The in silico structure-based docking method was used to measure the affinity of the ligand to the receptor by the smina program (version of AutoDock Vina) (http://pubs.acs.org/doi/abs/10.1021/ci300604z). The sklearn library and the Mini-Batch-Kmeans algorithm (Pedregosa et al., 2011) were used for statistical processing. The model of nAChR, ACR16 receptor of A. suum (see uniprot id from the Supplementary Figure S1) (The UniProt Consortium, 2023), was obtained by the Phyre2 service. Docking is limited to the subunit of the receptor with two domains, alpha and beta (Supplementary Figure S1) (Unwin, 2005; https://www.rcsb.org/structure/2BG9). Carveol XLogP3 2.1 (cyclohexene 2.9), carvacrol XLogP3 3.1 (benzene 2.1), and geraniol XLogP3 2.9 were chosen as ligands.

C. elegans, N2 wild-type is obtained from the Caenorhabditis Genetics Center (Charvet et al., 2018). Nematode Growth Medium was used for the cultivation of C. elegans (NGM, US Biological, Life Sciences, United States). After culturing on the NGM plates for 6 days, the worms were collected by cutting the medium into 3 × 3 mm squares and transferred to glass tubes with 2 mL of 0.1M NaCl. The tubes were placed in a shaker until complete dissolution of the substrate and release of the worms. The tubes were then centrifuged for 4 min at 2,500 r/min, and 400 μL of supernatant were transferred to a second test tube. This was followed by another minute of centrifugation, at 2000 r/min. After that, 20 μL of suspension of nematodes were inoculated on the Petri dish (diameter 3 cm) with 2.5 mL of NGM substrate and increasing concentrations of carveol (1, 3, 10, 30, 100, 300μM, 1 and 3 mM), The titer of adult worms was 21–35/20 μL and each concentration was tested on three Petri dishes. The three Petri dishes without the added test substances were untreated controls.

The plates inoculated with C. elegans were placed in a thermostat (Memmert IN30, Germany) at 20°C for 24 and 48 h. After incubation, the plates were observed under an inverted microscope (Motic AE 31, PRC) and the movement and pharyngeal pumping of C. elegans were recorded with a camera (Motic 5 MP, NRK) on the hard disk of a PC, for later analysis. The survival rate of the adult C. elegans was determined in the medium with carveol as well as in the untreated control medium. The lethality was determined by the cessation of movement and pharyngeal pumping. C. elegans was considered dead when it did not move and did not respond to repeated touching with a probe. Mortality was calculated for each treatment after 24 and 48 h and expressed in percentages.

Adult female A. suum were collected weekly from the slaughterhouse Ambar (Surčin, Serbia). After collection, the worms were placed and maintained in Locke’s solution of the following content (mM): NaCl 155, KCl 5, CaCl₂ 2, NaHCO₃ 1.5 and glucose 5. Temperature was maintained at 32°C–34°C using a water bath and Locke’s solution was changed twice a day. Each batch of worms was used for experiments within 4 days of collection.

Ascaris muscle flaps for contraction studies were prepared as described in Trailović et al. (2015). The preparations were allowed to equilibrate for 15 min under an initial tension of 0.5 g. Contractions were monitored after increasing concentrations of acetylcholine (1, 3, 10, 30 and 100 μM) and then in the presence of carveol or carveol and atropine. The maximum contraction was observed prior to washing and subsequent application of ACh, ACh and carveol, ACh, carvacrol and atropine. The interval between the application of increasing doses of ACh was 1 min and 2 min when the preparation was incubated with carveol or carveol with atropine. The responses for each concentration were expressed in grams (g), produced by each individual flap preparation. Contractions were monitored and recorded in real time on a PC computer, using a BioSmart interface, and eLAB 44 software (ElUnit, Belgrade). Sigmoidal concentration-response curves for ACh effects in the absence or presence of carveol/atropine were described by the Hill equation.

