In Silico and Ex Vivo Studies on the Spasmolytic Activities of Fenchone Using Isolated Guinea Pig Trachea

Abstract

Fenchone is a bicyclic monoterpene found in a variety of aromatic plants, including Foeniculum vulgare and Peumus boldus, and is used in the management of airways disorders. This study aimed to explore the bronchodilator effect of fenchone using guinea pig tracheal muscles as an ex vivo model and in silico studies. A concentration-mediated tracheal relaxant effect of fenchone was evaluated using isolated guinea pig trachea mounted in an organ bath provided with physiological conditions. Sustained contractions were achieved using low K⁺ (25 mM), high K⁺ (80 mM), and carbamylcholine (CCh; 1 µM), and fenchone inhibitory concentration-response curves (CRCs) were obtained against these contractions. Fenchone selectively inhibited with higher potency contractions evoked by low K⁺ compared to high K⁺ with resultant EC₅₀ values of 0.62 mg/mL (0.58-0.72; n = 5) and 6.44 mg/mL (5.86-7.32; n = 5), respectively. Verapamil (VRP) inhibited both low and high K⁺ contractions at similar concentrations. Pre-incubation of the tracheal tissues with K⁺ channel blockers such as glibenclamide (Gb), 4-aminopyridine (4-AP), and tetraethylammonium (TEA) significantly shifted the inhibitory CRCs of fenchone to the right towards higher doses. Fenchone also inhibited CCh-mediated contractions at comparable potency to its effect against high K⁺ [6.28 mg/mL (5.88-6.42, n = 4); CCh] and [6.44 mg/mL (5.86-7.32; n = 5); high K⁺]. A similar pattern was obtained with papaverine (PPV), a phosphodiesterase (PDE), and Ca²⁺ inhibitor which inhibited both CCh and high K⁺ at similar concentrations [10.46 µM (9.82-11.22, n = 4); CCh] and [10.28 µM (9.18-11.36; n = 5); high K⁺]. However, verapamil, a standard Ca²⁺ channel blocker, showed selectively higher potency against high K⁺ compared to CCh-mediated contractions with respective EC₅₀ values of 0.84 mg/mL (0.82-0.96; n = 5) 14.46 mg/mL (12.24-16.38, n = 4). The PDE-inhibitory action of fenchone was further confirmed when its pre-incubation at 3 and 5 mg/mL potentiated and shifted the isoprenaline inhibitory CRCs towards the left, similar to papaverine, whereas the Ca²⁺ inhibitory-like action of fenchone pretreated tracheal tissues were authenticated by the rightward shift of Ca²⁺ CRCs with suppression of maximum response, similar to verapamil, a standard Ca²⁺ channel blocker. Fenchone showed a spasmolytic effect in isolated trachea mediated predominantly by K⁺ channel activation followed by dual inhibition of PDE and Ca²⁺ channels. Further in silico molecular docking studies provided the insight for binding of fenchone with Ca²⁺ channel (-5.3 kcal/mol) and K⁺ channel (-5.7), which also endorsed the idea of dual inhibition.

Keywords: Ca++ channel blocker; K+ channel opener; PDE inhibitor; carbamylcholine; fenchone; papaverine.

Figure 1

Concentration-dependent inhibitory effects of (A) fenchone and (B) verapamil against low K⁺ (25 mM) and high K⁺ (80 mM)-induced contractions in isolated guinea pig tracheal tissues. Symbols represent mean ± SEM; n = 4–5.

Figure 2

Effect of (A) glibenclamide (Gb; 10 µM), (B) 4-aminopyridine (4-AP; 1 mM) and (C) tetraethylammonium (TEA; 1 mM) on the inhibitory effects of Fenchone against low K⁺ (25 mM)-induced contractions in isolated guinea pig tracheal tissues. Symbols represent mean ± SEM; n = 4–5.

Figure 3

Concentration-dependent inhibitory effects of (A) fenchone and (B) papaverine and (C) verapamil against carbachol (CCh; 1 µM) and high K⁺ (80 mM)-induced contractions in isolated guinea pig tracheal tissues. Symbols represent mean ± SEM; n = 4–5.

Figure 4

Inhibitory concentration–response curves of isoprenaline against carbachol (CCh; 1 µM)-induced contractions in the absence and presence of the increasing concentrations of (A) fenchone, (B) papaverine, and (C) verapamil in guinea pig tracheal preparations. Symbols represent mean ± SEM; n = 4–5.

Figure 5

Concentration–response curves of Ca++ in the absence and presence of the increasing concentrations of the (A) fenchone, (B) verapamil, and (C) papaverine in isolated guinea pig tracheal preparations. Symbols represent mean ± SEM; n = 4–5.

Figure 6

6A to 6D shows the ligand–receptor interactions. (A,B) show binding of PDE 4A(3ITU) with FNC and PPV, respectively. (C,D) show the binding modes of Ca++ ion channel (6JPA) bounded with FNC and VRP. Dark green circles and lines show hydrogen bonds, while the light green spheres show Van der Waal interaction between ligands and residues.

Figure 7

(A,B) Represent the 2D image of fenchone (FNC) and retigabine (RTG) docked within the voltage-gated potassium channel isoform 6EBM (Kv1.2-2.1). (C,D) Represent the 2D image of FNC and RTG interacting within the potassium channel isoform 7VPN (Human KCQN4).