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Animals : an Open Access Journal from MDPI logoLink to Animals : an Open Access Journal from MDPI
. 2020 Jan 29;10(2):219. doi: 10.3390/ani10020219

Anthelmintic Activity of Wormwood (Artemisia absinthium L.) and Mallow (Malva sylvestris L.) against Haemonchus contortus in Sheep

Dominika Mravčáková 1, Michaela Komáromyová 2, Michal Babják 2, Michaela Urda Dolinská 2, Alžbeta Königová 2, Daniel Petrič 1, Klaudia Čobanová 1, Sylwester Ślusarczyk 3, Adam Cieslak 4, Marián Várady 2,*, Zora Váradyová 1,*
PMCID: PMC7070545  PMID: 32013192

Abstract

Simple Summary

The gastrointestinal parasitic nematode Haemonchus contortus of small ruminants is an important target for chemoprophylaxis. Repeated use of anthelmintics in the form of synthetic drugs increases the risk of residues in food products and the development of anthelmintic resistance. However, the use of combinations of dry traditional medicinal plants as nutraceuticals is an alternative to chemotherapeutics for controlling haemonchosis in ruminants. Therefore, the aim of this study is to determine the effect of dietary supplementation with wormwood, mallow and their mix on parasitological status and inflammatory response in lambs experimentally infected with H. contortus. Simultaneously, the present study evaluated by the egg hatch test the in vitro anthelminthic effects of different concentrations (50–1.563 mg/mL) of the aqueous extracts of these plants. Our results revealed that the strong anthelmintic effect of both medicinal plants observed in vitro was not fully confirmed in vivo. This knowledge builds on our previously published findings and highlights that the effect of dry medicinal plants depends on the variety and synergy of plant polyphenols and the combination of bioactive compounds that together have an effect and contribute to a certain pharmacological efficacy.

Abstract

The objective of this study is to evaluate the effect of dry wormwood and mallow on the gastrointestinal parasite of small ruminants Haemonchus contortus. Twenty-four experimentally infected lambs were randomly divided into four groups of six animals each: unsupplemented lambs, lambs supplemented with wormwood, lambs supplemented with mallow and animals supplemented with a mix of both plants. Faecal samples from the lambs were collected on day 23, 29, 36, 43, 50, 57, 64 and 75 post-infection for quantification of the number of eggs per gram (EPG). The mix of both plants contained phenolic acids (10.7 g/kg DM) and flavonoids (5.51 g/kg DM). The nematode eggs were collected and in vitro egg hatch test was performed. The aqueous extracts of both plants exhibited strong ovicidal effect on H. contortus, with ED50 and ED99 values of 1.40 and 3.76 mg/mL and 2.17 and 5.89 mg/mL, respectively, in the in vitro tests. Despite the great individual differences between the treated lambs in eggs reduction, the mean EPG of the untreated and treated groups did not differ (p > 0.05). Our results indicate that using wormwood and mallow as dietary supplements do not have a sufficient effect on lambs infected with H. contortus.

Keywords: dietary treatments, plant bioactive compounds, egg counts, UHRMS, Haemonchus contortus

1. Introduction

The gastrointestinal nematode (GIN) infection haemonchosis is a prevalent parasitic disease associated with economic losses, lowered productivity, morbidity and mortality. Haemonchus contortus is highly prevalent in sheep and goats worldwide and is mainly controlled by chemoprophylaxis through the repeated application of chemotherapeutics with the risk of development of anthelmintic resistance [1].

