**3. Summary**

and young people with newly abnormal cerebellar or other central neurological function'. Furthermore, referral should be for urgent specialist assessment (and not a cancer pathway) in order to circumnavigate any issues with weekend cover, differences in local service con-

The guidelines point out that trade-off between net health benefits and resource utilisation are not supported by published economic analysis. It is likely that the above recommendations will result in an increase in MRI scanning with a subsequent reduction in GP attendance, because of fast-tracking medical clearance or diagnosis. In fact, the guidelines predict that

An evidence-based clinical guideline, 'Diagnosis of Brain Tumours in Children', was developed in 2010 by Wilne et al. [105]; it was informed by a systematic literature review, metaanalysis and cohort study [36]. As a result, six main categories of symptoms were identified: headache, nausea and vomiting, visual abnormalities, motor abnormalities, growth, development and behavioural abnormalities. Within the category of behavioural symptoms, lethargy and withdrawal were important neuropsychiatric signs of childhood CNS tumours. The importance of lethargy in diagnosing paediatric CNST has been highlighted by other studies [106, 107]. In fact, the prominence of lethargy in a series of 'sudden death from obstructive hydrocephalus due to intracranial lesions' prompted the authors to conclude that persistent lethargy should be considered a neurological symptom instead of a non-

The relevance of such symptoms in childhood CNST was also highlighted by an earlier study by Wilne et al. [36], in which 'behavioural and educational' symptoms were the presenting feature of brain tumours in children in 10% of cases, and were apparent in 44% of the patients. The behavioural symptoms included: lethargy (majority), irritability, personality change, aggression and emotional lability; and the educational symptoms included deterioration in reading and writing (majority), memory difficulty, poor concentration, global deterioration

Nevertheless, lethargy remains an unspecific symptom with many faces: it is prominent among physical syndromes relating to malignancy, infection, and inflammation; it is also a core psychiatric symptom in both affective (i.e., depression) and psychotic disorders (i.e., negative syndrome of schizophrenia). Despite such diagnostic opaqueness, the guidelines produced by Wilne et al. [105]—which recommend CNS imaging if lethargy or withdrawal persists for 4 weeks or more—have demonstrated promising results in initial UK studies. These successes include a reduction in median symptom interval, time between symptom onset, and

In addition to knowledge about when to suspect a brain tumour, it is important for those practicing in primary and secondary care to be familiar with the most comprehensive and current guidelines for childhood neuropsychiatric presentation of brain tumours, as outlined

such action will actually constitute a small decrease in overall costs [101].

figuration, etc.

186 Brain Tumors - An Update

specific clinical sign [108].

and decrease in school attendance.

below [26].

ultimately, the diagnosis of brain tumours in children [105].

#### **3.1. Red flags**

Recommendations for detecting malignancy in a childhood neuropsychiatric case:

	- internalising behaviour,
	- withdrawal,
	- social problems,
	- somatic complaints,
	- externalising problems,
	- depression and/or anxiety
	- hyperactivity.
	- eating disorder and
	- first episode of psychosis.
	- atypical psychiatric symptoms,
	- unexplained behavioural and/or mood changes,
	- unexplained deteriorating school performance or developmental milestones,
	- personality change,
	- disinhibited or inappropriate behaviour,
	- pathological laughter,
	- emotional lability,

○ especially emotional reactions disproportionate to significance or severity of trigger,

○ abnormal fundoscopy

• Visual assessment including fundoscopy:

• Growth and developmental abnormalities:

○ delayed, arrested or precocious puberty.

• Persistent back pain can be a symptom of cancer.

○ visual fields in school age children and older and

• Diabetes insipidus presenting with polyuria and polydipsia.

In children aged younger than 2 years, any of the following symptoms may suggest a CNS

Neuropsychiatry: Aspects of Childhood Cranial Tumours http://dx.doi.org/10.5772/intechopen.75679 189

○ proptosis.

○ acuity,

○ eye movements, ○ pupil responses,

○ optic disc appearance.

○ growth failure and

tumour and require following:

• Immediate referral:

• Urgent referral:

○ altered behaviour,

Following the diagnosis

○ abnormal eye movements, ○ lack of visual following and ○ poor feeding/failure to thrive.

○ new-onset seizures, ○ bulging fontanelle, ○ extensor attacks and ○ persistent vomiting.

○ abnormal increase in head size,

○ arrest or regression of motor development,

○ Urgency contingent on other factors: squint.

