Contribution of muscarinic receptors to in vitro and in vivo effects of Ruscus extract

•In vitro, Ruscus extract binds and activates different subtypes of muscarinic receptors;•Anti-inflammatory effects of Ruscus extract in vivo are, at least partially, mediated via muscarinic receptors;•Ruscus extract has binding affinity for muscarinic receptors particularly of M1 and M3 receptor subtypes.•The activity of Ruscus extract at muscarinic receptors may contribute to its vasoprotective and anti-inflammatory effects.

The objectives of this study were to evaluate, in vitro and in vivo, the contribution of muscarinic receptors to the effects of Ruscus extract.Ruscus extract was tested in competition binding experiments at recombinant human muscarinic receptors, heterologous expressed in Chinese Hamster Ovary (CHO) cells and in cellular assays measuring Ca2 + liberation and activator protein-1 (AP-1) reporter gene activation. The impact of muscarinic blockade on prolonged treatment outcome was evaluated using the hamster cheek pouch (HCP) microcirculation examining macromolecular permeability increase induced by histamine or ischemia/reperfusion (I/R), mean arteriolar and venular diameters, functional capillary density and I/R-induced leukocyte rolling and sticking.Ruscus extract exhibited affinities for muscarinic receptor subtypes at a range of 50-100 μg/ml and behaved as partial agonist at human recombinant M1 and M3 receptors for Ca2 + liberation, confirmed in an AP-1 reporter gene assay. In the HCP model, topical application of atropine completely or partially blocked Ruscus extract-induced reductions of histamine- and I/R-induced increases of macromolecular permeability and leukocyte-endothelium interaction.Our results showed that Ruscus extract in vitro binds and activates different subtypes of muscarinic receptors and in vivo its anti-inflammatory effects are, at least partially, mediated via muscarinic receptors.