Antispasmodic Effect of Valeriana pilosa Root Essential Oil and Potential Mechanisms of Action: Ex Vivo and In Silico Studies

Infusions of Valeriana pilosa are commonly used in Peruvian folk medicine for treating gastrointestinal disorders. This study aimed to investigate the spasmolytic and antispasmodic effects of Valeriana pilosa essential oil (VPEO) on rat ileum. The basal tone of ileal sections decreased in response to accumulative concentrations of VPEO. Moreover, ileal sections precontracted with acetylcholine (ACh), potassium chloride (KCl), or barium chloride (BaCl2) were relaxed in response to VPEO by a mechanism that depended on atropine, hyoscine butylbromide, solifenacin, and verapamil, but not glibenclamide. The results showed that VPEO produced a relaxant effect by inhibiting muscarinic receptors and blocking calcium channels, with no apparent effect on the opening of potassium channels. In addition, molecular docking was employed to evaluate VPEO constituents that could inhibit intestinal contractile activity. The study showed that α-cubebene, β-patchoulene, β-bourbonene, β-caryophyllene, α-guaiene, γ-muurolene, valencene, eremophyllene, and δ-cadinene displayed the highest docking scores on muscarinic acetylcholine receptors and voltage-gated calcium channels, which may antagonize M2 and/or M3 muscarinic acetylcholine receptors and block voltage-gated calcium channels. In summary, VPEO has both spasmolytic and antispasmodic effects. It may block muscarinic receptors and calcium channels, thus providing a scientific basis for its traditional use for gastrointestinal disorders.


Introduction
Gastrointestinal disorders are related to motility disturbances, visceral hypersensitivity, altered mucosal and immune functions, gut microbiota dysbiosis, and impaired regulation by the central nervous system [1]. Gastrointestinal dysfunction is associated with significant global healthcare costs [2,3] and decreased quality of life [4]. Existing synthetic antispasmodic drugs may cause unpleasant side effects such as dizziness, blurred

Animals
The experiments in this study were performed following the procedures of the American Veterinary Medical Association (AVMA) [21] and the Ethics Committee of Pharmacy and Biochemistry Faculty of the National University of Trujillo (COD.N • : P 012-19/CEIFYB). Twenty male rats (8-10 weeks old Rattus norvegicus Holtzman, 170-200 g) were housed in cages (22-25 • C, 12 h light/dark cycles) with ad libitum access to standard rat chow (Molinorte S.A.C., Trujillo, Peru) and water.

Preparation of Rat Ileum
Animals were sacrificed by cervical dislocation. A portion of the ileum (2.5 cm), without considering the 10 cm nearest to the ileocecal valve, was removed and placed into a petri dish containing Tyrode's solution (concentrations in mM): NaCl 136.9; KCl 2.68; CaCl 2 1.8; MgCl 2 1.05; NaHCO 3 11.9; NaH 2 PO 4 0.42; and D-glucose 5.55 [22]. Ileum fragments were placed into an isolated organ chamber filled with 25 mL of Tyrode s solution. The chamber was kept at 37 • C continuously gassed with a mixture of 95% O 2 and 5% CO 2 (pH 7.4). Resting tension was fixed at 1 g. The experimental data were recorded using a Power Lab 26T system (AD-Instruments Pty Ltd., Bella Vista, NSW, Australia) with the LabChart 8 program for Windows (Colorado Springs, CO, USA).

Ex Vivo Experimental Protocol 2.6.1. Effect of VPEO on the Basal Tone of Rat Ileum
The contractility of the rat ileum in response to VPEO was assessed through cumulative dose-response experiments. A series of seven distinct concentrations of VPEO (1, 10, 100, 250, 500, 750, and 1000 µg/mL) were administered in 5 min intervals [22].

Antispasmodic VPEO Effect in the Dose-Response Curves of ACh, KCl, and BaCl 2
The effect of VPEO on the contractibility in response to ACh was assessed as follows: dose-response experiments (ACh, 10 −10 to 10 −3 M) before and after VPEO (250 and 500 µg/mL) administration were performed in the same experiment. Similar experiments to those described above but contracting the ileal sections with KCl (10 −4 to 10 −1 M) or BaCl 2 (10 −8 to 10 −2 M) were performed.
Dose-response data were fitted to the standard Hill's function using Graphpad.

