In vitro spasmolytic effects of eugenol on bovine abomasal smooth muscle: a focus on calcium-mediated mechanisms

Document Type : Original Article

Authors

Department of Internal Medicine and Clinical Pathology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran

Abstract
Abomasal motility disorders, such as displacement and volvulus, represent significant clinical and economic challenges in dairy cattle. The use of synthetic prokinetics is limited by residue concerns. This study investigated the in vitro effects of eugenol on bovine abomasal smooth muscle contractility. Muscle strips from healthy cattle were mounted in organ baths containing oxygenated Krebs solution. Basal tone and contractions induced by carbachol (1.00 and 4.00 µM), barium chloride (30.00 mM), and potassium chloride (30.00 and 80.00 mM) were recorded. Cumulative eugenol concentrations (1.00 - 600 mg L-1) caused a significant, concentration-dependent inhibition of both basal tone and spasmogen-induced contractions (Friedman test followed by Dunnett's test). The inhibition was particularly abrupt in high- potassium chloride -induced contractions. The effects were fully reversible upon washout. These results demonstrated that eugenol acted as a non-selective spasmolytic agent on bovine abomasal smooth muscle, likely via inhibition of calcium influx. This supported its potential as a natural therapeutic candidate for managing gastrointestinal hypermotility in ruminants, warranting further in vivo studies.

