The chemicals utilized in this study were obtained from Sigma Company, located in St. Louis, MO, USA, including carbachol (CCh), acetylcholine perchlorate (ACh), and verapamil. The physiological buffer solution (Tyrode’s) was prepared using salts acquired from Merck (Darmstadt, Germany). Castor oil was acquired from a local pharmacy in Al-Kharj. All chemicals used were of analytical quality.

Wistar albino rats (weighing 200–250 g) were used for the ex vivo study, while Swiss albino mice (weighing 30–35 g) were used for the in vivo study. These animals were obtained from the Animal Care Unit at the College of Pharmacy, Prince Sattam bin Abdulaziz University (PSAU), Saudi Arabia. They were kept under optimal conditions, with a temperature of 22 $\pm$ 1 °C, relative humidity of 55 $\pm$ 5%, and equal exposure to light and dark cycles. All animals were given a standardized pellet meal and unrestricted access to water. Prior to conducting the ex vivo studies, the rats underwent a 24-hour fasting period. Their cervical dislocation was performed after administering light anesthesia using diethyl ether, and the cessation of ear reflexes was used to certify their death. All tests, both in vivo and ex vivo, were conducted with meticulous attention to detail, adhering strictly to the directions outlined in National Research Council [18]. The study protocol has received approval from the Bio-Ethical Research Committee (BERC) at PSAU, with the reference number BERC-004-12-19.

O. majorana leaves were purchased from the local market of Dammam and authenticated (PL/0445/2020-21/P-009) by the Department of Pharmacognosy, College of Clinical Pharmacy, Taif University, Saudi Arabia. The plant material was dried in the shade. All plant materials were crushed into a coarse size and later subjected to extraction.

The plant matter was crushed and macerated to prepare the extract. Ten grams of each sample were weighed into 500 mL Erlenmeyer flasks, and 100 mL of methanol was added to the plant samples. Extraction was carried out by the maceration technique with frequent agitation at room temperature for 5 days. After filtration through filter paper, the extracts of every sample were evaporated until the constant weight and respective extractives were calculated. The final residues were used in this study.

The phytochemical composition of the methanolic leaf extract of O. majorana was analyzed by Agilent GC-MS (7890A) using HP 5MS column (30 m $\times$ 0.25 mm $\times$ 0.25 µm film thickness) for the separation on split mode 1:20 with helium being used as the gaseous mobile phase at a flow rate of 1.0 mL per min. The injector temperature was 280 °C, the source temperature of MS was kept at 230 °C, and the Quad temperature was kept at 150 °C. The column oven temperature was increased from 150 °C to 300 °C at a gradual rise of 10 °C/min, and the total run time was 40 min. The scanning was carried out in the range of 40 to 600 mass while electron energy was kept at 70 eV. A solvent delay of 3 minutes was employed during the analysis. Identification of components was done by comparing the spectra of the sample with that of the standard spectra available with National Institute of Standards and Technology and Wiley library [19, 20].

Altogether, twenty mice were assigned randomly to four groups, each containing an equal number of animals. After fasting for 18 hours, the mice in the first group were given a saline solution (10 mL/kg) through oral gavage and were labeled as the negative control group. Following the pilot screening to determine the appropriate dosage, the second and third groups (referred to as test groups) were administered orally with two escalating doses of 200 and 400 mg/kg of methanolic leaves extract of O. majorana, respectively. The fourth group of mice was administered loperamide at a dosage of 10 mg/kg and designated as the positive control group. Following one hour, all mice were administered castor oil orally at a dosage of 10 mL/kg. Each animal was then placed in an individual cage with a blotting sheet on the floor. This allowed a blind observer to determine the presence or absence of diarrhea. After four hours, the blotting sheet from each cage was examined for the presence of characteristic diarrheal droppings. The protective effect of the extract was evident when no instances of diarrhea were seen, as previously documented by Jebunnessa et al. [21].

The study rats were euthanized, and the terminal portion of the small intestine (ileum) was extracted following the protocol described by Rehman et al. [22]. After being isolated, specific sections of ileal tissues measuring 2–3 cm in length were thoroughly cleaned to remove nearby tissues and fecal matter. These sections were then placed in emkaBath (France) and connected to a transducer and IOX software (version 2.9.10.6, emka technologies, SAS, Paris, France). The Tyrod solution (pH 7.4) was freshly prepared [22] and added to tissue baths with a volume of 20 mL. The baths were then filled with carbogen gas and maintained at a temperature of 37 °C. A stress of 0.7 grams was exerted by turning the transducer knob in a clockwise direction. The tissues were then allowed to stabilize for 30 minutes, during which they were exposed to acetylcholine numerous times at a concentration of 0.3 µM. Following stabilization, continuous muscle contractions were induced using CCh, and high doses of K${}^{+}$ and O. majorana were gradually introduced into the bathing solution, with concentrations increasing to a maximum final bath concentration (FBC) of 10 mg/mL.