	- new-onset seizures—including focal (with/without loss of awareness) and generalised onset
	- altered consciousness,
	- cranial nerve abnormalities,
	- regression in motor skills,
	- gait abnormalities and/or abnormal motor co-ordination,
	- focal motor weakness and
	- swallowing difficulties.
	- papilledema,
	- reduced visual acuity,
	- reduced visual fields,
	- new-onset nystagmus,
	- new-onset paralytic squint,
	- optic atrophy,

○ especially emotional reactions disproportionate to significance or severity of trigger,

• Psychiatric symptoms (including behavioural) with a headache (i.e., advised to directly enquire about presence of a headache) constitute a red flag. Persistent headache that can occur at any time of the day or night requires a neurological examination. Younger children and those with communication difficulties may not be able to report headache, thus watch for behavioural representation, e.g., pointing at, holding or

• Headache and vomiting that cause early morning waking or occur on awakening are classical signs of raised intracranial pressure and an immediate referral should be made. In cases of persistent vomiting, exclude pregnancy where appropriate. Psychiatric symptoms (including behavioural) with neurological signs warrants a neurological examination. How-

○ new-onset seizures—including focal (with/without loss of awareness) and generalised

ever, a normal neurological examination does not exclude a brain tumour.

○ gait abnormalities and/or abnormal motor co-ordination,

○ flattening or blunting of affect, ○ impairment of working memory,

○ impaired executive function and

• Neurological and motor symptoms:

○ altered consciousness,

○ cranial nerve abnormalities,

○ regression in motor skills,

○ focal motor weakness and

• Visual symptoms and signs including:

○ swallowing difficulties.

○ reduced visual acuity, ○ reduced visual fields, ○ new-onset nystagmus,

○ new-onset paralytic squint,

○ papilledema,

○ optic atrophy,

○ poor attention.

188 Brain Tumors - An Update

squeezing head.

onset

○ agrammatism and dysprosody of speech,

	- acuity,
	- eye movements,
	- pupil responses,
	- visual fields in school age children and older and
	- optic disc appearance.
	- growth failure and
	- delayed, arrested or precocious puberty.

In children aged younger than 2 years, any of the following symptoms may suggest a CNS tumour and require following:

	- new-onset seizures,
	- bulging fontanelle,
	- extensor attacks and
	- persistent vomiting.
	- abnormal increase in head size,
	- arrest or regression of motor development,
	- altered behaviour,
	- abnormal eye movements,
	- lack of visual following and
	- poor feeding/failure to thrive.
	- Urgency contingent on other factors: squint.

Following the diagnosis

	- Externalising problems are less common than internalising problems but, if missed, will have significant implications for educational abilities.

profiling will enrich the clinical pre-operative prognostication and determine treatment goals

Neuropsychiatry: Aspects of Childhood Cranial Tumours http://dx.doi.org/10.5772/intechopen.75679 191

[1] Ostrom QT, Gittleman H, Fulop J, Liu M, Blanda R, Kromer C, et al. CBTRUS statistical report: Primary brain and central nervous system tumors diagnosed in the United States in 2008-2012. Neuro-Oncology. Oxford University Press. 2015;**17**(Suppl 4):iv1-iv62. DOI:

[2] Siegel RL, Miller KD, Jemal A. Cancer statistics, 2017. CA: a Cancer Journal for Clinicians.

[3] Louis DN, Perry A, Reifenberger G, von Deimling A, Figarella-Branger D, Cavenee WK, et al. The 2016 World Health Organization classification of Tumors of the central nervous system: A summary. Acta Neuropathologica. Springer Berlin Heidelberg.

[4] Ramkissoon SH, Bandopadhayay P, Hwang J, Ramkissoon LA, Greenwald NF, Schumacher SE, et al. Clinical targeted exome-based sequencing in combination with genome-wide copy number profiling: Precision medicine analysis of 203 pediatric brain tumors. Neuro-Oncology. Oxford University Press. 2017;**19**(7):now294. DOI: 10.1093/

[5] Komori T. The 2016 WHO classification of tumours of the central nervous system: The major points of revision. Neurologia Medico-Chirurgica (Tokyo). 2017;**57**(7):301-311. DOI:

[6] Reifenberger J, Reifenberger G, Liu L, James CD, Wechsler W, Collins VP.Molecular genetic analysis of oligodendroglial tumors shows preferential allelic deletions on 19q and 1p. The American Journal of Pathology. American Society for Investigative Pathology. 1994;

[7] Hartmann C, Meyer J, Balss J, Capper D, Mueller W, Christians A, et al. Type and frequency of IDH1 and IDH2 mutations are related to astrocytic and oligodendroglial differentiation and age: A study of 1,010 diffuse gliomas. Acta Neuropathologica. 2009;

post-operatively.