Role of Extracellular Ca 2+ Influx in the Intestinal VPEO-Mediated Relaxation
In addition to the Tyrode s solution, a calcium-free solution was prepared for this experiment. The solution had the following composition (concentrations in mM): KCl 50; NaCl 91.04; MgCl 2 1.05; NaHCO 3 11.87; NaH 2 PO 4 0.41; glucose 5.55; and EDTA 0.1 [23]. Initially, the tissue was stabilized in normal Tyrode s solution and then replaced with a Ca 2+ -free Tyrode s solution. Then, 10 min after the addition of calcium-free solution, the intestinal sections were contracted with ACh 10 −5 M, followed by the addition of increasing CaCl 2 concentrations (0.1 mM; 0.3 mM; 0.6 mM; and 1 mM). Then, the tissue was washed with normal Tyrode's solution for at least 10 min, followed by the addition of VPEO (250 and 500 µg/mL) for 20 min [22].
The tension was re-adjusted in the middle of the experiment to 1 g when necessary [22].

Effects of VPEO on Voltage-Gated Calcium Channel
To determine whether the effect of VPEO was related to the inhibition of voltage-gated calcium channel, experiments were performed in the absence and presence of verapamil (a voltage-gated calcium channel blocker). Initially, the tissue was stabilized for 1 h and then washed in 15 min intervals (4-5 times). The tension was adjusted again to 1 g if necessary.

Effects of VPEO on Potassium Channel Blockers
The role of K + channels in VPEO-induced relaxation was investigated by pre-incubating the ileum rat for 20 min with two K + channel blockers, namely, glibenclamide [28] (ATP sensitive K + channel blocker), and barium chloride [29] (an inward rectifier K + channel blocker). The tissue was stabilized for 1 h and then washed in 15 min intervals (4-5 times). The tension was adjusted again to 1 g if necessary. The tissue was pre-incubated with glibenclamide (10 µM) and barium chloride (1 mM) for 20 min. Then, VPEO was added (1, 10, 100, 250, 500, 750, and 1000 µg/mL) in intervals of 5 min for each successive dose, and the response was recorded.

In Silico Studies
Molecular modelling analysis of compounds 1-47 (see the SMILES in supplementary materials) of VPEO [20] to M 2 Muscarinic Acetylcholine Receptor [30], M 3 Muscarinic Acetylcholine Receptor [31], and Ca v 1.2 L-type voltage-gated calcium channel [32] were performed using AutoDock (v 4.2.1), AutoDock Vina (v 1.0.2) [33], and AutoDockTools packages [34]. The crystal structures considered for these docking studies had the following PDB Codes: 3UON (M 2 Muscarinic Acetylcholine Receptor), 4DAJ (M 3 Muscarinic Acetylcholine Receptor), and 5V2P (L-type voltage-gated calcium channel). Data was obtained from the Protein Data Bank [35]. The three-dimensional coordinates of all structures were optimized using MOPAC2016 software by the PM6-D3H4 semi-empirical method [36,37]. The crystal structures were treated with Schrödinger's Protein Preparation Wizard [38]; polar hydrogen atoms were added, nonpolar hydrogen atoms were merged, and charges were assigned. Docking was treated as rigid and performed using the empirical free energy function and Lamarckian Genetic Algorithm provided by AutoDock Vina [39]. The grid map dimensions were 20 × 20 × 20 Å 3 . The center of the binding site were the following coordinates for each of the proteins studied (Table 1). All other parameters were set to their default values as defined by AutoDock Vina. Dockings were repeated 10 times, with the space search exhaustiveness set to 100. The best interaction binding energy (kcal·mol −1 ) was selected for evaluation. Docking results in 3D representations were obtained using Discovery Studio [40] 3.1 (Accelrys, San Diego, CA, USA) molecular graphics system. The co-crystallized ligands were removed from their proteins and saved separately in the PDB format, which was used for redocking their respective protein active domains to validate our docking methodology [20].

Statistical Analysis
GraphPad Prism 8.0.2 software (San Diego, CA, USA) was used for the statistical analyses. Non-linear regression analysis (three-parameter Hill function) was generated to compare dose-response curves. Two-way ANOVA analysis followed by the Bonferroni post hoc test was used to evaluate the significance between different groups.