Keywords

Subjects

1.     Ivanova S, Sukhikh S, Popov A, et al. Medicinal plants: a source of phytobiotics for the feed additives. J Agric Food Res 2024; 16: 101172. doi: 10.1016/j.jafr. 2024.101172.
2.     Alem WT. Effect of herbal extracts in animal nutrition as feed additives. Heliyon 2024; 10(3): e24973. doi: 10.1016/j.heliyon.2024.e24973.
3.     Wang J, Deng L, Chen M, et al. Phytogenic feed additives as natural antibiotic alternatives in animal health and production: a review of the literature of the last decade. Anim Nutr 2024; 17: 244-264.
4.     Mountzouris KC, Brouklogiannis I. Phytogenics as natural gut health management tools for sustainable poultry production. Livest Sci 2024; 286: 105525. doi: 10.1016/j.livsci.2024.105525.
5.     Kuralkar P, Kuralkar SV. Role of herbal products in animal production - an updated review. J Ethnopharmacol 2021; 278: 114246. doi: 10.1016/ j.jep.2021.114246.
6.     Silva MV, de Lima AdCA, Silva MG, et al. Clove essential oil and eugenol: a review of their significance and uses. Food Biosci 2024; 62: 105112. doi: 10.1016/ j.fbio.2024.105112.
7.     Kathiravan S, Immadi S, Kurbet PS, et al. In vitro evaluation of acaricidal activity of eugenol and benzaldehyde against Rhipicephalus annulatus. Vet Parasitol 2024; 331: 110285. doi: 10.1016/j.vetpar. 2024.110285.
8.     Chen M, Nie YK, Liu XY, et al. Antiviral properties of the natural product eugenol: a review. Fitoterapia 2025; 185: 106674. doi: 10.1016/j.fitote.2025.106674.
9.     Martín-Martín C, Ruiz-Rico M, Barat JM, et al. Decoding the antiviral potential of eugenol, thymol and vanillin against human cytomegalovirus infection. J Gen Virol 2026; 107(3): 002248. doi: 10.1099/jgv.0.002248.
10. Damasceno ROS, Pinheiro JLS, Rodrigues LHM, et al. Anti-inflammatory and antioxidant activities of augenol: an update. Pharmaceuticals (Basel) 2024; 17 (11): 1505. doi: 10.3390/ph17111505.
11. Noman AM, Sultan MT, Mazhar A, et al. Eugenol: an insight into the anticancer perspective and pharmacological aspects. Food Sci Nutr 2025; 13(8): e70727. doi: 10.1002/fsn3.70727.
12. Bernacchi A, Valerii MC, Spigarelli R, et al. Investigating the molecular mechanisms of the anticancer effects of eugenol and cinnamaldehyde against colorectal cancer (CRC) cells in vitro. Int J Mol Sci 2026; 27(2): 649. doi: 10.3390/ijms27020649.
13. Khwaza V, Aderibigbe BA. Antibacterial activity of selected essential oil components and their derivatives: A review. Antibiotics (Basel) 2025; 14(1): 68. doi: 10.3390/antibiotics14010068.
14. Friedman M, Henika PR, Mandrell RE. Bactericidal activities of plant essential oils and some of their isolated constituents against Campylobacter jejuni, Escherichia coli, Listeria monocytogenes, and Salmonella enterica. J Food Prot 2002; 65(10): 1545-1560.
15. Yu B, Li C, Gu L, et al. Eugenol protects against Aspergillus fumigatus keratitis by inhibiting inflammatory response and reducing fungal load. Eur J Pharmacol 2022; 924: 174955. doi: 10.1016/ j.ejphar.2022.174955.
16. Shahina Z, Dahms TES. A comparative review of eugenol and citral anticandidal mechanisms: partners in crimes against fungi. Molecules 2024; 29(23): 5536. doi: 10.3390/molecules29235536.
17. Kakkar A, Singh H, Anand A, et al. Neuroprotective effects of eugenol in Alzheimer's disease: mitigating oxidative stress, inflammation and amyloid plaques. Curr Pharm Des 2026; 32(7): 493-504.
18. Baraka SM, Hussien YA, Ahmed-Farid OA, et al. Neuroprotective potential of eugenol against acrylamide-induced brain toxicity by regulating Nrf2/NQO1/HO-1 and NLRP3/NF-κB/IL-1β signaling cascades. Sci Rep 2026; 16: 3265. doi: 10.1038/ s41598-025-34650-8.
19. Devi S, Chauhan S, Mannan A, et al. Targeting cardiovascular risk factors with eugenol: an anti-inflammatory perspective. Inflammopharmacology 2024; 32(1): 307-317.
20. Khaki A, Youssefi MR, Nasrabadi NT. Comparative efficacy of eugenol, eugenol nanoemulsion, and metronidazole against Trichomonas gallinae: an experimental study. Open Vet J 2024; 14(10): 2618-2627.
21. Geng T, Yang H, Liu J, et al. Transcriptomic and untargeted metabolomic studies on the anticoccidial activity of eugenol in broilers. Poult Sci 2025; 104(7): 105205. doi: 10.1016/j.psj.2025.105205.
22. Liu XY, Nie YK, Liu Y, et al. Cardiovascular protective properties of the natural product eugenol. Eur J Pharmacol 2025; 1003: 177929. doi: 10.1016/ j.ejphar.2025.177929.
23. Peixoto-Neves D, Wang Q, Leal-Cardoso JH, et al. Eugenol dilates mesenteric arteries and reduces systemic BP by activating endothelial cell TRPV4 channels. Br J Pharmacol 2015; 172(14): 3484-3494.
24. Silva AFB, Lima LF, Morais ANP, et al. Oocyte in vitro maturation with eugenol improves the medium antioxidant capacity and total cell number per blastocyst. Theriogenology 2022; 192: 109-115.