The screening of traditional medicinal plants containing promising contents of bioactive compounds with anthelmintic activity has great potential as an alternative source of natural anthelmintics and antioxidants that may be sustainable and environmentally acceptable. Various bioactive compounds (i.e., polyphenols, flavonoids, condensed tannins) that possess an anthelmintic effect [2,3] and antibacterial and antioxidant activities have been isolated from wormwood (Artemisia absinthium L.) [4,5]. Many authors have reported the antioxidant and antimicrobial properties of wormwood essential oils [6,7] and the anthelmintic activity of the flavonoids quercetin and apigenin [3]. Diets containing dried wormwood as a 5%–10% replacement for rice straw also provide better quality roughage with a considerable content of crude protein [8,9,10]. The high pharmacological activity of the medicinal plant mallow (Malva sylvestris L.), due to the presence of amino acids, flavonoids, mucilages, terpenoids, phenol derivatives, enzymes, coumarins and sterols, is known [11,12]. Mallow has antimicrobial, antifungal and anti-inflammatory properties [13,14].

In traditional medicine, whole plants or mixtures of plants are used rather than isolated compounds, and therefore more research is needed on all types of interaction between plant constituents. The ultra-high-resolution mass spectrometry (UHRMS) analyses of dry medicinal plants or plant mixtures in our recent studies with H. contortus [15,16,17] identified a wide range of bioactive compounds with important pharmacological activities, mainly flavonoids, phenolic acids, diterpenes, and alkaloids. These experiments, which combined chromatographic analyses with the determination of antioxidant capacity, are helpful in identifying plants with consistent concentrations of anthelmintic and antioxidant compounds for in vitro and in vivo studies. Our previous studies showed that medicinal plant mixtures are multicomponent mixes that possess effects via a multitarget additive and synergistic mode [16,17,18].

Therefore, in the present study, we hypothesize that some medicinal plants from these mixtures are by themselves multicomponent mixes and can elicit effects via pharmacological activity on H. contortus infected lambs. The medicinal plants, wormwood and mallow, were chosen based on their previously described best phytotherapeutic properties and anthelmintic activity in vitro [15]. The goal is to determine the effect of dietary supplementation with wormwood and mallow on parasitological status and inflammatory parameters of lambs experimentally infected with H. contortus.

2. Material and Methods

2.1. Ethics Statement

All procedures and animals were cared for under European Community guidelines (EU Directive 2010/63/EU). The experimental protocol was approved by the Ethical Committee of the Institute of Parasitology of the Slovak Academy of Sciences, in accordance with national legislation in Slovakia.

2.2. Analysis of Bioactive Compounds

Wormwood and mallow were ground to a fine powder, and 100 mg were extracted three times in 80% MeOH for 30 min at 40 °C. The extracts were evaporated to dryness, dissolved in 2 mL of Milli-Q water (acidified with 0.2% formic acid) and purified by Solid Phase Extraction (SPE) using Oasis HLB 3cc Vac Cartrige (60 mg, Waters Corp., Milford, MA, USA). The cartridges were washed with 0.5% methanol to remove carbohydrates and then washed with 80% methanol to elute phenolics. The phenolic fraction was re-evaporated and dissolved in 1 mL of 80% methanol (acidified with 0.1% formic acid). The sample was then centrifuged (23,000× g, 5 min) before spectrometric analysis. All analyses were performed in triplicate for three independent samples and stored in a freezer at −20 °C before analysis. The phenolic acids and flavonoids of the plant materials were analysed by a ultra-high-resolution mass spectrometry (Dionex UltiMate 3000RS, Thermo Scientific, Darmstadt, Germany) system with a charged aerosol detector interfaced with a high-resolution quadrupole time-of-flight mass spectrometer (HR/Q-TOF/MS, Compact, Bruker Daltonik GmbH, Bremen, Germany). The metabolomes of the samples were chromatographically separated, as was described [19]. The flow rate, spectra, operating parameters, collision energy, data calibration and spectra processing were previously described [20]. The amount of the particular phenolic acids in the samples was calculated as the chlorogenic acid (CAS 327-97-9, 3-Caffeoylquinic acid) equivalent, and hyperoside (CAS 482-36-0, quercetin 3-galactoside) was used for calculating the amount of identified flavonoids. Stock solutions of hyperoside and chlorogenic acids were prepared in MeOH, as was described previously [16].

