Cuminaldehyde, carbamylcholine (CCh), loperamide, acetylcholine perchlorate (ACh), isoprenaline, verapamil, and papaverine were obtained from Sigma (St. Louis, MO, USA). Reagents (salts) to prepare a physiological buffer solution (Tyrode) were procured from Merck (Darmstadt, Germany). All chemicals used were of analytical grade, with the exception of castor oil, which was procured from a local pharmacy.

The Wistar albino rats (180–200 g) and Swiss albino mice (26–30 g) were obtained from the Animal Care Unit at the College of Pharmacy, Prince Sattam bin Abdulaziz University (PSAU), Saudi Arabia. They were kept at an optimal temperature of 22 $\pm$ 1 °C, with a relative humidity of 55 $\pm$ 5%, and were exposed to equal periods of light and darkness. All animals were given conventional pellet food and unlimited access to water. Stringent precautions were observed, and all studies (in vivo and ex vivo) adhered to the directions outlined in the National Research Council (NRC) [23]. The project has obtained approval from the Bio-Ethical Research Committee (BERC) at PSAU, with the reference number BERC-004-12-19.

A previously reported method was followed to test the possible antidiarrheal effect of cuminaldehyde [24]. A total of thirty-five mice were divided randomly into groups, ensuring that each group consisted of five mice. After fasting for twenty-four hours, the mice in the first group were given a saline solution (10 mL/kg) through oral gavage, and they were labeled as the negative control group. Following the pilot screening to determine the effective dose, the second and third groups (test groups) were subjected to two doses of cuminaldehyde (10 and 20 mg/kg). The mice in the fourth group were exposed to cromakalim (10 mg/kg). The mice in the fifth, sixth, and seventh groups were given a pretreatment of glibenclamide (3 mg/kg) one hour before receiving cuminaldehyde or cromakalim. One hour after administration, all mice were orally administered castor oil (10 mL/kg) using a 1 mL syringe. Each mouse was then placed in an individual cage with a blotting sheet on the floor. After 4 hours, each cage blotting sheet was examined for the presence or absence of characteristic diarrheal droppings by a blind observer. The presence of protection was observed when no instances of diarrhea were detected, as previously documented by Jebunnessa et al. [25].

Prior to conducting ex vivo studies, rats underwent a 24-hour fasting period. Subsequently, their cervical dislocation was carried out under mild anesthesia. The ileum tissue was isolated using a previously described procedure [26]. After being isolated, specific sections of ileum tissues measuring 2–3 cm in length were thoroughly cleansed to remove nearby tissues and fecal matter. These cleaned tissues were then placed in an emkaBath apparatus (Paris, France) and connected to a transducer and IOX software (version 2.9.10.6, emka technologies, SAS, Paris, France). The tissue baths were filled with 20 mL of fresh tyrode solution (pH 7.4) and gassed with carbogen. The temperature was adjusted to 37 °C. A force of 1 gram was exerted by turning the transducer knob in a clockwise direction, and the tissues were allowed to stabilize for 30 minutes while being exposed to numerous doses of acetylcholine (0.3 µM). Following stabilization, sustained contractions were induced using low (25 mM) and high (80 mM) K${}^{+}$ final bath concentrations (FBC). Additionally, cuminaldehyde was gradually introduced into the bath solution at concentrations ranging from 0.1 to 30 mM (FBC). The inhibitory effects of cuminaldehyde against K${}^{+}$ (25 mM) and K${}^{+}$ (80 mM)-mediated contractions have different meanings regarding the pharmacodynamics, including K${}^{+}$ channel activation and/or voltage-gated Ca${}^{++}$ channel blockade. Godfraind et al. [27] found that high concentrations of K${}^{+}$ ($>$30 mM) cause depolarization in various smooth muscles by activating Ca${}^{++}$ channels of the L-type. This leads to sustained contractions. Therefore, any substance that specifically and effectively inhibits the excitations mediated by low levels of K${}^{+}$ is considered a K${}^{+}$ channel activator. These types of assays are capable of distinguishing K${}^{+}$ channel activators from Ca${}^{++}$ antagonists [28, 29]. To know the possible involvement of the subtype, ATP-dependent K${}^{+}$ channels, the inhibitory CRC of cuminaldehyde against low K${}^{+}$ was reconstructed in the presence of glibenclamide, a known ATP-dependent potassium channel antagonist [30]. Parallel experiments were run with a positive control drug, cromakalim, a selective opener of ATP-dependent K${}^{+}$ channels [31].

After examining the relaxation effect of cuminaldehyde on high levels of K${}^{+}$, further confirmation of calcium channel blocking (CCB) was achieved by incubating ileum tissues in a calcium-free Tyrode’s solution with ethylenediaminetetraacetate (EDTA) (0.1 mM) for 45 minutes to remove calcium ions. In order to further reduce the levels of Ca${}^{++}$ within the cells, a solution without Ca${}^{++}$ was replaced with another solution known as K${}^{+}$-rich and Ca${}^{++}$-free Tyrode’s solution. After incubating the tissue in this solution for 45 minutes, CaCl${}_2$ CRCs were achieved in the absence and presence of increasing concentrations of cuminaldehyde. The results were then compared to the CRCs of a conventional CCB drug, verapamil [28].

