Warionia saharae Ben-them ex Benth. & Coss. is an endemic plant of North Africa regions. This plant is native to Morocco, and it is the sole species in the genus Warionia (Asteraceae) [1]. The plant may reach a height of one to three meters. The shrub has a strong trunk covered in a gray peel, a structure of highly wavy terminal leaf bouquets, and a profusion of yellow flowers. The latex that pours out of peel injuries glues to hands in a very tenacious way; picking the leafed stems of this bush clears a very strong and spicy odor [2]. Afessas, abessas, or tazart nîfiss are some of the native Berber names for this plant [3]. W. saharae leaves are used in traditional medicine to treat inflammatory disorders, gastrointestinal problems, and epileptic seizures [4]. Until recently, W. saharae had not sparked the curiosity of scientists, and just a few articles on this species had been published. The hydromethanolic extract of W. saharae exhibits significant glucosidase inhibitory activities, according to Rechek et al. [4]. The essential oil and crude extracts of this plant were found to have antibacterial, cytotoxic, antioxidant, and anti-inflammatory properties against the cancer cell line “KB cells” [4, 5].

Essential oils and their constituents have been utilized to treat a wide range of human ailments since ancient times. Essential oils are used in aromatherapy, medicine, fragrances, and cosmetics. Their use is limited to their varied biological functions [6]. Heghes et al. [7] 2019 identified 39 plant species containing essential oils with antispasmodic activities. They found that the main mechanisms of antispasmodic action were achieved via inhibiting voltage-dependent calcium channels, modifying potassium channels, and intracellularly controlling AMPc. Several investigations have identified various biological activities of essential oils [8], but the antispasmodic effect, while mentioned in traditional medicine literature, has received far less experimental attention.

The objectives of this study were to investigate at the chemical composition of essential oils extracted from aerial parts of W. saharae in the southern region of Morocco and to study their antispasmodic activities.

The essential oil was extracted and analyzed using GC-MS from the dried areal parts of W. saharae. The essential oil yields were about 0.5% (w/w) on a dry weight basis. Sellam et al. [10] found a 1.1% essential oil extraction yield from W. saharae. For Mezhoud, the extraction yield was around 0.85% (plant grown in Algeria) [11]. These yield variations might be attributed to extraction techniques, ecotype, or aerial component employed (stem, leaf or flower). The composition of the EoWs was presented as a ratio of numerous molecules from the monoterpene, sesquiterpene, terpene esters, and monoterpenol groups. The oil’s distinctive and aromatic odor may be explained by its high concentration of identified oxygenated compounds (Table 1). Several investigations have found nerolidyl acetate, $\beta$-eudesmol, and linalool to be among the primary compounds found in EoWs, although with varied chemical profiles and quantities in comparison to our data [10, 11, 12, 13]. Ramaut et al. [12] published the first study on the chemical composition of W. saharae essential oils extracted from the leaves. Only three compounds have been identified by the authors: $\beta$-eudesmol (42.25%), nerolidol (8.63%), and linalool (17.26%). On the other hand, according to Essaqui et al. [13], $\beta$-eudesmol (52.7%), trans-nerolidol (17.4%), linalool (5.1%), were the main components of the essential oil. Other compounds were identified so far: terpinen-4-ol and 1, 8-cineole, and guaiol [14]. These variations were explained by the plant genotype, ecology, and technique of extraction.

To assess a substance’s potential risk to humans, toxicity studies in animal models are commonly conducted. The study of effect EoWs at concentrations of 0.5, 1.0, 1.5, and 2.0 g/kg body weight showed no signs of toxicity or mortality over a 14-day observation period, nor did the mice’s weight change, and even after the animal was euthanized, the weight of vital organs such as the kidneys, liver, and heart, as well as the aspect of the stomach and intestine, did not change. After investigating these factors, we detected no evidence of toxicity. Based on these results, we may infer that our plant is relatively safe in the case of acute intake of up to 2 g/kg body weight of albino mice.

The myorelaxant activity of W. saharae essential oil has been examined in the rabbit jejunum, which is characterized by contractions whose amplitude and frequency are rhythmic (do not change over time), owing to the slow waves generated by the Cajal interstitial cells [15]. Furthermore, the waves are clearer and larger than the rat’s, making them easier to examine and comprehend. As a result, we chose to concentrate our studies on the myorelaxant impact of EoWs, specifically on the rabbit jejunum. With an IC${}_{50}$ of 16.80 $\pm$ 0.63 $\mu$g/mL, EoWs at doses of 10, 30, and 50 $\mu$g/L suppressed basal jejunal contractions in rabbits in a dose-dependent and reversible manner. This action is more effective than the myorelaxant effect found for extracts of Origanum majorana, with an IC${}_{50}$ of 59.22 $\pm$ 2.42 $\mu$g/mL [16] and for Psidium guajava, with an IC${}_{50}$ of 0.84 $\pm$ 1.08 $\mu$g/mL [17].

