Isolated guinea pig trachea relaxation assay

Male Dunkin–Hartley guinea-pigs weighing 250–350 g were provided by the Experimental Animal Center of Shenyang Pharmaceutical University. The experiment has been approved by the Animal Care and Use Committee of Shenyang Pharmaceutical University. The methods for the isolation of tracheal smooth muscle strips and muscle tone measurements have been described [36]. The strips were equilibrated in Krebs–Henseleit solution (composition in mM: NaCl 118, KCl 5.4, CaCl₂ 2.5, MgSO₄ 0.6, NaH₂PO₄ 2.94, NaHCO₃ 25 and D-glucose 11.7) and aerated with 95% O₂ and 5% CO₂ for 2 h. Histamine (10 μmol/L) was added to produce maximum contraction. The compound was then added to produce relaxation. The percentage of relaxation was calculated as follows: