are synthesized by a wide variety of plant species, and many of these have been used also as animal feeds.50 However, the distribution of

anthelmintic activity could also be linked to the capability of an HT to bind and precipitate proteins. Furthermore, as the protein precipitation activity and oxidative activity (ease of oxidation) have been shown to be inversely proportional,35,36,39 it could be concluded that the HTs that are weakest anthelmintics are oxidatively active compounds. Even if this conclusion is correct for many of the studied compounds with high protein precipitation capacity (4−6, 9)37 or high oxidative activities (10, 15−18), not all of the studied compounds followed this simplified logic. For example, 21 has been shown to be easily oxidized at high pH,21 but it was still one of the best egg hatching inhibitors. To understand the activity patterns observed in the present study, one must consider that tannin− protein interactions are not limited to reversible, noncovalent binding resulting in protein precipitation. Additionally, irreversible, covalent interactions may occur if the conditions are favorable for auto-oxidation, or if external oxidants are present. As the results from the incubation tests showed, oxidation occurred for the easily oxidized compound in the slightly alkaline test conditions (pH 7.2). Altogether, it can be concluded that both high protein precipitation capacity, and a favorable combination of structural flexibility (to reach to the target egg and larvae structures) and oxidative activity, as in the case of 21, result in effective inhibition of egg hatching. To make our results more generally utilizable for researchers in the field of parasitology, we traced the differences in the anthelmintic activities of HTs back to their structural characteristics. This enabled us to create an equation by which in vitro anthelmintic activities of known HT structures can be estimated without the time-consuming biological activity measurements with purified HTs. Even though the equation took into account only six of the most important structural features together with the different linkage types of the oligomeric HTs, a strong correlation was observed for the measured and calculated anthelmintic activities of the 33 HTs. Thus, the created equation provides a tool that can be used with most HTs, if not to calculate their absolute activities but especially to estimate these and to divide plant HTs into compounds with low, moderate, or high anthelmintic activities. The prediction of the cause of the witnessed in vitro anthelmintic activity of the studied HTs gets more complicated when considering that the incubation tests indicated that the studied compounds underwent degradation via hydrolysis during the 48 h incubation in egg solution. Thus, the origin of the observed activity depends on the concentration of the original HT and its hydrolysis products, which again depends on the rate of the hydrolysis of each specific HT. Depending on the original structure, the resulting hydrolysis products can be less or more active than the initial HT. For example, 6, heptagalloyl glucose, was rather inactive in EHA but inhibited motility efficiently even at low concentrations. This difference in activity in the two tests could be explained by a different mode of action of 6 against H. contortus egg and larvae. Alternatively, the partial hydrolysis of 6 to produce 4 during the 48 h incubation may have influenced this result. In this scenario, at the time point of egg hatching, the concentration of 4 would be high enough to ensure high activity against the motility of the hatched larvae. A similar HT effect could be expected also in vivo as hydrolysis of the HTs has been witnessed to occur also in the digestive track of ruminants.40,41 However, if it is assumed that the anthelmintic effect is based on the tannin−protein interactions, then the conditions and the encountering species determine if any interaction takes place, J



■

ABBREVIATIONS USED EHA, egg hatch assay; ET, ellagitannin; GG, galloylglucose; DHHDP, dehydrohexahydroxydiphenoyl; HHDP, hexahydroxydiphenoyl; NHTP, nonahydroxytriphenoyl; PA, proanthocyanidin; PGG, pentagalloylglucose; PPC, protein precipitation capacity; SEM, scanning electron microscopy; UPLC-MS, ultraperformance liquid chromatography mass spectrometry; UPLC-MS/MS, ultraperformance liquid chromatography tandem mass spectrometry

gallotannins is rather limited in nature, while ellagitannins are widespread in many plant families.51 Furthermore, as parasitic infections are not the only problem present in ruminant production, also the other plausible positive effects of plant species rich in HTs should be considered when searching for the best choice of ruminant feeds. For example, Niderkorn et al.52 tested 156 grassland plant species for their capacity to prevent methane and ammonia emissions from ruminants in vitro. They found 17 species that were able to simultaneously produce at least 30% less methane and 80% less ammonia than perennial ryegrass. We have studied the polyphenol composition of nine of these 17 species and know them to be rich in HTs.25,27,53−55 The obvious next step would be to utilize the equation produced in this study to estimate the anthelmintic activities of the HTs present in these bioactive plant species. As a result, it might be possible to find plant species that reduce both the parasitic infections and the methane and ammonia emissions in ruminants. This will be the effort of future studies as methods for HT characterization and quantitative analysis are readily available.27,30,31