Several investigations have indicated that intestine smooth muscle contraction occurs as a result of an increase in free calcium in the cytoplasm, which subsequently activates the contractile elements [18]. The Ca${}^{2+}$ ion accumulation in the cytoplasm is caused by either inflow through voltage-gated calcium (VGC) channels or release from the cytoplasmic reticulum. Membrane depolarization facilitates the control of spontaneous intestine movements, and when it is intensified, it generates an action potential of the cell, which manifests as a fast influx of Ca${}^{2+}$ ions through VGC channels [19]. The myorelaxant action of the W. saharae extract, as demonstrated in our investigation, is most likely due to EoWs interfering with Ca${}^{2+}$ ion influx through VGC channels or release from the cytoplasmic reticulum. On the other hand, we established that the muscle relaxant action generated by this plant is reversible, meaning that the essential oil of this plant extract did not injure smooth muscle cells or alter their functioning in an irreversible way. Several studies have reached similar conclusions. Essential oil isolated from the flowers of Anthemis mauritiana (Maire & Sennen) displayed an inhibitory impact on the dose-response curves caused by CaCl${}_2$, but at greater concentrations (100 g/mL), most likely attributable to $\alpha$-pinene [20]. The same effect was achieved using an essential oil extracted from the aerial portion of Artemisia herba alba (Asso.), albeit at a lower concentration (200 ng/mL). This latter spasmolytic effect might be related to the presence of camphor, terpinene, 1,8-cineol, $\alpha$- and $\beta$-pinene, all of which have been reported to be smooth muscle relaxants [21]. Bergapten, a hydrophobic substance isolated from the fruits of Heracleum leskovii (Grossch.) (Apiaceae), produced myorelaxation of intestinal preparations at concentrations ranging from 0.0001 to 1 mM. Bergapten induced smooth muscular relaxation or contraction at higher doses [22]. As a result, in order to avoid the opposite effect, the dosages must be carefully established.

Previously, it was established that catecholamines and adrenergic drugs reduced spontaneous contractions of the rabbit jejunum [23]. For this reason, we selected three adrenergic inhibitors: prazosin, yohimbine, and propranolol, which are recognized for their competitive action on $\alpha$1, $\alpha$2 and $\beta$ adrenergic receptors, respectively. Adrenaline (epinephrine) is a catecholamine neurotransmitter that acts on receptors coupled with G proteins [24]. This compound is added to ensure that all adrenergic receptors have been blocked by the adrenergic inhibitors listed above. Treatment with EoWs (30 $\mu$g/mL) reduced the amplitude of rabbit jejunal contractions, indicating that EoWs do not operate via adrenergic receptors.

Carbachol, a structural analogue of acetylcholine, was employed to evaluate the cholinergic receptor pathway, since it has the advantage of not being degraded in the physiological medium by the action of acetylcholinesterases. In the presence of EoWs, we examined two different cholinergic inhibitors: atropine, a muscarinic antagonist, and hexametonium, a nicotinic inhibitor. EoWs appear to operate via muscarinic receptors but not nicotinic receptors. Then this oil may therefore induce the activation of M2 and M3 muscarinic receptors found in the intestine [25]. Furthermore, muscarinic receptor activation can stimulate non-selective calcium (NSC) channels in the plasma membrane, resulting in a membrane potential and Ca${}^{2+}$ entry via VGC channels [26]. EoWs significantly reduced the tone of the rat jejunum caused by 1 $\mu$M CCh in a dose-dependent manner, with an IC${}_{50}$ of 12.04 $\pm$ 0.30 $\mu$g/mL. As a consequence, we can postulate that one or more active plant compounds in EoWs inhibit M2 and M3 muscarinic receptors. We examined the dose response effect of Carbachol in the presence or absence of EoWs to corroborate this hypothesis (Fig. 4). The EoWs has a significant inhibitory action on CCh-induced maximal contraction. Papaverine (10 $\mu$M) used as a positive control, relaxes intestinal muscle by inhibiting adenosine monophosphate phosphodiesterase (AMPC), resulting in an increase of this molecule in the intracellular medium, which negatively influences the phosphorylation of the myosin light chains and, as a result, contraction [27].

We obtained the same results, when we progressively added calcium in the presence of EoWs or verapamil. These results, thus, suggest that EoWs operated as an agonist through one of the two muscarinic receptor contraction pathways. Similar observations were done with plant extracts from Nepeta cataria L. [28], Satureja hortensis [29], and Satureja obavata [30].