**Author details**

**References**

Alexander Hegarty1,2 and Yulia Furlong1,2\*

10.1093/neuonc/nov189

neuonc/now294

**145**(5):1175-1190

10.2176/nmc.ra.2017-0010

1 Perth Children's Hospital, Perth, WA, Australia

2017;**67**(1):7-30. DOI: 10.3322/caac.21387

2016;**131**(6):803-820. DOI: 10.1007/s00401-016-1545-1

**118**(4):469-474. DOI: 10.1007/s00401-009-0561-9

2 University of Western Australia, Crawley, WA, Australia

\*Address all correspondence to<EMAIL_ADDRESS>
• In those for whom the symptoms represent an adjustment reaction, there is an improved outcome if a truthful, complete and consistent approach to communication is taken.

When considering posterior fossa tumour resection

• The complication of cerebellar mutism is rarely mentioned to parents during the consent for surgery of a posterior fossa tumour. It is a common syndrome and, reassuringly, to the authors' knowledge there have been no reported cases of a child with cerebellar mutism not returning to some functional speech.


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

**Dominika Mravˇcáková 1 , Michaela Komáromyová 2 , Michal Babják 2 , Michaela Urda Dolinská 2 , Alžbeta Königová 2 , Daniel Petriˇc 1 , Klaudia Cobanov ˇ á 1 , Sylwester Slusarczyk ´ 3 , Adam Cieslak 4 , Marián Várady 2,\* and Zora Váradyová 1,\***

Received: 8 December 2019; Accepted: 26 January 2020; Published: 29 January 2020

**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 [8–10]. 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* [15–17] 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 forin 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 [16–18].

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.

(+): 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.

*Animals* **2020**, *10*, 219

**Table 2.** *Cont.*

*Animals* **2020**, *10*, 219

**Table 2.** *Cont.*

*Animals* **2020**, *10*, 219

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)* respectively, against *H. contortus* in the egg hatch test (EHT) are shown in Figure 1a,b. Both aqueous

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. 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.

*Animals* **2020**, *10*, 219 9 of 15

The dose-response relationships of aqueous extracts of *A. absinthium* or *M. sylvestris*,

**Figure 1.** (**a,b**) Dose-response relationship of plant aqueous extracts against *Haemonchus contortus* in **Figure 1.** (**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.

#### the egg hatch test (EHT) after 24 h of incubation at 26 °C. *3.3. Parasitological Status*

*Animals* **2020**, *10*, x; doi: www.mdpi.com/journal/animals 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

*3.3. Parasitological Status*

*3.3. Parasitological Status*

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). 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). 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).

from D36 were statistically compared and used to determine the reduction in egg output for ART,

from infection (*p* < 0.05), and for all groups, EPGs increased until D50 or D57, respectively. The EPGs

*Animals* **2020**, *10*, 219 10 of 15

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,

*Animals* **2020**, *10*, 219 10 of 15

**Figure 2.** 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 2.** 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. (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.** Abomasal worm counts of *Haemonchus contortus* in the lambs of each treatment at the end of **Figure 3.** 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: **Figure 3.** 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.

#### the experiment (*p* > 0.05). UNS: unsupplemented; ART: *A. absinthium*; MAL: *M. sylvestris*; ARTMAL: ART plus MAL. *3.4. Inflammatory Response*

ART plus MAL. 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).

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 [16–18]. 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 [16–18] 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 [16–18]. 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.

**Author Contributions:** Conceptualization, Z.V., M.V.; Investigation, D.M., Z.V., M.V.; Supervision, M.V.; Formal analysis, D.M., M.K., K.C., M.B., M.U.D., A.K., D.P., S. ˇ S.; 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).

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

**Conflicts of Interest:** The authors declare no conflict of interest.

### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*


**5. Conclusions**

We concluded that, although exist many orientations and recommendations to use and apply the electromyography method, sometimes these components can be a path too complex to understand and to respect with closed eyes. In a considerable perspective of study we were able to show with a model of exercise in high intensity, which was capable to produce a lot of noises and variations on the signal, that different methods of process to achieve the muscular activity do not change the final result if used the complete signal or just the burst parts, or still using all sequence of treatment with filtration, rectification and smoothing in many combinations of analyses. Moreover, should be noted that only filtration was sufficient to improve the quality of EMG signal, making us think in keeping the use at least the filtration in electromyography analyses, still this procedure is used to at least maintain the signal inside the muscle activity range, so, it should not be took out just because no significant differences were founded, we have to consider all the process, as said before, like the devices used and the investigator experience. These outcomes show us that we have remained with a critical knowledge to many things and test the main recommendations to use some techniques. In order to make those results clearer and give us more confidence when use the treatments in EMG analyzes. Some studies creating different noises in computer should be made. This way we can be more secure about the removing of noises, securing that the absence of difference is not because a good pre-acquisition was made, securing not enough noises to be cut.