The functional expression of A. suum ACR-16 nAChR in Xenopus laevis oocytes was carried out as described previously (Abongwa et al., 2016). Briefly, defolliculated Xenopus laevis oocytes (Ecocyte Bioscience) were micro-injected with 36 nL of Asu-ACR-16 cRNA at 50 ng/μL using the Nanoject II microinjector (Drummond). Two micro-electrode voltage-clamp experiments were performed 3 days after micro-injections as previously described (Trailović et al., 2021). Data were collected and analyzed using the pCLAMP 10.4 package (Molecular Devices).

White male Wistar rats, weighing 150–200 g, were housed under standard conditions for laboratory animals in groups of four in home cages (42.5 × 27 × 19 cm) under standard conditions: temperature of 21°C ± 1°C, relative humidity of 55%–60% and 12/12 h light/dark cycle. Food and water were provided ad libitum, except during experimental procedures. All procedures in the study conformed to EEC Directive 86/609 and were approved by the Ethics Committee of the Faculty of Veterinary Medicine, University of Belgrade.

Male Wistar rats were anesthetized with thiopentone and decapitated. Thiopentone sodium (PANPHARMA, France) was administered using an intravenous cannula (OptivaTM 2, Johnson & Johnson, Arlington, TX, United States) via the lateral tail vein at a dose of 35 mg/kg body weight. Diaphragm strips for contraction studies were prepared as described in Trailović et al. (2015). Contractions were monitored after Electrical Field Stimulation (EFS). The pair of platinum electrodes were placed parallel to the muscles and EFS was performed by application of tetanic pulses (10 μs widths, 50 Hz frequency, 30 V voltage, and 2 s duration) in trains of five pulses every 30 s, with a resting period of 3 min between trains. EFS was generated by the BioSmart digital stimulator with software control (El Unit, Belgrade). Isometric muscle force production was determined after 30 min of equilibration time (control). The amplified signals were recorded online by an eLAB 44 software (ElUnit, Belgrade). To confirm that the electrical field stimulation technique induced muscle contraction only via the neuromuscular end-plate, the nicotinic antagonist mecamylamine (100 μM) was used (Papke et al., 2001). The effect of carveol (10, 30, 100 and 300 μM) on contractions of the isolated diaphragm caused by EFS was tested. Tissues were washed each time after recording effects of carveol or mecamylamine.

Ileum segments were prepared for testing contractions as described in (Trailović et al., 2017). After 30 min of the equilibration period, until a stable baseline was attained, increasing concentrations of ACh (1, 3, 10, 30 and 100 μM) were administrated and the maximum contraction was observed before washing and subsequent application of the next concentration of ACh. Afterwards the contractile effect of the same concentrations of ACh was tested in the presence of 100, 300 and 1,000 μM of carveol or 3, 10, 30 and 100 nM of atropine.

In a separate series of tests, ileum preparations were subjected to EFS by packages containing 5 stimulations every 60 s (EFS: 50 Hz, for 2 s, 1.0 ms pulse duration, 50 V intensity), with resting periods of 5 min between each package. EFS was generated by the digital stimulator BIOSMART 150 with software control (El Unit, Belgrade), while EFS was delivered by a pair of platinum electrodes, placed parallelly to the preparation. After two series of stable contractions, the response was observed in the presence of carveol. The amplified signals have been recorded online by SMARTPLUS 150 software (El Unit, Belgrade).

Acetylcholine, atropine, mecamylamine and carveol were obtained from Sigma-Aldrich Co. (St Louis, MO, United States). Acetylcholine, mecamylamine, and atropine were dissolved in the APF-Ringer and Tyrode solution. Carveol, was dissolved in ethanol, with a final concentration of ethanol in the APF-Ringer and Tyrode Solution of 0.1 %v/v.

The results of muscle contraction assay are expressed as means ± S.E. in grams (g) of contractions. Sigmoid concentration dose-responses were described by the equation as follows: % response = 1/1 + [EC₅₀/Xa]nH, where EC₅₀ is the concentration of the agonist (Xa) producing 50% of the maximum response and nH is the Hill coefficient (slope). Prism 6.0 (GraphPad Software, San Diego, CA.) was used to estimate the constants EC₅₀ and nH, by non-linear regression for each preparation. We determined the mean contraction responses to each concentration of ACh (control dose-response: CR[ACh]) and compared with responses in presence of carveol. Other data was analyzed by ANOVA using Prism 6.0, and the differences were considered significant when the p-value is <0.05.