■

■

ASSOCIATED CONTENT

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b05691. Effect of the studied compounds on the egg hatching of H. contortus (Figures S1−S5); inhibition versus concentration plots of the effect of studied compounds for the motility of hatched L1 and L2 stage H. contortus larvae (Figure S6); and the studied 33 hydrolyzable tannins and their hydrolysis product gallic acid with information on the original plant material, the purity by UPLC at 280 nm, and the ESI−MS identification (Appendix S1) (PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel.: +358 2 333 6755. E-mail: marica.engstrom@utu.fi. Funding

These investigations were supported by the University of Turku Doctoral Programme in Physical and Chemical Sciences, by the European Commission (PITN-GA-2011-289377, “LegumePlus” project), by the Academy of Finland (Grant no. 258992 to J.-P.S. and 251388 to M.K.) and by the Svenska tekniska vetenskapsakademien i Finland (STV) to M.T.E. The Strategic Research Grant (Ecological Interactions) to J.-P.S. enabled the use of the UPLC-MS/MS instrument. Notes

The authors declare no competing financial interest.

■

REFERENCES

(1) Hoste, H. Adaptive physiological processes in the host during gastrointestinal parasitism. Int. J. Parasitol. 2001, 31, 231−244. (2) Hoste, H.; Torres-Acosta, J. F. J.; Sandoval-Castro, C. A.; Mueller-Harvey, I.; Sotiraki, S.; Louvandini, H.; Thamsborg, S. M.; Terrill, T. H. Tannin containing legumes as a model for nutraceuticals against digestive parasites in livestock. Vet. Parasitol. 2015, 212, 5−17. (3) Sykes, A. R. Parasitism and production in farm animals. Anim. Prod. 1994, 59, 155−172. (4) Jackson, F.; Bartley, D.; Bartley, Y.; Kenyon, F. Worm control in sheep in the future. Small Rumin. Res. 2009, 86, 40−45. (5) Saeed, M.; Iqbal, Z.; Jabbar, A.; Masood, S.; Babar, W.; Saddiqi, H. A.; Yaseen, M.; Sarwar, M.; Arshad, M. Multiple anthelmintic resistance and the possible contributory factors in beetal goats in an irrigated area (Pakistan). Res. Vet. Sci. 2010, 88, 267−272. (6) Taylor, M. A.; Learmount, J.; Lunn, E.; Morgan, C.; Craig, B. H. Multiple resistance to anthelmintics in sheep nematodes and comparison of methods used for their detection. Small Rumin. Res. 2009, 86, 67−70. (7) Beynon, S. A. Potential environmental consequences of administration of anthelmintics to sheep. Vet. Parasitol. 2012, 189, 113−124. (8) De Gives, P. M.; Arellano, M. L.; Hernández, E. L.; Marcelino, L. A. Plant extracts: a potential tool for controlling animal parasitic nematodes. In The Biosphere; Ishwaran, N., Ed.; InTech: Rijeka, 2012; pp 119−131. (9) Rochfort, S.; Parker, A. J.; Dunshea, F. R. Plant bioactives for ruminant health and productivity. Phytochemistry 2008, 69, 299−322. (10) Acharya, J.; Hildreth, M. B.; Reese, R. N. In vitro screening of forty medicinal plant extracts from the United States Northern Great Plains for anthelmintic activity against Haemonchus contortus. Vet. Parasitol. 2014, 201, 75−81. (11) Athanasiadou, S.; Kyriazakis, I.; Jackson, F.; Coop, R. L. Direct anthelmintic effects of condensed tannins towards different gastrointestinal nematodes of sheep: In vitro and in vivo studies. Vet. Parasitol. 2001, 99, 205−219. (12) Hoste, H.; Martinez-Ortiz-De-Montellano, C.; Manolaraki, F.; Brunet, S.; Ojeda-Robertos, N.; Fourquaux, I.; Torres-Acosta, J. F. J.; Sandoval-Castro, C. A. Direct and indirect effects of bioactive tanninrich tropical and temperate legumes against nematode infections. Vet. Parasitol. 2012, 186, 18−27. (13) Ketzis, J. K.; Vercruysse, J.; Stromberg, B. E.; Larsen, M.; Athanasiadou, S.; Houdijk, J. G. M. Evaluation of efficacy expectations for novel and non-chemical helminth control strategies in ruminants. Vet. Parasitol. 2006, 139, 321−335. (14) Min, B. R.; Hart, S. P. Tannins for suppression of internal parasites. J. Anim. Sci. 2003, 81, 102−109. (15) Butter, N. L.; Dawson, J. M.; Wakelin, D.; Buttery, P. J. Effect of dietary condensed tannins on gastrointestinal nematodes. J. Agric. Sci. 2001, 137, 461−469. (16) Novobilský, A.; Stringano, E.; Hayot Carbonero, C.; Smith, L. M. J.; Enemark, H. L.; Mueller-Harvey, I.; Thamsborg, S. M. In vitro effects of extracts and purified tannins of sainfoin (Onobrychis viciifolia) against two cattle nematodes. Vet. Parasitol. 2013, 196, 532−537. (17) Quijada, J.; Fryganas, C.; Ropiak, H. M.; Ramsay, A.; MuellerHarvey, I.; Hoste, H. Anthelmintic activities against Haemonchus contortus or Trichostrongylus colubriformis from small ruminants are