These results and conclusion takes in consideration only cyclic exercise with the intensity used in the studies. Exercises such as isometric or acyclic have different signal waves and so, could have different results to the same treatments. Also, exercises with lower load could change mainly the results in the Burst + Silence (Second Study) results, once that a task such as 10 km in low intensity, would be realized with less intense movements, creating not only different power signals but also different silence and burst time duration.

Still, a more accuracy statistic method could be used, such as The Smallest Worthwhile Change [23], capable to find minimal and almost invisible differences between different methods, that can contribute with good perspective to sports domain when obscure changes exist among several techniques to data process in EMG analysis and if we use a classical statistic we may not identify with probabilities these modulations.


*3.3. R1234yf–R152a–RE170*

The third mixture has a completely different composition to the previous ones. It was decided to check the working parameters of the mixture, which in addition to fluorinated HFC/HFO refrigerants also contains dimethyl ether, known as RE170. The proportion of dimethyl ether in the mixture is likely to influence the toxicity and flammability class of the new refrigerant. The mixture's flammability class is assumed to be 3, as to be expected from a mixture with a high proportion of RE170. The representative from the HFC group is R152a. This is often used in existing mixtures because of its low GWP and its relatively high normal boiling point, thus prompting its use in medium to high temperature circuits. The last mixture component is the most common and most easily available fluid from the HFO group—R1234yf. As in the R161–R41–R1234ze(E) mixture, all the constituents have a GWP lower than 150; therefore, the GWP of the mixture itself does not exceed this value (Figure 11a). The flaws of this blend is that normal boiling point never reached −30 ◦C, which proves third mixture is not predisposed for use in low-temperature units (Figure 11b). The device's operation under negative pressure is associated with certain dangers and requires the use of a larger compressor. Considering all of the above, in the case of the R1234yf–R152a–RE170 mixture, it was decided to analyze only the high-temperature cycle.

**Figure 11.** Summary of the basic properties for the R1234yf–R152a–RE170 mixture: (**a**) GWP and (**b**) normal boiling point.

When analyzing the third mixture (R1234yf–R152a–RE170) and the temperature glides obtained (Figure 12), the conclusion is that the temperature difference during the phase change does not exceed 1 K for the assumed evaporating pressure level regardless of the composition; thus, it can be considered at least as near-azeotropic. The achieved volumetric cooling capacity and COP play the greatest role in the assessment (Figure 13a,b). The highest COP value determined was 5.67, but it was for pure RE170. Given the importance of the fluid share from the A3 group being as small as possible, this point was not analyzed. It is worth noting that, with a binary and ternary mixture, as many as 54 points out of 63 have a COP > 5.48, which is the reference value obtained by R134a. There are two ternary mixtures that meet all the assumptions of the work and appear to be good potential substitutes for phased-out fluorinated greenhouse gases. The first is the 0.1/0.1/0.8 mixture, which has a COP of 5.65. The second mixture with a very similar COP has a mass fraction of 0.1/0.5/0.4, but is additionally characterized by a lower normal boiling point and temperature glide well below 0.1 K. In this term, it can be considered as azeotropic. The second composition also has a volumetric cooling capacity that is 124 kJ/m3 higher than that of the first mixture.

**Figure 12.** Temperature glide at the evaporation pressure for air-conditioning system (*t*e/*t*c = 0/30 ◦C) with R1234yf–R152a–RE170 mixture as refrigerant.

**Figure 13.** Volumetric cooling capacity (**a**) and (**b**) the COP for air-conditioning system (*t*e/*t*c = 0/30 ◦C) with R1234yf– R152a–RE170 mixture as refrigerant.


**Christine Erbe**

Christine Erbe holds an M.Sc. degree in Physics (University of Dortmund, Germany) and a Ph.D. in Geophysics (University of British Columbia, Canada). She worked as a Research Scientist at Fisheries and Oceans Canada, was Director of JASCO Applied Sciences Australia, and after a brief stint in high-school education, returned to academia as Director of the Centre for Marine Science and Technology at Curtin University (Perth, WA, Australia). Christine's interests are underwater sound (biotic, abiotic, and anthropogenic), sound propagation, signal processing, and noise effects on marine fauna. She's a John Curtin Distinguished Professor, Fellow of the Acoustical Society of America, former Chair of the Animal Bioacoustics Technical Committee of the Acoustical Society of America, and former Chair of the international conference series on The Effects of Noise on Aquatic Life.