In molecular docking analyses we used the model of homomeric A. suum nAChR, ACR16 (uniprot: A0A0K0QSL7_ASCSU). The model contains an alpha and a beta domain, namely: alpha domain (alpha subunit, containing 7 alpha helices, alpha 7 domain, transmembrane) and beta domain (beta subunit, extracellular domain containing loops B and C, outer vestibule) as described in PDB entry 2BG9, nicotinic aceylcholine receptor T. marmarota at 4Å resolution (Unwin, 2005). The tested ligands were carveol, carvacrol and geraniol, and the common property for all ligands is diffusion through the membrane. Therefore, the affinity at several places in the alpha domain for each of the ligands must not be ignored. However, carveol and carvacrol probably participate in the activation mechanism in the beta domain. Only the organization of interactions of geraniol in the alpha domain shows distinctive binding patterns. Furthermore, the differences are indicated by the lower affinity of geraniol for the receptor. On the other hand, unique to carvacrol is the possibility of pi-stacking and cation-pi interactions due to benzene in the structure. This may explain the different affinities compared to carveol in the beta domain. Carvacrol shows affinity for an allosteric binding site in the beta domain (a possible allosteric site composed of two sub-sites located close to each other). Geraniol potentially binds to the receptor at two sites in the alpha domain with lower affinity than carveol and carvacrol. While, carveol has an affinity for two binding sites that are distant, i.e. one in the beta and one in the alpha domain. The geometry of the binding sites is different from those of carvacrol and geraniol. In the beta domain, carveol shows the highest affinity for the orthosteric binding site (Figure 1).

To verify the results of docking testing of the cholinergic properties of carveol we examined its effect on the free-living nematode C. elegans. The Median Lethal Concentration (LC₅₀) of carveol after 24 h of exposure was 490.10 ± 1.40 μM, and it did not change significantly after 48 h of exposure (430.50 ± 1.35 μM).

By analyzing real-time motility recordings, we noticed that exposure to carveol resulted in spastic paralysis. Also, when carveol was in the medium, pharyngeal pumping stopped first (Supplementary Figure S2), while the worms still responded by moving to the touch stimulus.

We checked the results obtained on the free-living nematode C. elegans on the contraction model of the neuromuscular preparation of the parasitic nematode A. suum (Figure 2A). Initially, we tested the effects of 1, 3 and 10 μM of carveol on ACh-induced contractions. Carveol concentrations of 1 and 3 μM had no or very weak effect on the amplitude of contractions and the EC₅₀ value of acetylcholine. Therefore, we continued testing with 10 μM of carveol. The EC₅₀ value of ACh for control contractions was 15.97 ± 1.56 μM, while in the presence of carveol 10μM, the EC₅₀ was 6.63 ± 1.82μM, which is a significant decrease (p = 0.0008). After washing, the EC₅₀ of ACh was further significantly reduced to 6.14 ± 1.80 μM (p = 0.0004) (Figure 2B). At the same time, carveol increased the maximal contractile response (E_max) to ACh from the control 1.37 ± 013 g to 1.52 ± 0.12 g. The E_max continued to increase after washing and reached 1.72 ± 0.14 g (Figures 2A, B).