2.3. In Vitro Test

The in vitro egg hatch test (EHT) was performed in order to assess the ovicidal effect of aqueous extracts of wormwood and mallow and compared with the chemotherapeutic effect of thiabendazole anthelmintic drug. The nematode eggs for in vitro EHT were obtained from the untreated UNS group. The concentrations of aqueous extracts used and EHT has been previously described [15].

Chemical tests for the screening of main constituents in the medicinal plants under study were carried out in the aqueous extracts using standard procedures [21,22]. Qualitative phytochemical screening revealed the active compounds mainly tannins, flavonoids, glycosides, saponins, alkaloids, and terpenoids (Table 1).

Table 1.

Chemical composition of the aqueous plant extracts.

Plant Species Tannins Flavonoids Glycosides Saponins Alkaloids Terpenoids
A. absinthium + - - + - +
M. sylvestris + + + - + +
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(+): the presence of phytochemicals; (-): the absence of phytochemicals.

2.4. Experiment In Vivo

The experiment was conducted on 24 3–4-month-old female lambs (Improved Valachian) with initial body weights of 18.67 ± 0.55 kg. The lambs were housed in common stalls on a sheep farm (Hodkovce, Slovak Republic) with free access to water. After a period of adaptation, all parasite-free lambs were infected by L3 larvae of H. contortus MHCo1 strain [16]. The diet of each animal consisted of oats (500 g DM/d) and meadow hay (ad libitum). Four groups of six animals based on their live-weight were established: unsupplemented lambs (UNS), lambs supplemented with stem of A. absinthium (ART, 1 g DM/d/lamb), lambs supplemented with flower of M. sylvestris (MAL, 15 g DM/d/lamb) and animals supplemented with mix of A. absinthium and M. sylvestris (ARTMAL, 16 g DM/d/lamb). The doses of plant supplements were based on the plant proportions used in our previous study [18]. The dry plants from commercial sources (AGROKARPATY, Plavnica, Slovak Republic) were mixed daily with the oats during the experimental period (75 days, D). The lambs were weighed on D0, D15, D30, D45 and D70. Faecal samples from the rectum of lambs were collected on D23, D29, D36, D43, D50, D57, D64 and D75 post-infection for quantification of the eggs per gram (EPG). The detection of strongylid eggs was performed by McMaster technique, as was previously described [23]. The blood sera samples of each animal were obtained from D15, D30, D45 and D70 [18]. Helminthological autopsy were done after 75 days of infection [16].

2.5. Blood Sera Analysis

Sheep immunoglobulin G (IgG), sheep immunoglobulin A (IgA) and sheep eosinophil peroxidase (EPX) were measured by ELISA kits (MyBioSource Ltd., San Diego, CA, USA). The sensitivity of the IgG, IgA and EPX kits were 0.938 ng/mL, 1.875 ng/mL and 1.0 ng/mL, respectively.

2.6. Statistical Analysis

Statistical analysis was performed using analysis variance (GraphPad Prism 8, GraphPad Software, Inc., San Diego, CA, USA) as repeated-measures mixed models representing the four animal groups (UNS, ART, MAL, ARTMAL) and sampling days. Differences between the animal groups were analysed by a two-way ANOVA with a Bonferroni post hoc test. Differences between the arithmetic EPG means between groups and between worm counts at dissection were analysed by Student’s t-tests. A logistic regression model was used to determine the ED50 and ED99 [24].

3. Results

3.1. Bioactive Compounds

The A. absinthium contained 6.48 g/kg DM of phenolic acids and 0.35 g/kg DM of flavonoids with greater concentrations of chlorogenic acid (3.42 g/kg DM) and 1,5-dicaffeoylquinic acid (2.12 g/kg DM) (Table 2). The M. sylvestris contained 0.65 g/kg DM of phenolic acids and 6.48 g/kg DM of flavonoids with higher concentrations of delphinidin-5-glucoside 3-lathyroside (1.64 g/kg DM), kaempferol-3-O-rutinoside (0.82 g/kg DM), apigenin-o-hex (1.56 g/kg DM) and coumarinic acid (0.47 g/kg DM). The mix of both plants contained 10.7 g/kg DM phenolic acids and 5.51 g/kg DM of flavonoids with greater concentrations of methyl-4-O-beta-d-glucopyranosylcaffeate (2.23 g/kg DM), 1,5-dicaffeoylquinic acid (1.64 g/kg DM), kaempferol-O-Hex (1.40 g/kg DM), apigenin-O-Hex (1.29 g/kg DM) and luteolin-O-Hex (0.70 g/kg DM).

