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Pharmaceuticals (Basel)
2021 May 26;146:. doi: 10.3390/ph14060505.
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Action of Carvacrol on Parascaris sp. and Antagonistic Effect on Nicotinic Acetylcholine Receptors.
Trailovic SM
,
Rajkovic M
,
Marjanovic DS
,
Neveu C
,
Charvet CL
.
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Parascaris sp. is the only ascarid parasitic nematode in equids and one of the most threatening infectious organisms in horses. Only a limited number of compounds are available for treatment of horse helminthiasis, and Parascaris sp. worms have developed resistance to the three major anthelmintic families. In order to overcome the appearance of resistance, there is an urgent need for new therapeutic strategies. The active ingredients of herbal essential oils are potentially effective antiparasitic drugs. Carvacrol is one of the principal chemicals of essential oil from Origanum, Thymus, Coridothymus, Thymbra, Satureja and Lippia herbs. However, the antiparasitic mode of action of carvacrol is poorly understood. Here, the objective of the work was to characterize the activity of carvacrol on Parascaris sp. nicotinic acetylcholine receptor (nAChR) function both in vivo with the use of worm neuromuscular flap preparations and in vitro with two-electrode voltage-clamp electrophysiology on nAChRs expressed in Xenopus oocytes. We developed a neuromuscular contraction assay for Parascaris body flaps and obtained acetylcholine concentration-dependent contraction responses. Strikingly, we observed that 300 µM carvacrol fully and irreversibly abolished Parascaris sp. muscle contractions elicited by acetylcholine. Similarly, carvacrol antagonized acetylcholine-induced currents from both the nicotine-sensitive AChR and the morantel-sensitive AChR subtypes. Thus, we show for the first time that body muscle flap preparation is a tractable approach to investigating the pharmacology of Parascaris sp. neuromuscular system. Our results suggest an intriguing mode of action for carvacrol, being a potent antagonist of muscle nAChRs of Parascaris sp. worms, which may account for its antiparasitic potency.
Figure 1. Contraction of Parascaris sp. muscle strips produced by acetylcholine. (a) Adult female Parascaris sp. collected from horses and used in this study; (b) photograph of a single worm indicating the location of the body muscle flap (1 cm length between the two red arrows), within the anterior part of the worm (3–4 cm caudal to the head), to be dissected for isometric contraction measurements; (c) isometric contractions of Parascaris sp. muscle flap produced by increasing concentrations of acetylcholine (ACh) from 1 to 100 µM (short bars); (d) concentration–response plot for ACh fitted with non-linear regression, with mean contraction in g ± SE (n = 5).
Figure 2. Effect of carvacrol on contractions of Parascaris sp. muscle strips produced by acetylcholine. Isometric contractions of Parascaris sp. muscle flap produced by increasing concentrations of acetylcholine (ACh) from 1 to 100 µM (left panel, short bars) and inhibition of contractions mediated by 300 µM carvacrol (middle panel, full line). Absence of ACh-induced response recovery after washing the preparation (right panel).
Figure 3. Concentration–response relationships of ACh on the Parascaris sp. ACR-26/27 M-AChR expressed in Xenopus laevis oocytes in absence of carvacrol (a) or in presence of carvacrol (b). Representative current traces for single oocytes. The concentrations of ACh and carvacrol (µM) are indicated above each trace. Bars indicate drug applications: ACh was applied for 10 s. (c) Concentration–response curves. All responses are normalized to 100 µM ACh. Results are shown as the mean ± SE (n = 5–6).
Figure 4. Concentration–inhibition relationship of carvacrol on the Parascaris sp. ACR-26/27 M-AChR expressed in Xenopus oocytes. Representative current traces for single oocytes challenged with acetylcholine (ACh) in the presence of increasing concentration of carvacrol from 30 to 300 (a) and 1000 µM (b). The concentrations of ACh and carvacrol (µM) are indicated above each trace. ACh was applied for 10 s (black bars), and carvacrol was applied for 20 s (red bars). (c) Concentration–inhibition response curve of carvacrol. All responses are normalized to 100 µM ACh. Results are shown as the mean ± SE (n = 7).
Figure 5. Carvacrol effect on the ACh concentration–response relationships for the Parascaris sp. ACR-16 N-AChR expressed in Xenopus oocytes in absence of carvacrol (a) or in presence of carvacrol (b). Representative current traces for single oocytes. The concentrations of ACh and carvacrol (µM) are indicated above each trace. Bars indicate drug applications: ACh was applied for 10 s, and carvacrol was applied for 11 min (red bar). (c) Concentration–response curves. All responses are normalized to 100 µM ACh. Results are shown as the mean ± SE (n = 6–10).
Figure 6. Concentration–inhibition relationship of carvacrol on the Parascaris sp. ACR-16 N-AChR expressed in Xenopus oocytes. (a) Representative current traces for single oocytes challenged with acetylcholine (ACh) in the presence of increasing concentration of carvacrol from 10 to 1000 µM. The concentrations of ACh and carvacrol (µM) are indicated above each trace. ACh was applied for 30 s intervals (black bars), and carvacrol was applied for 10 s (red bars). (b) Concentration–inhibition response curve of carvacrol. All responses are normalized to 100 µM ACh. Results are shown as the mean ± SE (n = 6).
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