Previous research has found that the antispasmodic activity of medicinal plants is generally mediated by blocking calcium channels [31, 32]. Similarly, we aimed to determine if our essential oil’s antispasmodic activity is driven by similar pathways. The effect of EoWs on rat jejunal contractions generated by 25 mM KCl was studied for this purpose, and the results showed that this essential oil significantly reduced the maximum tone of the jejunum created by the latter in a dose-dependent manner, with an IC${}_{50}$ = 15.76 $\pm$ 0.37 $\mu$g/mL (Fig. 3B). To determine if our essential oil acted through calcium channels, we investigated the dose-response impact of CaCl${}_2$ in the presence and absence of EoWs. CaCl${}_2$, like KCl, can induce plasma membrane depolarization by activating l-type calcium channels, which causes muscle contraction [33, 34]. Furthermore, a voltage-gated calcium channel inhibitor is defined as a chemical that prevents these contractions. For this, verapamil (1 $\mu$M) was employed as a positive control. This chemical has been identified as a calcium channel blocker for l-type dependent VGC channels [35]. Our results indicate that EoWs prevented calcium entry through the VGC channels and, as a result, calcium release from the endoplasmic reticulum. In general, the chemical composition of aromatic and medicinal plants can fluctuate from year to year depending on climate, soil, altitude, and other factors. The antispasmodic activity of these essential oils can be attributed to one or more components, which can operate alone or in combination. For instance, other essential oils, such as Origanum majorana essential oil [16] containing terpinenes, terpineols, limonene, linalool and carvacrol, Artemisia herba-alba essential oil [21] containing camphor, terpinene, 1,8-cineol, $\alpha$- and $\beta$-pinene, or crude extract of Lavandula stoechas [36] are described for their spasmolytic activities.

We also explored whether the activity of our plant essential oil is mediated by nitric oxide (NO) or guanylate cyclase to better understand the mechanism of action. l-NAME (NG-nitro-l-arginine methyl ester) [37, 38] and methylene blue [39] were used as selective inhibitors of these pathways, respectively. The administration of both inhibitors had a significant effect on EoWs-induced relaxation. As a result of these observations, our extract is likely to function directly through the NO and guanylate cyclase signaling pathways.

This antispasmodic activity can be linked to the chemical components found in this essential oil that have spasmolytic properties, such as Cineole [40], 1-terpinen-4-ol [41], terpineols [42], and linalool [43]. It should also be mentioned that $\beta$-eudesmol, which is particularly abundant (15.8%) in our essential oil, stimulated small intestine motility and gastric emptying by inhibiting both the 5-HT 3 receptor and the dopamine D2 receptor [44]. To our knowledge, except our previous work conducted with the total aqueous extract [45], no study on the antispasmodic action of W. saharae essential oil has been done. Although the results of this study were comparable, far higher dosages (in mg of extract) were required to achieve comparable action, demonstrating that the essential oil fraction is certainly the most bioactive and allowing for a better understanding of the phytochemicals that may be responsible for this activity. Similarly, in another study, we observed that the essential oil fraction was more active than the aqueous extract of Origanum majorana [16]. The myorelaxing effect might be attributed to a variety of EoWs compounds that need to be identified. This action was most likely caused by different EoWs components acting independently or synergistically. Nonetheless, we cannot rule out the possibility that compounds derived from the aqueous extract of W. saharae might possibly explain its antispasmodic effect.

The antispasmodic activity of W. saharae essential oil (EoWs) on isolated rabbit and rat small intestine was here studied. The EoWs demonstrated a significant spasmolytic effect by acting on several pharmacological pathways such as NO, guanylate cyclase, VGC channels, and muscarinic receptors. These results support the traditional usage of this plant to treat intestinal disorders and will allow us to explore future prospects for the treatment of intestinal spasms using natural chemicals derived from this medicinal plant.

MA (Mohammed Aziz) conceived and designed research. OA, MM, HM conducted the experiments. OA, MA (Mohamed Addi), MA (Mohammed Aziz) wrote the manuscript. MA (Mohamed Addi), MM contributed to software analysis. CA, AK analyzed the data. CH, JTC critical analysis of the data, review and editing manuscript. All authors contributed to editorial changes in the manuscript.

The experiments were carried out ethically in conformity with the principles outlined in the Declaration of Helsinki, and the study was authorized by the Faculty of Sciences institutional review board in Oujda, Morocco (01/20-LBBEH-04 and 09/01/2020).

Mustapha Badraoui, Karim Ramdaoui, and Mohammed Joudar are acknowledged for technical support and animal breeding. The authors would like to thank the head of the chemistry department, Abdelmonaem Talhaoui of the Faculty of Sciences of Oujda (Mohamed Premier University).

This work was funded by the budget allocated to research at Mohamed the First University by the Ministry of National Education, Vocational Training, Higher Education and Scientific Research.

The authors declare no conflict of interest. JTC is serving as one of the Guest Editor of this journal. We declare that JTC had no involvement in the peer review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to GA.