Table 2.

Contents of the flavonoids and phenolic acids (g/kg DM) identified in the plants and mix.

No. RT (min) λmax (nm) m/z
[M-H]- 		MS2 MS2 Fragments Formula Compound Flavonoids Phenolic Acids
Artemisia absinthium
1 2.80 189.0759 127/0759 171/145/115 C8H14O5 L-(-)Malic acid diethyl ester 0.22
2 4.10 215.325 353.0877 191/0567 179/161/135 C16H18O9 Chlorogenic acid 3.42
3 7.80 281.1023 C14H18O6 ND 0.01
4 8.00 367.1031 191/0546 173 C17H20O9 3-O-Feruloylquinic acid 0.08
5 8.90 279.1223 234/1009 261/217/177/199 C15H20O5 Artabsinolide
6 9.10 325.1283 163 279/235 C16H22O7 ND 0.03
7 9.20 327.1440 279 235 C16H24O7 ND
8 10.00 289.000 263.1282 201/1271 245/219/149/161/177 C15H20O4 Tanacetin
9 10.20 281.1386 219/373 263/237/201 C16H24O7 Artabsinolide D
10 11.00 515.1193 353/0867 191/179/135 C25H24O12 1,5-Dicaffeoylquinic acid 2.12
11 11.20 653.1719 345/0595 330/302 C29H34O17 Spinacetin 3-rutinoside 0.24
12 11.40 477.1032 314/0415 357 C22H22O12 Isorhamnetin 7-glucoside 0.10
13 11.70 515.1192 353/0869 173/179/191/155 C25H24O12 4,5-Dicaffeoylquinic acid 0.61
14 14.90 507.1502 413/1246 101/324/259 C24H28O12 Hedycoryside B
15 15.00 511.2698 467/2775 405 C30H40O7 Anabsin
16 15.50 345.1344 301/1433 257/213/187 C19H22O6 Diosbulbin E
17 15.60 511.2698 245/1175 263/201 C30H40O7 Anabsin
18 16.50 329.2323 211/1324 229/171/183/139 C18H34O5 Pinellic acid
Total flavonoids and phenolic acids 0.35 6.48
Malva sylvestris
1 1.60 250.320 517.1195 355/0667 193 C21H26O15 Ferullo-O-Hex-O-Hex 0.02
2 1.80 250.301 206.0443 144/0437 C10H9NO4 ND 0.17
3 7.00 523 757.1846 347/0761 329/261/509 C32H39O21 Delphinidin 5-glucoside 3-lathyroside 1.64
4 7.90 308 163.0381 119/0502 C9H8O3 Coumaric acid 0.47
5 8.00 288 465.1046 303/0505 285/275/177 C21H22O12 Xeractinol 0.17
6 8.20 520 449.1094 287/0555 259/243 C21H22O11 Cyanidin-O-Hex 0.28
7 8.50 518 593.1645 431/0982 269/0460 C27H31O15 Pelargonidin-O-Hex-O-Hex 0.14
8 8.70 283 687.1784 507/1142 345/0629/165 ND 0.18
9 9.00 283 525.1246 345/0815 165/197/139 C23H25O14 ND 0.07
10 9.20 287 303.0498 153/0169 125/217 C15H12O7 ND 0.04
11 9.50 283 773.1781 507/1124 345/165 C32H38O22 ND 0.22
12 10.00 609.1458 301/0330 C27H31O16 Quercetin-3-O-rutinoside 0.40
13 10.20 268.343 447.0928 285/0386 C21H20O11 Kaempferol-O-Hex 0.49
14 10.50 346 505.0981 343/0442 C23H22O13 Quercetin 3’-glucoside-7-acetate 0.03
15 10.90 266.343 593.1504 285/0395 C27H30O15 Kaempferol-3-O-rutinoside 0.82
16 11.10 291.346 433.1124 271/0599 151 C21H22O10 Naringenin-O-Hex 0.13
17 11.25 291 287.0550 259/0596 152/201/243 C15H12O6 Tetrahydroxyflavone 0.30
18 11.40 268.336 431.0978 269/0435 C21H20O10 Apigenin-O-Hex 1.56
19 15.00 285.340 271.0595 151/0012 177/119 C15H12O5 Naringenin 0.01
20 15.40 327.2169 C18H32O5 (E)-10-(8-Hydroxyoctanoyloxy)-enoic acid
21 15.70 215.334 269.0443 151/0016 225 C15H10O5 Trihydroxyflavone 0.02
Total flavonoids and phenolic acids 6.50 0.66
Mix of A. absinthium and M. sylvestris