In order to get a molecular understanding of the predominant involvement of potassium channel activation by cuminaldehyde observed in the ex vivo assays, in silico molecular modeling studies were performed on three distinct isoforms of ATP-dependent potassium channels with Protein Data Bank (PDB) IDs 7MJP, 7S61, and 6C3O. The protein structures of the receptors under consideration for study were obtained from the RCSB Protein Data Bank (RCSB PDB) website (https://www.rcsb.org/). The co-crystalline ligands of each receptor were isolated and redocked to validate the docking process. As indicated in our prior study work [32], Autodock Vina [33] was utilized for docking. The interaction between the docking confirmations and potassium channel receptors was observed using the Biovia Solutions Discovery Studio 4 programs [34]. The downloaded proteins were treated to remove water molecules, insert polar hydrogens, add Coleman charges, and store them in PDBQT format for docking. The structures of cuminaldehyde and cromakalim were retrieved in Stromal cell-derived factor (SDF) format from PubChem. In the next step, they were converted from SDF to PDB format and then written in the appropriate PDPQT format.

A chemical’s ADMET (absorption, distribution, metabolism, excretion, and toxicity) characteristics are critical for developing it as a lead medicine in the drug discovery cascade. The ADMET characteristics of cuminaldehyde were determined using the pkCSM software (https://biosig.lab.uq.edu.au/pkcsm/prediction), a web-based application [35]. Because this software allows smiles, the smiles were copied from PubChem and then sent to pkCSM for comprehensive profile prediction of the cuminaldehyde molecule.

The results were provided as the mean $\pm$ SEM and the number of experiments conducted is denoted by “n”. The median effective concentration (EC${}_{50}$) values were evaluated with 95% confidence intervals (CI). The statistical analysis involved the use of Student’s t-test or two-way ANOVA, followed by Bonferroni’s post-test, to compare concentration-response curves (CRCs) with a control group. The efficacy of diarrhea protection was assessed by a statistical analysis, namely by comparing all groups to the saline control group using the Chi-square ($\chi$${}^2$) test. A p-value less than 0.05 is regarded as statistically significant. The regression analysis of CRCs was performed using GraphPad Prism (version 4, GraphPad Software, Inc., San Diego, CA, USA).

In mice, cuminaldehyde exhibited dose-dependent protection from diarrhea evoked by castor oil, while the saline group did not produce any protection. Preincubated mice with cuminaldehyde (10 and 20 mg/kg) exhibited 60% and 80% protection, whereas cromakalim (positive control group) produced 100% protection from diarrhea at 10 mg/kg. Glibenclamide (3 mg/kg) preincubation suppressed the antidiarrheal actions of cuminaldehyde and cromakalim, as detailed in Table 1.

When checked against low and high K${}^{+}$-mediated excitations, cuminaldehyde (Fig. 1A) suppressed the low K${}^{+}$-mediated excitations with significantly higher potency (p 0.05) with EC${}_{50}$ values of 3.14 mM (2.78–3.92, 95% CI, n = 5) while low potency was observed in its inhibitory effect against the high K${}^{+}$-mediated contractions [13.56 mM (12.20–14.42, 95% CI, n = 5)]. Cromakalim also selectively suppressed the low K${}^{+}$-mediated excitations (Fig. 1B) with an EC${}_{50}$ value of 0.0008 mM (0.0002–0.0010, 95% CI, n = 5) while slightly suppressed the high K${}^{+}$-mediated excitations with the highest % inhibition of (13.5 $\pm$ 1.5) whereas verapamil suppressed both types of low K${}^{+}$ and high K${}^{+}$-mediated excitations with comparable EC${}_{50}$ values of 0.00028 mM (0.00018–0.00045, 95% CI, n = 5) and 0.00024 µM (0.00017–0.00035) completely, respectively (Fig. 1C).

Fig. 1.

Inhibitory concentration-response curves showing the comparison of (A) cuminaldehyde, (B) cromakalim, and (C) verapamil for the inhibitory effect against low K${}^{\mathrm{+}}$ (25 mM) and high K${}^{\mathrm{+}}$ (80 mM)-induced contractions in isolated rat ileum preparations. Values shown are mean $\pm$ SEM, n = 5.

The inhibitory actions of cuminaldehyde and cromakalim against low K${}^{+}$ were markedly reversed when the segments were preincubated with glibenclamide (Gb), resulting in 12.5 $\pm$ 2.5 and 7.0 $\pm$ 3.0% suppression, respectively (Fig. 2A,B), whereas verapamil was completely insensitive to Gb (Fig. 2C).

Fig. 2.

Comparisons of the inhibitory concentration-response curves of (A) cuminaldehyde, (B) cromakalim, and (C) verapamil against low K${}^{\mathrm{+}}$ in the absence and presence of glibenclamide (Gb, 10 µM) in isolated rat ileum preparations. Values shown are mean $\pm$ SEM, n = 5.