Following the first relaxation of O. majorana in response to high K${}^{+}$ levels, additional verification of Ca${}^{++}$ channel blocking (CCB) was achieved by incubating the ileal tissues in a solution of Ca${}^{++}$-free Tyrode’s solution combined with EDTA (0.1 mM) for 45 minutes. This solution was used to chelate Ca${}^{++}$ ions. To further reduce the amount of Ca${}^{++}$ inside the cells, the solution that did not contain Ca${}^{++}$ was replaced with another solution called K${}^{+}$-rich and Ca${}^{++}$-free Tyrode’s solution. The concentrations of the different components in this solution were as follows (in mM): potassium chloride: 50, sodium chloride: 91.03, sodium dihydrogen phosphate dehydrates: 0.32, sodium bicarbonate: 11.9, magnesium chloride hexahydrate: 0.50, glucose: 5.05. After incubating the solution for 45 minutes, CaCl${}_2$- Concentration response curves (CRCs) were created with and without varying concentrations of O. majorana. The findings were then compared to the typical CCB drug, verapamil [23].

The results have been reported as the mean $\pm$ standard error of the mean (SEM), and the number of experiments conducted has been denoted by “n”. The EC${}_{50}$ values were determined, and their 95% confidence intervals (CI) were calculated. The statistical analysis involved two-way ANOVA, followed by Bonferroni’s post-test, to compare concentration-response curves (CRCs) with a control group. The effectiveness of diarrhea prevention was assessed using a statistical analysis, comparing all groups to a control group, using the Chi-square ($\chi$${}^2$) test. A p-value less than 0.05 has been regarded as statistically significant. The software GraphPad Prism (version 4, GraphPad Software, Inc., San Diego, CA, USA) was utilized to perform regression analysis of CRCs.

The leaves of O. majorana yielded 14.7% of methanolic crude extract.

O. majorana was analyzed by GC-MS (Fig. 1). The phytoconstituents identified are listed in Table 1. GC-MS identified twenty-one constituents representing 96.27% of the extract. Alpha-terpineol (35.18%) and linoleic acid (13.3%) were characterized as major components of the methanolic extract. Sabinene, a bicyclic monoterpene found in the essential oils of various plant species, was also identified in the profiling.

Fig. 1.

Gas chromatography–mass spectrometry (GC-MS) chromatogram of Origanum majorana (O. majorana) leaves extract.

Both the increasing doses of orally administered O. majorana extract in mice exhibited significant antidiarrheal effects in comparison to the control group (Table 2). When administered a dose of 200 mg/kg, two out of five mice exhibited protection, indicating a 40% protection rate. In contrast, a higher dose of 400 mg/kg resulted in 80% protection. The positive control drug, loperamide, showed 100% protection at 10 mg/kg, as detailed in Table 2.

When tested against CCh and high K${}^{+}$-mediated spasm in rat-isolated ileal tissues, O. majorana resulted in complete inhibition with a resultant EC${}_{50}$ value of 1.38 mg/mL (1.06–1.82, 95% CI, n = 4) recorded against high K${}^{+}$ whereas the potency against CCh was found weak with a resultant EC${}_{50}$ of 7.14 mg/mL (6.85–7.84, 95% CI, n = 4), as shown Fig. 2A. Similarly, verapamil resulted in distinctly higher potency to inhibit high K${}^{+}$compared to CCh-evoked spasms with EC${}_{50}$ values of 0.24 µM (0.19–0.32, 95% CI, n = 4) and 2.28 µM (1.84–2.92, 95% CI, n = 4), respectively as shown in Fig. 2B.

Fig. 2.

The concentration-response curves compare the inhibitory effect of the methanolic Origanum majorana (O. majorana) leaf extract (A) and verapamil (B) against carbachol (CCh, 1 µM) and high K${}^{\mathrm{+}}$ (80 mM)-induced contractions in isolated rat ileum preparations. The values presented are the mean $\pm$ Standard error of the mean (SEM), n = 4.

To further confirm the Ca${}^{++}$ inhibitory effect, ileal tissues preincubated with O. majorana methanolic extract shifted the Ca${}^{++}$ CRCs curves at tested concentrations of 0.3 and 1.0 mg/mL (Fig. 3A), similar to that caused by verapamil at concentrations of 0.03 and 0.10 µM (Fig. 3B).

Fig. 3.

The concentration-response curves of Ca${}^{\mathrm{+}\mathrm{+}}$ were measured in isolated rat ileum preparations in the absence and presence of increasing concentrations of (A) the methanolic Origanum majorana (O. majorana) leaf extract and (B) verapamil. The values presented are the mean $\pm$ SEM, n = 4.