1 4.00 215.325 353.0883 191/0561 173/179 C16H18O9 4-O-Caffeoylquinic acid 0.61
2 4.10 215.325 353.0883 191/0561 179/173 C16H18O9 3-O-Caffeoylquinic acid 0.74
3 5.90 215.287 355.1035 193/0498 149/134 C16H20O9 1-O-2’-Hydroxy-4’-
methoxycinnamoyl
-b-D-glucose		 0.38
4 6.10 215.302 355.1038 149/0598 193/134 C16H20O9 1-O-Feruloylglucose 0.70
5 7.90 161.0225 133/0282 C9H6O3 Umbeliferone 0.40
6 8.00 323.0760 161/0221 C15H16O8 Mahaleboside 0.02
7 8.10 225.287 465.1033 303/177 285/0399 C21H22O12 Xeractinol 0.04
8 8.30 520.000 449.1094 287/0555 259/243 C21H22O11 Cyanidin-O-Hex 0.03
9 8.50 367.1025 173/0433 193/155/134 C17H20O9 Feruloylquinic acid 0.25
10 9.00 233.294.318 355.1034 193/0507 149/134 C16H20O9 Methyl-4-O-beta-D-
glucopyranosylcaffeate		 2.23
11 9.80 255.354 463.0882 301/0337 343 C21H20O12 Quercetin O-Hex 0.44
12 9.90 252.351 609.1472 301/0331 285/0415 C27H30O16 Isoquercitrin O-Dhex 0.42
13 10.30 257,4 447.0920 285/0386 C21H20O11 Kaempferol-O-Hex 1.40
14 10.70 217.291.325 515.1189 353/0877 179/191 C25H24O12 3,5-Dicaffeoylquinic acid 0.80
15 10.80 187.0958 125/0968 169 C9H16O4 ND
16 10.90 221.329 593.1520 285/0397 C27H30O15 Kaempferol-3-O-rutinoside 0.37
17 11.10 217.291.325 515.1197 353/0869 191/179 C25H24O12 1,5-Dicaffeoylquinic acid 1.64
18 11.15 291.346 433.1124 271/0599 151 C21H22O10 Naringenin-O-Hex 0.19
19 11.40 266.3 431.0976 269/0434 C21H20O10 Apigenin O-Hex 0.56
20 11.50 268.343 447.0928 285/0386 C21H20O11 Luteolin O-Hex 0.70
21 11.60 266.3 431.0976 269/0434 C21H20O10 Apigenin O-Hex 0.73
22 11.70 215.290.325 515.119 353/0868 173/179/191 C25H24O12 4,5-Dicaffeoylquinic acid 0.68
23 12.40 325.0 517.1342 355/1022 353/193/149/161 C25H26O12 3-caffeoyl-4-dihydrocaffeoyl quinic acid 0.35
24 12.90 268.320 639.3176 519/2604 476/373/145 C37H44N4O6 Tris-trans-p-coumaroylspermine 0.50
25 13.10 218.268.339 473.1083 269/0426 413 C23H22O11 Apigenin -O-(Hex-Ac) 0.12
26 13.70 325.0 517.1330 323/0759 353/193/149/161 C25H26O12 4-caffeoyl-3-dihydrocaffeoyl quinic acid 0.07
27 14.20 218.268.339 473.1083 269/0426 413 C23H22O11 Apigenin -O-(Hex-Ac) 0.22
28 14.40 266.336 515.1187 269/0444 C25H24O12 Formononetin 7-O-glucoside-6’’-malonate 0.22
29 15.00 285.340 271.0595 151/0012 177/119 C15H12O5 Naringenin 0.07
30 15.40 327.2169 C18H32O5 (E)-10-(8-Hydroxyoctanoyloxy)dec-2-enoic acid 0.03
31 15.70 215.334 269.0443 151/0016 225 C15H10O5 Trihydroxyflavone 0.03
32 17.10 222.309 785.3554 545/2397 665/502/399/145 C46H50N4O8 Tetra-trans-p-coumaroylspermine 0.47
33 19.00 373.0914 358/0681 343/329/315 C19H18O8 Dihydroxy—tetramethoxyflavone 0.03
34 20.30 267.334 559.1069 269/0443 515/1172 C26H24O14 Apigenin 7-(2’’-acyl-6”maloylglycosyl) 0.13 0.65
Total flavonoids and phenolic acids 5.51 10.7
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No: peak numbers from UV chromatograms; RT: retention time; λmax: wavelengths of maximum absorption in the visible region; MS2: main ion; ND: not determined.

