Ex-vivo Studies on Isolated Guinea-Pig Trachea

The trachea was isolated from guinea pig sacrificed by cervical dislocation and exsanguinated. Approximately, 2–3 cartilaginous rings, constituting one tracheal tissue, were opened by a longitudinal cut opposite to the smooth muscle layer. The opened tissue was then mounted in a 20 ml organ tube containing Kreb’s solution (37°C) and bubbled with carbogen. A tension equal to 1 g was constantly maintained in each of the tracheal tissues during the experiment. During the equilibration of tissue for 1 h, the Krebs solution in the bath was replaced every 15 min, while CCh (1 µM) and K⁺ (80 mM) were repeatedly used to stabilize the tissue until constant contractions of each agonist were achieved. After stabilization, the relaxant effect of TS Oil was determined by adding it cumulatively every 10 min to get the concentration-dependent effects. Tracheal contraction and relaxation were recorded by using isometric transducers attached to emkaBath with IOX software (France).

To investigate the possible effect of the plant oil to exhibit inhibitory action on Ca⁺⁺ channels, the tracheal tissues were depolarized with high K⁺ (80 mM). K⁺ > 30 mM is known to depolarize the tissue by opening of voltage-gated Ca⁺⁺ channels and/or other transporters and channels such as; Ca-Na exchanger and TRP channels and thus, causing smooth muscle contractions (Wray, 2010). Meanwhile, the substance inhibiting high K⁺-induced contraction might in some way leads to a reduction in the Ca⁺⁺ influx through these pathways and might be considered as a Ca⁺⁺ channel blockers (Hussain et al., 2008). In the next step, to endorse the Ca⁺⁺ channel blockade (CCB) effect of TS Oil, the tracheal tissues stabilized in normal Kreb’s solution were immersed in Ca⁺⁺-free Kreb’s solution containing EDTA (0.1 mM) where the tissues were incubated for 30 min to remove Ca⁺⁺ from the tissue. Thereafter, the Ca⁺⁺-free Kreb’s solution was replaced with K⁺-rich and Ca⁺⁺-free Kreb’s solution. After incubation of tracheal tissue for an hour in K⁺-rich and Ca⁺⁺-free Kreb’s solution, Ca⁺⁺ induced contractions were recorded to obtained control CRCs. When the Ca⁺⁺-CRCs were found to be superimposable, the tracheal tissues were incubated with TS Oil for 1 h. The CRCs of Ca⁺⁺ were repeated in the presence of increasing concentrations (1 and 3 mg/ml) of the TS Oil and the results were compared using verapamil (0.03 and 0.1 µM) as standard (positive control).

To test whether the tracheal relaxant effect of the TS Oil also involves any additional mechanism(s), such as anticholinergic like effects, the CRCs of CCh were constructed by adding CCh in cumulative manner to the bath and the tissues were washed with fresh Krebs solution after achieving CCh maximum peaks. The CCh CRCs were repeated in the presence of pre-incubated tissues with increasing concentrations (0.3 and 1 mg/ml) of TS Oil for 1 h (Shah and Gilani, 2010).

Usually, plant materials that produce tracheal relaxation also show inhibitory actions on the PDE-enzyme, hence the method described by (Gilani et al., 2005) was followed to assess the presence of possible PDE inhibitory effects in our plant essential oil. Briefly, inhibitory CRCs of isoprenaline were constructed against CCh-induced contractions in the absence (control) and in the presence of TS Oil.