S Supporting Information *

■

Article

ACKNOWLEDGMENTS

Sanjib Saha, Jussi Suvanto, and Milla Leppä are acknowledged for their valuable help in compound purification. We address special thanks to Jessica Quijada, Israel Chan Pérez, Elodie Gaudin, Ramzi El Korso, and Jack Pot for the introduction to the world of parasites and for fruitful discussions. Liza Fonsou is acknowledged for obtaining the egg matrix used in the incubation tests. K


Article

Journal of Agricultural and Food Chemistry influenced by structural features of condensed tannins. J. Agric. Food Chem. 2015, 63, 6346−6354. (18) Williams, A. R.; Fryganas, C.; Ramsay, A.; Mueller-Harvey, I.; Thamsborg, S. M. Direct anthelmintic effects of condensed tannins from diverse plant sources against Ascaris suum. PLoS One 2014, 9, e97053. (19) Williams, A. R.; Ropiak, H. M.; Fryganas, C.; Desrues, O.; Mueller-Harvey, I.; Thamsborg, S. M. Assessment of the anthelmintic activity of medicinal plant extracts and purified condensed tannins against free-living and parasitic stages of Oesophagostomum dentatum. Parasites Vectors 2014, 7, 518. (20) Barbehenn, R. V.; Jaros, A.; Lee, G.; Mozola, C.; Weir, Q.; Salminen, J.-P. Hydrolyzable tannins as “quantitative defenses”: Limited impact against Lymantria dispar caterpillars on hybrid poplar. J. Insect Physiol. 2009, 55, 297−304. (21) Moilanen, J.; Salminen, J.-P. Ecologically neglected tannins and their biologically relevant activity: chemical structures of plant ellagitannins reveal their in vitro oxidative activity at high pH. Chemoecology 2008, 18, 73−83. (22) Roslin, T.; Salminen, J.-P. Specialization pays off: contrasting effects of two types of tannins on oak specialist and generalist moth species. Oikos 2008, 117, 1560−1568. (23) Katiki, L. M.; Ferreira, J. F. S.; Gonzalez, J. M.; Zajac, A. M.; Lindsay, D. S.; Chagas, A. C. S.; Amarante, A. F. T. Anthelmintic effect of plant extracts containing condensed and hydrolyzable tannins on Caenorhabditis elegans, and their antioxidant capacity. Vet. Parasitol. 2013, 192, 218−227. (24) König, M.; Scholz, E.; Hartmann, R.; Lehmann, W.; Rimpler, H. Ellagitannins and complex tannins from Quercus petraea bark. J. Nat. Prod. 1994, 57, 1411−1415. (25) Moilanen, J.; Koskinen, P.; Salminen, J.