3.2. In Vitro Test (EHT)

The dose-response relationships of aqueous extracts of A. absinthium or M. sylvestris, respectively, against H. contortus in the egg hatch test (EHT) are shown in Figure 1a,b. Both aqueous plant extracts exhibited a strong ovicidal effect on H. contortus in in vitro EHT. The ED50 and ED99 values were 1.40 and 3.76 mg/mL in A. absinthium (Figure 1a) and 2.17 and 5.89 mg/mL in M. sylvestris (Figure 1b), respectively. Thiabendazole at a concentration of 1.0 µg/mL has a 100% ovicidal effect.

Figure 1.

Figure 1

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(a,b) Dose-response relationship of plant aqueous extracts against Haemonchus contortus in the egg hatch test (EHT) after 24 h of incubation at 26 °C.

3.3. Parasitological Status

The patterns of egg shedding for UNS, ART, MAL and ARTMAL are shown in Figure 2. Data from D36 were statistically compared and used to determine the reduction in egg output for ART, MAL and ARTMAL relative to UNS. Mean faecal eggs per gram (EPGs) were influenced by time from infection (p < 0.05), and for all groups, EPGs increased until D50 or D57, respectively. The EPGs in the lambs treated with MAL, ART and ARTMAL compared with UNS group did not differ (p > 0.05). The necropsy on D75 found a numerical decrease (p > 0.05) in the abomasal worm counts for the ARTMAL groups compared to the other groups (Figure 3).

Figure 2.

Figure 2

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Mean faecal egg counts for the groups of lambs infected with Haemonchus contortus (Treatment: p > 0.05; time: p < 0.001; treatment × time: p > 0.05). UNS: unsupplemented; ART: A. absinthium; MAL: M. sylvestris; ARTMAL: ART plus MAL.

Figure 3.

Figure 3

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Abomasal worm counts of Haemonchus contortus in the lambs of each treatment at the end of the experiment (p > 0.05). UNS: unsupplemented; ART: A. absinthium; MAL: M. sylvestris; ARTMAL: ART plus MAL.

3.4. Inflammatory Response

Table 3 shows the inflammatory IgG, IgA and EPX response. Serum IgG and IgA values were not influenced by treatment, time and treatment × time (p > 0.05). Mean serum EPX values ranged in the treated groups from 24.1 to 73.4 ng/mL, and the values were influenced by treatment and time (p < 0.05).