It was further interesting to check whether this potentiating effect of carveol on ACh-evoked contractions is sensitive to atropine (Figure 3A). The EC₅₀ value for ACh in the control series of contractions was 8.52 ± 2.34 μM, while in the presence of carveol (10 μM) it was reduced by more than 40% (but not significant, p = 0.791) to 4.99 ± 2.33 μM. Atropine 1 μM insignificantly increased the EC₅₀ of ACh to 5.76 ± 2.63 μM (p = 0.887), but after removal of carveol and atropine, the EC₅₀ value was slightly higher than the control value, reaching 10.52 ± 3.31 μM (Figure 3B). As in the previous study, carveol increased the E_max value of ACh-evoked contractions from the control 1.01 ± 0.13g to 1.22 ± 0.13 g (for 20%), while in the presence of atropine E_max decreased to 1.11 ± 0.13 g. After washing, the E_max of contractions was 1.02 + 0.21 g, which corresponds to the control value. However, these differences did not reach the level of significance. Apparently, the effect of atropine is reversible, as we observed in previous studies with the neuropeptide AF2 (Trailovic and all, 2005) and the muscarinic agonist 5-methylfurmethiodide (Trailovic and all, 2008). Unfortunately, there is still not much data on the more detailed characteristics of the muscarinic (M) receptor in the neuromuscular system of parasitic nematodes, which represents a special challenge for further research.

Following the results obtained on the A. suum neuromuscular preparations, we hypothesized that the effect of carveol 10 µM could be mediated through the nicotine-sensitive acetylcholine receptors (nAChR) of A. suum. In order to assess carveol at the receptor level, we expressed the A. suum nAChR in Xenopus laevis oocytes and carried out electrophysiological studies as recently achieved for carvacrol (Trailovic et al., 2021). Indeed, the A. suum ACR-16 subunit has been shown to give rise to a robust functional homomeric nAChR in the Xenopus oocyte heterologous expression system (Abongwa et al., 2016). To check for the functional expression of the nAChRs in Xenopus oocytes micro-injected with Asu-ACR-16 cRNAs, acetylcholine 100 µM was first applied, leading to the recording of large currents in the µA range (Figure 4A). The application of ACh 100 µM was repeated a second time, giving a similar current amplitude. When carveol 10 µM (same concentration used in neuromuscular contraction experiments) was perfused in the recording chamber, we observed no direct agonistic action on the nAChR (Figure 4A). To investigate a possible antagonistic effect, the nAChRs were challenged by increasing concentrations of ACh ranging from 1 to 100 μM, without carveol or in the continued presence of carveol 10 µM. With current amplitudes normalized to the maximal response to 100 μM, we found that the ACh EC₅₀ value of A. suum nAChR was 4.9 ± 1.2 µM (n = 11) in the absence of carveol (Figure 4B). In the presence of carveol 10 μM, the ACh concentration–response curve was characterized by an EC₅₀ of 7.8 ± 1.1 µM (n = 4) which was not significantly different from the ACh EC₅₀ value obtained without carveol (p = 0.077). Interestingly, the maximal response amplitude elicited by ACh 100 µM was increased by 7% in the presence of carveol 10 µM (p = 0.142) and continued to increase by 8.75% after removal of carveol (wash). Thus, carveol 10 µM non-significantly increased the ACh efficacy for the homomeric ACR16 A. suum nAChR, what could be one of the possible mechanisms accounting for the potentiating effect of ACh-induced contractions.

Carveol showed qualitatively different pharmacological effects on the neuromuscular system of nematodes compared to carvacrol in our earlier studies. In order to further analyze pharmacological characteristics of carveol, we tested its effects on contractions of the isolated rat ileum (Figure 5A). Increasing concentrations of ACh caused dose-dependent contractions of the ileum, where the EC₅₀ was 6.51 ± 2.75 μM. Carveol applied in concentrations of 100, 300 and 1000 μM did not significantly affect the EC₅₀ value of ACh, 4.31 ± 1.94, 6.51 ± 2.75 and 5.49 ± 3.1 μM (Figure 5B). However, incubation of ileum preparations with carveol caused a decrease of maximal contractile response to ACh. The control E_max was 1.57 ± 0.05 g, after incubation with 100 μM carveol it decreased to 1.30 ± 0.07 g, while after 300 and 1000 μM of carveol the E_max significantly further decreased to 1.05 ± 0.12 g (p = 0.001) and 0.91 ± 0.10 g (p < 0.0001), respectively. This inhibitory effect on ACh-induced contractions was reversible and after washing E_max increased to the control level, 1.31 ± 0.10 g (Figures 5A, B).