-P. Distribution and content of ellagitannins in Finnish plant species. Phytochemistry 2015, 116, 188−197. (26) Salminen, J.-P.; Ossipov, V.; Pihlaja, K. Distribution of hydrolysable tannins in the foliage of finnish birch species. Z. Naturforsch., C: J. Biosci. 2002, 57, 248−256. (27) Baert, N.; Karonen, M.; Salminen, J.-P. Isolation, characterisation and quantification of the main oligomeric macrocyclic ellagitannins in Epilobium angustifolium by ultra-high performance chromatography with diode array detection and electrospray tandem mass spectrometry. J. Chromatogr. A 2015, 1419, 26−36. (28) Matsuo, Y.; Wakamatsu, H.; Omar, M.; Tanaka, T. Reinvestigation of the stereochemistry of the C-glycosidic ellagitannins, vescalagin and castalagin. Org. Lett. 2015, 17, 46−49. (29) Coles, G. C.; Simpkin, K. G. Resistance of nematode eggs to the ovicidal activity of benzimidazoles. Res. Vet. Sci. 1977, 22, 386−387. (30) Engström, M. T.; Pälijärvi, M.; Salminen, J.-P. Rapid fingerprint analysis of plant extracts for ellagitannins, gallic acid, and quinic acid derivatives and quercetin-, kaempferol- and myricetin-based flavonol glycosides by UPLC-QqQ-MS/MS. J. Agric. Food Chem. 2015, 63, 4068−4079. (31) Moilanen, J.; Sinkkonen, J.; Salminen, J.-P. Characterization of bioactive plant ellagitannins by chromatographic, spectroscopic and mass spectrometric methods. Chemoecology 2013, 23, 165−179. (32) Yoshida, T.; Hatano, T.; Ito, H.; Okuda, T. Structural diversity and antimicrobial activities of ellagitannins. In Chemistry and Biology of Ellagitannins: An Underestimated Class of Bioactive Plant Polyphenols; Quideau, S., Ed.; World Scientific: Singapore, 2009; pp 55−93. (33) Hördegen, P.; Cabaret, J.; Hertzberg, H.; Langhans, W.; Maurer, V. In vitro screening of six anthelmintic plant products against larval Haemonchus contortus with a modified methyl-thiazolyl-tetrazolium reduction assay. J. Ethnopharmacol. 2006, 108, 85−89. (34) Martínez-Ortíz-de-Montellano, C.; Arroyo-Ló p ez, C.; Fourquaux, I.; Torres-Acosta, J. F. J.; Sandoval-Castro, C. a.; Hoste, H. Scanning electron microscopy of Haemonchus contortus exposed to tannin-rich plants under in vivo and in vitro conditions. Exp. Parasitol. 2013, 133, 281−286. (35) Salminen, J.-P. The chemistry and chemical ecology of ellagitannins in plant-insect interactions: From underestimated