Table 3.

Inflammatory responses of the experimental groups (n = 6).

Item Day UNS ART MAL ARTMAL SD Significance of Effects
Treatment (T) Time T × Time
IgG 15 0.627 2.15 2.31 2.23 2.54 NS NS NS
(ng/mL) 30 1.03 0.780 0.986 1.61 1.61
45 1.33 0.639 0.378 1.04 1.07
70 1.16 0.689 3.53 1.39 1.66
IgA 15 0.434 1.28 0.811 0.767 0.522 NS NS NS
(ng/mL) 30 0.825 0.666 0.519 0.589 0.245
45 0.529 0.438 0.787 0.485 0.233
70 0.813 0.696 0.726 0.626 0.195
EPX 15 37.5 66.6 52.2 50.0 17.9 * * NS
(ng/mL) 30 44.0 36.9 29.4 44.8 13.9
45 31.4 38.6 24.1 51.4 17.5
70 49.2 33.4 37.8 73.4 21.1
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UNS: unsupplemented; ART: A. absinthium; MAL: M. sylvestris; ARTMAL: ART plus MAL; EPX: eosinophil peroxidase; NS: not significant; SD: standard deviation. * p < 0.05.

4. Discussion

In the present study, phenolic compounds, flavonoids and phenolic acids among them, were detected in wormwood, mallow and a mix of both plants. Phenolic acids, including chlorogenic acid, caffeoylquinic acid derivatives, coumaric acid and methyl 4-O-beta-D-glucopyranosylcaffeate, were identified in a range from 0.65 to 10.7 g/kg DM. Chlorogenic acid and 1,5- and 4,5-dicaffeoylquinic acid possess well-known antibacterial, anthelmintic, anti-inflammatory, and antioxidant biological activities in vitro and in vivo [25,26]. Similarly, coumaric acid in Senegalia gaumeri leaf extract compounds has shown potential anthelmintic effects against H. contortus larvae [27]. The phenolic acid contents for wormwood and the mix (but not for mallow) were within the range of 3.6 to 57.3 g/kg DM, as was reported for plant mixtures used previously in infected lambs [16,17]. In relation to flavonoids with antioxidant properties, we identified mainly flavones (apigenin and luteolin), flavonols (kaempferol and quercetin) and flavanones (naringenin) [28], which may also have anthelmintic activity [3,29]. However, the total content of flavonoids in wormwood in the present study was lower (0.35 g/kg DM) versus mallow or the mix (6.48 or 5.51 g/kg DM, respectively) or compared to previous studies (9.96 and 29.5 or 41.5 g/kg DM, respectively) [16,17].

It is clear that medicinal plants that have an anthelmintic effect in vitro are often not equally effective in vivo, because there is different bioavailability, pharmacology of host animals, metabolism of bioactive compounds by rumen microflora and experimental conditions [30]. In the present experiment, the aqueous plant extracts of both medicinal plants, A. absinthium and M. sylvestris, exhibited a strong ovicidal effect on H. contortus in vitro, similar to the extracts of the species Artemisia against sheep nematodes [31,32]. However, the mean egg outputs of the UNS group compared to ART, MAL and ARTMAL groups showed no significant differences in egg reduction in lambs. Egg production by H. contortus females remained high (i.e., thousands EPG) until D50 post-inoculation and then decreased similarly as during the patent period of H. contortus in sheep [33]. The rapid reduction in egg excretion after D50 (MAL and ARTMAL) or D57 (ART), respectively, was accompanied by a lower number (not statistically) of adult H. contortus worms in the ARTMAL group. No significant differences in egg excretion in the treated groups may have been due to the lower content of plant biologically active compounds, especially flavonoids, compared to our previous studies [16,17]. However, relatively high SD of the means of the treated groups in the present experiment point to a potentially different treatment effect between lambs. This suggests that these plant materials could have an indirect antiparasitic effect and may promote a host’s resistance to parasitic infection in the longer term. However, the anthelmintic mechanism of action is unknown. It seems that flavonoids with antioxidant capacity, in particular, contribute to the indirect antiparasitic activity [34,35]. However, not only the content of flavonoids appears to play an important role in the anthelmintic activity of medicinal plants and their mixtures. Our results indicate that wormwood and mallow themselves are not responsible for egg and worm reduction in the lambs but probably acting in synergy with other medicinal plants and their bioactive compounds, as was done in previous mixes [17,18]. The effect of dry medicinal plants on the health of animals depends on the source of the multitarget complex bioactive compounds that work synergistically [36,37] and antagonistically [38]. An increase in the resistance of infected lambs to H. contortus infection was shown after the administration of mixtures of dry medicinal plants composed already of 9–13 species [16,17,18]. However, a generally great contribution to the discovery and development of new drugs is a widely applicable strategy for identifying the combinatory compounds responsible for a certain pharmacological activity of plant medicines followed by in vitro and in vivo validation [39].