To further confirm the Ca${}^{++}$ inhibitory effect, cuminaldehyde preincubated ileum tissues shifted the Ca${}^{++}$ CRCs curves at 3 and 10 mM towards the right, similar to that caused by verapamil at concentrations of 0.00003 and 0.0001 mM (Fig. 3A,B).

Fig. 3.

Concentration-response curves of Ca${}^{\mathrm{+}\mathrm{+}}$ in the absence and presence of the increasing concentrations of (A) cuminaldehyde and (B) verapamil in isolated rat ileum preparations. Values shown are mean $\pm$ SEM, n = 5.

ATP-dependent potassium channel receptors are complex macromolecules consisting of sulfonylurea receptor (SUR) and inward rectifier Kir subunits, and they have been reported in RCSb in propeller and quatrefoil forms. The pore opening in Kir is linked to coordinated structural changes in the ATP-binding site and the channel gate. The first chosen protein, 7S61, when subjected to molecular docking with cuminaldehyde and cromakalim, exhibited binding energies of –5.8 and –6.8 kcal/mol, respectively. For further confirmation, another human KATP (ATP-sensitive potassium channel) bound to ATP and ADP in quatrefoil form was downloaded and docked with the same two compounds considered in the study. The binding energies exhibited by cuminaldehyde and cromakalim this time were –6.8 and –7.8 kcal/mol, respectively. For further legitimization of this study, another ATP-dependent potassium channel, 7MJP, was considered for the docking study of the molecules in question, and they exhibited binding energies of –5.0 and –6.9 kcal per mole. The pattern of binding energy among all three receptors was found to be more or less similar. Cuminaldehyde and cromakalim have exhibited various forms of molecular interactions with the KATP proteins, as shown in Table 2 (Conventional H-Bond, Vander Waal, pi-pi stacking, Amide-pi stacking, Sigma-Pi stacking). Cuminaldehyde exhibited three conventional hydrogen bond interactions with the protein 7S61, while cromakalim exhibited only one. The hydrogen bond distance is also considered a crucial parameter for strong binding between ligand and receptor, the lesser the stronger. Cuminaldehyde showed shorter H-bond distances with 7S61 {a-1.91 (Ser721), b-2.32 (Lys720), c-2.48 (Ser722)}, on the other hand, cromakalim had longer {2.67 (Ser409)}. As shown in Fig. 4A–D, cuminaldehyde showed interactions with 6 residues (Trp-E689, Ser-E1483, Phe-E1482, Ser-E722, Ser-E721, Lys-E720), and cromakalim showed interactions with only 5 residues (Trp-E689, Ser-E1483, Phe-E1482, Ser-E409, Ser-E721). The KATP protein 6C3O, when overviewed for such interaction, cuminaldehyde exhibited interactions (Vander Waal, Sigma-Pi stacking) with 6 residues (Val679, Ile703, Ala726, Arg766, Phe851, Val784), while cromakalim showed interactions (Conventional H-Bond, carbon H-Bond, pi-pi stacking, Anion-Pi stacking) with 4 residues (Trp688, Glu1479, Ser1482, Gln1483), as shown in Fig. 5A–D. Interestingly, cuminaldehyde made two Vander Waal interactions with bond distances of 2.07 and 2.93 with Arg766. At the same time, cromakalim exhibited a conventional H-bond but with a larger distance of 2.99 with Gly1483. Likewise, when the KATP protein 7MJP was overviewed cuminaldehyde exhibited interaction (Conventional H-Bond, Vander Waal, Sigma-Pi stacking) with 7 residues (Ile199, Leu206, Ile271, Leu277, Ile280, Phe208, Ala355) while cromakalim shown interaction (Conventional H-Bond and Sigma-Pi stacking) with 4 residues (Thr190, Leu191, Arg216, Ile220) as shown in Fig. 6.

Fig. 4.

Representation of the molecular interaction of cuminaldehyde (A,B) and cromakalim (C,D) with KATP (7S61). (A,C) 2-Dimensional and (B,D) 3-Dimensional.

Fig. 5.

Representation of the molecular interaction of cuminaldehyde (A,B) and cromakalim (C,D) with KATP (6C3O). (A,C) 2-Dimensional and (B,D) 3-Dimensional.

Fig. 6.

Representation of the molecular interaction of cuminaldehyde (A,B) and cromakalim (C,D) with KATP (7MJP). (A,C) 2-Dimensional and (B,D) 3-Dimensional.

As shown in Table 3, cuminaldehyde has exhibited acceptable ADMET profiling. ADMET profiling is a must for establishing the drug-like properties of any molecule. The ADMET properties of cuminaldehyde were calculated by PKCSM software and exhibited an intestinal absorption close to 95%, and volume distribution (steady state) was found to be 0.324 (log L/kg). The log BB quotient for blood-brain barrier permeability was found to be 0.438, showing good BBB permeability. Cuminaldehyde does not inhibit any family of CYP450 enzymes except CYP1A2 and has a safe toxicity profile, except for being sensitive to the skin.