Tracheal relaxation through calcium channel blockade of Achillea millefolium hexanic extract and its main bioactive compounds

Abstract Ethnopharmacological importance Achillea millefolium L. (Asteraceae) is used for the treatment of respiratory diseases, diabetes, and hypertension. Aim to explore its tracheal relaxant properties and clarify its functional mechanism of action on smooth muscle cells, which allow us to propose it as a potential anti-asthmatic drug. Material and methods organic and hydro-alcoholic extracts from A. millefolium were obtained by macerations, then their relaxing effect on ex vivo isolated rat trachea rings was determined. Most active extract (hexanic extract, EHAm) was studied to determine its functional mechanism of action using synergic, antagonist and inhibitor agents related with the contraction/relaxation process of the smooth muscle. Also, EHAm was subjected to bio-guided fractionation by open-column chromatography (on silica gel) using cyclohexane-EtOAc (80:20) in an isocratic way to isolate main bioactive compounds. Results organic and hydro-alcoholic extracts showed relaxant effect in a concentration-response dependent manner, being EHAm the most active. The functional mechanism of action indicates that EHAm induced a non-competitive antagonism to the muscarinic receptors ; in addition, the NO/cGMP pathway is involved in the relaxation process of the tracheal smooth muscle. However, the most important mechanism of action showed by EHAm was related with the calcium channel blockade influx into the smooth muscle cells. On the other hand, epimeric sesquiterpene lactones leucodin (1) and achillin (2) were isolated and purified, which are responsible for the observed smooth muscle relaxant activity of the extract. Conclusion hexanic extract of A. millefollium induced a significant relaxant effect on tracheal rat rings by calcium channel blockade and NO release.