Ruminal and intestinal fermentation parameters can be manipulated by supplementing a diet with medicinal plants [40]. No adverse effects of wormwood and mallow on the ruminal fermentation parameters (i.e., pH, ammonia N, methane, gas production, and volatile fatty acids) were found [18]. Mainly, pH and ammonia N can affect the release of phenolic compounds from plant materials and the growth of ruminal microbes for microbial protein synthesis [41,42]. Additionally, phenolic compounds, especially flavonoids, can improve body weight gain, growth and the quality of animal products [43]. In the present experiment, polyphenols as dietary supplements did not significantly affect the body weights or live-weight gains of the infected lambs. Dry medicinal plants in the diets of the infected lambs may [18] or may not have influenced the body weights or live-weight gains, which is consonant with a meta-analysis of gastro-intestinal nematode infection in sheep [44].

Our recent studies [16,17,18] of lambs infected with the gastrointestinal nematode H. contortus showed the potential value of medicinal plant mixtures to decrease egg output and worm numbers in parasitic infections of the digestive tract. However, this effect is probably not a consequence of a direct anthelmintic impact on the viability of nematodes, but an increase in the resistance of lambs to nematode infections. Additionally, a recent study also showed that a complementary vegetable mixture of plants belonging to the Compositae, Cesalpinacae, Liliacae, Bromeliaceae and Labiatae families used as feed at two different dosages was ineffective against gastrointestinal nematode infection [45]. It seems that mainly a combination of medicinal plants belonging to different botanical families with beneficial bioactive compounds probably contributes to slowing the dynamics of infection. In the present study, because of the low variety and synergy of plant polyphenols and the combination of bioactive compounds of wormwood and mallow, the reduction in parasitic infection intensity in the treated infected lambs was not sufficient during the 75 days of infection compared to previous studies [16,17,18]. However, it seems that mixtures of dry medicinal plants may affect the host over the longer term. Therefore, more research is needed on combinations of medicinal plants and interactions between compositions of plant mixtures for longer (90-120 days) experimental periods.

5. Conclusions

The data in the present study showed additional new knowledge on the anthelmintic effects of dry medicinal plants as dietary supplements. Our results indicate that using medicinal plants, even those with the best anthelmintic properties in vitro, may not have sufficient effects in vivo on H. contortus infected lamb.

Acknowledgments

The authors are grateful to Valéria Venglovská, Silvia Spišáková, Peter Jerga and Gabriel Benkovský for laboratory and technical assistance.

Author Contributions

Conceptualization, Z.V., M.V.; Investigation, D.M., Z.V., M.V.; Supervision, M.V.; Formal analysis, D.M., M.K., K.Č., M.B., M.U.D., A.K., D.P., S.Ś.; Data Curation, Z.V., M.V., A.C.; Draft Preparation, Z.V.; Writing-Review & Editing, Z.V., M.V. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by funds from the Slovak Research and Development Agency (APVV 18-0131).

Conflicts of Interest

The authors declare no conflict of interest.

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