In addition to the effect of carveol on contractions of the isolated ileum induced by ACh, we also examined the effect of this monoterpenoid on the contractions of the ileum induced by EFS. The mean value of control contractions was 2.70 ± 0.27 g, while the incubation of the preparation with carveol 100 μM resulted in a significant inhibition of contractions, with a mean value of 1.60 ± 0.25 g (ANOVA p = 0.0015). Furthermore, this inhibitory effect of carveol was reversible and after washing it exceeded the control value, which was 2.80 ± 0.26 g (ANOVA p = 0.009) (Figures 6A, B).

In the next series of experiments, we tested the effect of increasing concentrations of atropine on ACh-induced ileal contractions, in order to compare the result with the previously observed inhibitory effect of carveol. The control EC₅₀ of ACh was 3.75 ± 2.50μM, while in the presence of atropine 3, 10, 30 and 100 nM this value significantly increased to 7.99 ± 1.62 μM, 17.61 ± 1.65 μM (p = 0.0001), 32.85 ± 1.77 μM (p = 0.0001) and 28.73 ± 1.69 μM (p = 0.0001). On the other hand, incubation of preparations with atropine 3, 10 and 30 nM reduced the E_max of ACh from the control 1.68 ± 0.09 g to 1.41 ± 0.09 g, 1.26 ± 0.12 g, 1.20 ± 0.18 g, but only the highest tested concentration (100 nM) caused a significant reduction, it was 0.93 ± 0.13 g (p = 0.0004) (Figures 7A, B). The effect of atropine was reversible and after washing the EC₅₀ and E_max of ACh returned to the control level (6.71 ± 1.99nM and 1.60 ± 0.11 g).

Finally, the effect of carveol on a skeletal muscle model, i.e. neuromuscular preparation of rat diaphragm was tested. Contractions were induced by EFS, and preparations were incubated in increasing concentrations of carveol 1, 3, 10, 30 and 100 μM (Figure 8A). Carveol caused a slight increase in amplitude of contractions from control 1.60 ± 0.14 g to 1.64 ± 0.17 g, 1.66 ± 0.17 g and 1.65 ± 0.17 g. Carveol concentration of 30 μM caused a significant increase of the contraction’s amplitude to 1.80 ± 0.17 g (p = 0.0063). On the other hand, 100 μM of carveol led to a reduction of the amplitude back to the control level, 1.62 ± 0.21 g (Figures 8A,B).

In the next series of experiments, we compared the effect of 30 μM of carveol and 100 μM of mecamylamine, a non-competitive nicotinic receptor antagonist. Carveol 30 μM significantly increased contractions of the isolated diaphragm induced by EFS, from control 1.79 ± 0.34g to 1.93 ± 0.34 g (p = 0.045). On the other hand, compared to the control, mecamylamine significantly reduced the amplitude of contractions to 1.29 ± 0.23 g (p = 0.046). When the diaphragm preparations were incubated simultaneously with 30 μM of carveol and 100 μM of mecamylamine, the inhibition of contractions was even more pronounced and the mean amplitude was 1.01 ± 0.16 g (p = 0.049) (Figures 9A, B).

We emphasize that the ratio (R) between the tension at the end (B) and the maximum tension at the beginning of individual tetanic contractions (A) was also analyzed (R = A/B). Increasing concentrations of carveol (1, 3, 10 and 30 μM), in addition to the potentiation of contractions, also led to an increase in the R value. However, carveol 100 μM, in addition to reducing the amplitude, also reduced the R value (Figure 9A). Furthermore, in the next series of contractions, carveol 30 μM increased the amplitude of contractions as well as the R value, while mecamylamine led to inhibition of the contraction amplitude and caused a tetanic fade. When we used carveol at the same time with mecamylamine, there was no tetanic fade, but the inhibition of the amplitude of contractions was more pronounced. This effect was reversible and contractions slightly increased after washing (Figure 9A).