25. Benchaar C, Hassanat F, Petit HV. Dose-response to eugenol supplementation to dairy cow diets: methane production, N excretion, ruminal fermentation, nutrient digestibility, milk production, and milk fatty acid profile. Anim Feed Sci Technol 2015; 209: 51-59.
26. Wall EH, Doane PH, Donkin SS, et al. The effects of supplementation with a blend of cinnamaldehyde and eugenol on feed intake and milk production of dairy cows. J Dairy Sci 2014; 97(9): 5709-5717.
27. Yang WZ, Benchaar C, Ametaj BN, et al. Dose response to eugenol supplementation in growing beef cattle: ruminal fermentation and intestinal digestion. Anim Feed Sci Technol 2010; 158(1-2): 57-64.
28. Ahmad AA, Maurice MN, Monib MEM, et al. Eugenol essential oil and nanoemulsion as antihydatic agents with antifibrotic and immunomodulatory effects in cystic Echinococcosis. Trop Med Infect Dis 2023; 8(5): 253. doi: 10.3390/tropicalmed8050253.
29. Jandhyam H, Mohanty BP, Parija SC. The vasodilator effect of eugenol on uterine artery - potential therapeutic applications in pregnancy-associated hypertension. J Recept Signal Transduct Res 2024; 44(2): 54-62.
30. Trailović MS, Robertson PA, Nedeljković-Trailović J. Inhibitory effect of eugenol on rat ileal motility in vitro. Acta Veterinaria (Beograd) 2009; 59(2-3): 123-131.
31. Araújo MC, Pereira-Gonçalves Á, Cardeal Dos Santos AN, et al. Effect of eugenol on detrusor muscle: potential for overactive bladder treatment. Int Neurourol J 2024; 28(4): 253-263.
32. Lima FC, Peixoto-Neves D, Gomes MD, et al. Antispasmodic effects of eugenol on rat airway smooth muscle. Fundam Clin Pharmacol 2011; 25(6): 690-699.
33. de Alencar Silva A, de Morais LP, de Sena Bastos CM, et al. Vasorelaxant effect of phenylpropanoids: methyl eugenol and eugenol in human umbilical cord vein. Biomed Pharmacother 2024; 178: 117227. doi: 10.1016/j.biopha.2024.117227.
34. Seo H, Li HY, Perez-Reyes E, et al. Effects of eugenol on T-type Ca2+ channel isoforms. J Pharmacol Exp Ther 2013; 347(2): 310-317.
35. Sensch O, Vierling W, Brandt W, et al. Effects of inhibition of calcium and potassium currents in guinea-pig cardiac contraction: comparison of beta-caryophyllene oxide, eugenol, and nifedipine. Br J Pharmacol 2000; 131(6):1089-1096.
36. Mohseni M, Maham M, Dalir-Naghadeh B, et al. Does Achillea millefolium extracts possess prokinetic effects on the bovine abomasum thourgh M3 muscarinic receptors? Vet Res Forum 2017; 8(2): 115-120.
37. Hamilton TC, Weir SW, Weston AH. Comparison of the effects of BRL 34915 and verapamil on electrical and mechanical activity in rat portal vein. Br J Pharmacol 1986; 88(1): 103-111.
38. Sanders KM, Koh SD, Ro S, et al. Regulation of gastrointestinal motility - - insights from smooth muscle biology. Nat Rev Gastroenterol Hepatol 2012; 9(11): 633-645.
39. Bolton TB. Mechanisms of action of transmitters and other substances on smooth muscle. Physiol Rev 1979; 59(3): 606-718.
40. Damiani CE, Moreira CM, Zhang HT, et al. Effects of eugenol, an essential oil, on the mechanical and electrical activities of cardiac muscle. J Cardiovasc Pharmacol 2004; 44(6): 688-695.
41. Leal-Cardoso JH, Lahlou S, Coelho-de-Souza AN, et al. Inhibitory actions of eugenol on rat isolated ileum. Can J Physiol Pharmacol 2002; 80(9): 901-906.
42. Karaki H, Satake N, Shibata S. Mechanism of barium-induced contraction in the vascular smooth muscle of rabbit aorta. Br J Pharmacol 1986; 88(4): 821-826.
43. Ahmadnejad-Asl-Gavgani M, Maham M, Dalair-Naghadeh B. In vitro effects of essential oils of Tanacetum balsamita and carvone on the contractility of bovine ileum smooth muscles. Vet Res Forum 2022; 13(1): 29-37.
44. Ehlert FJ, Griffin MT, Abe DM, et al. The M2 muscarinic receptor mediates contraction through indirect mechanisms in mouse urinary bladder. J Pharmacol Exp Ther 2005; 313(1): 368-378.
45. Damiani CE, Rossoni LV, Vassallo DV. Vasorelaxant effects of eugenol on rat thoracic aorta. Vascul Pharmacol 2003; 40(1): 59-66.
46. Heller MC, Chigerwe M. Diagnosis and treatment of infectious enteritis in neonatal and juvenile ruminants. Vet Clin North Am Food Anim Pract 2018; 34(1): 101-117.
47. Niederberger MD, Hirsbrunner G, Steiner A, et al. In vitro effects of bethanechol on abomasal and duodenal smooth muscle preparations from dairy cows with left displacement of the abomasum and from healthy dairy cows. Vet J 2010; 184(1): 88-94.
48. Cobellis G, Trabalza-Marinucci M, Yu Z. Critical evaluation of essential oils as rumen modifiers in ruminant nutrition: a review. Sci Total Environ 2016; 545-546: 556-568.
49. Marchese A, Barbieri R, Coppo E, et al. Antimicrobial activity of eugenol and essential oils containing eugenol: a mechanistic viewpoint. Crit Rev Microbiol 2017; 43(6): 668-689.
Volume 17, Issue 10
October 2026
Pages 731-737

  • Receive Date 25 May 2026
  • Accept Date 07 June 2026