molecules to bioactive plant constituents. In Recent Advances in Polyphenol Research; Romani, A., Lattanzio, V., Quideau, S., Eds.; John Wiley & Sons, Ltd: Chichester, UK, 2014; pp 83−113. (36) Barbehenn, R. V.; Jones, C. P.; Hagerman, A. E.; Karonen, M.; Salminen, J.-P. Ellagitannins have greater oxidative activities than condensed tannins and galloyl glucoses at high pH: Potential impact on caterpillars. J. Chem. Ecol. 2006, 32, 2253−2267. (37) Kilkowski, W.; Gross, G. G. Color reaction of hydrolyzable tannins with Bradford reagent, Coomassie brilliant blue. Phytochemistry 1999, 51, 363−366. (38) Haslam, E. Natural polyphenols (vegetable tannins) as drugs: Possible modes of action. J. Nat. Prod. 1996, 59, 205−215. (39) Salminen, J.-P.; Karonen, M. Chemical ecology of tannins and other phenolics: We need a change in approach. Funct. Ecol. 2011, 25, 325−338. (40) Murdiati, T.; McSweeney, C.; Lowry, J. Metabolism in sheep of gallic acid, tannic acid and hydrolysable tannin from Terminalia oblongata. Aust. J. Agric. Res. 1992, 43, 1307−1319. (41) Shimada, T. Salivary proteins as a defense against dietary tannins. J. Chem. Ecol. 2006, 32, 1149−1163. (42) Appel, H. M. Phenolics in ecological interactions: The importance of oxidation. J. Chem. Ecol. 1993, 19, 1521−1552. (43) Nursten, H. Practical Polyphenolics: From structure to molecular recognition and physiological action. Trends Food Sci. Technol. 1999, 10, 339. (44) Hagerman, A. E. Fifty years of polyphenol-protein complexes. In Recent Advances in Polyphenol Research; Cheynier, V., Sarni-Manchado, P., Quideau, S., Eds.; Wiley-Blackwell: Oxford, UK, 2012; pp 71−97. (45) Hoste, H.; Jackson, F.; Athanasiadou, S.; Thamsborg, S. M.; Hoskin, S. O. The effects of tannin-rich plants on parasitic nematodes in ruminants. Trends Parasitol. 2006, 22, 253−261. (46) Molan, A.-L. Effect of purified condensed tannins from pine bark on larval motility, egg hatching and larval development of Teladorsagia circumcincta and Trichostrongylus colubriformis (Nematoda: Trichostrongylidae). Folia Parasitol. 2014, 61, 371−376. (47) Molan, A.-L.; Faraj, A. M. The effects of condensed tannins extracted from different plant species on egg hatching and larval development of Teladorsagia circumcincta (Nematoda: Trichostrongylidae). Folia Parasitol. 2010, 57, 62−68. (48) Molan, A. L.; Alexander, R.; Bbookes, I. M.; Mcnabb, W. C. Effects of Sulla condensed tannins on the degradation of ribulose-1,5bisphosphate carboxylase/oxygenase (rubisco) and on the viability of three sheep gastrointestinal nematodes in vitro. J. Anim. Vet. Adv. 2004, 3, 165−174. (49) Molan, A. L.; Waghorn, G. C.; Min, B. R.; McNabb, W. C. The effect of condensed tannins from seven herbages on Trichostrongylus colubriformis larval migration in vitro. Folia Parasitol. 2000, 47, 39−44. (50) Mueller-Harvey, I. Analysis of hydrolysable tannins. Anim. Feed Sci. Technol. 2001, 91, 3−20. (51) Okuda, T.; Ito, H. Tannins of constant structure in medicinal and food plants− hydrolyzable tannins and polyphenols related to tannins. Molecules 2011, 16, 2191−2217. (52) Niderkorn, V.; Macheboeuf, D. Identification of bioactive grassland plants for reducing enteric methane production and rumen proteolysis using an in vitro screening assay. Anim. Prod. Sci. 2014, 54, 1805−1809. (53) Rauha, J.-P.; Wolfender, J.-L.; Salminen, J.-P.; Pihlaja, K.; Hostettmann, K.; Vuorela, H. Characterization of the polyphenolic composition of Purple Loosestrife (Lythrum salicaria). Z. Naturforsch., C: J. Biosci. 2001, 56, 13−20. (54) Tuominen, A.; Toivonen, E.; Mutikainen, P.; Salminen, J.-P. Defensive strategies in Geranium sylvaticum. Part 1: Organ-specific distribution of water-soluble tannins, flavonoids and phenolic acids. Phytochemistry 2013, 95, 394−407. (55) Agrawal, A. A.; Hastings, A. P.; Johnson, M. T. J.; Maron, J. L.; Salminen, J.-P. Insect herbivores drive real-time ecological and evolutionary change in plant populations. Science 2012, 338, 113−116.

L


Chemical Structures of Plant Hydrolyzable ... - ACS Publications

Recommend Documents