Extracellular Ca 2+ Dependence of VPEO Effect
The relation between the relaxant effect of VPEO (250 and 500 µg/mL) and extracellular calcium was assessed. The contraction induced by the cumulative concentration of extracellular Ca 2+ ions (0 to 1 mM) in ileum rats precontracted with ACh (10 −5 M) maintained in Ca 2+ -free Tyrode's solution (containing 0.1 mM EDTA) in the presence and absence of VPEO was determined ( Figure 5A). The cumulative addition of calcium ions in Ca 2+ -free Tyrode increased in the contraction of ileal sections in a concentrationdependent manner. Pre-treatment with VPEO at concentrations of 500 µg/mL (171 ± 29.1% vs. 249.1 ± 23.8 control; p < 0.01) significantly decreased the contraction induced by CaCl 2 0.6 mM. However, the concentration-response curve produced by the different concentrations of calcium ions was significantly decreased in the presence of VPEO compared to the control ( Figure 5B). Ca 2+ -free Tyrode increased in the contraction of ileal sections in a concentration-dependent manner. Pre-treatment with VPEO at concentrations of 500 µg/mL (171 ± 29.1% vs. 249.1 ± 23.8 control; p < 0.01) significantly decreased the contraction induced by CaCl2 0.6 mM. However, the concentration-response curve produced by the different concentrations of calcium ions was significantly decreased in the presence of VPEO compared to the control ( Figure 5B).  The relaxant effect produced by VPEO was decreased in the presence of verapamil 1 µM (p < 0.05, Figure 5C). For instance, relaxation induced by VPEO in ileal sections treated with 100 µg/mL VPEO was 29.37 ± 4.82 vs. 6.73 ± 1.16, control vs. verapamil, respectively. Taken together, the results suggest that essential oils might decrease contraction by reducing the entry of calcium ions into smooth muscle cells produced by extracellular calcium.

Effect of Two K + Channel Blockers on the Relaxation in Precontracted Ileum
The antispasmodic effect of VPEO in the rat ileum pre-incubated for 20 min with two K + channel blockers, glibenclamide (an ATP-sensitive K + channel blocker, 3 µM) and barium chloride (inward rectifier K + channel blocker, 1 mM) was studied. The relaxation induced by VPEO in the rat ileum was unaffected by glibenclamide ( Figure 6A) or BaCl 2 ( Figure 6B) treatment. Therefore, we conclude that Valeriana pilosa root essential oil does not induce opening of potassium channels in rat ileum.
The antispasmodic effect of VPEO in the rat ileum pre-incubated for 20 min with two K⁺ channel blockers, glibenclamide (an ATP-sensitive K⁺ channel blocker, 3 µM) and barium chloride (inward rectifier K⁺ channel blocker, 1 mM) was studied. The relaxation induced by VPEO in the rat ileum was unaffected by glibenclamide ( Figure 6A) or BaCl2 ( Figure 6B) treatment. Therefore, we conclude that Valeriana pilosa root essential oil does not induce opening of potassium channels in rat ileum.
Molecular docking was performed to identify possible VPEO constituents that could inhibit some proteins involved in intestinal contractile activity. Based on the results obtained in the current study, we selected M2 and M3 Muscarinic Acetylcholine Receptor and Cav1.2 (L-type voltage-gated calcium channel). Table 2 shows a heat map of intermolecular docking energy values of 47 VPEO compounds. The values appear in a three-color scheme (red-yellow-green), where red represents the most stable binding energies and green represents the least stable ones. Table 2 shows a clear trend of compounds acting as putative inhibitors of the M2 and M3 muscarinic acetylcholine receptors.
Molecular docking was performed to identify possible VPEO constituents that could inhibit some proteins involved in intestinal contractile activity. Based on the results obtained in the current study, we selected M 2 and M 3 Muscarinic Acetylcholine Receptor and Ca v 1.2 (L-type voltage-gated calcium channel). Table 2 shows a heat map of intermolecular docking energy values of 47 VPEO compounds. The values appear in a three-color scheme (redyellow-green), where red represents the most stable binding energies and green represents the least stable ones. Table 2 shows a clear trend of compounds acting as putative inhibitors of the M 2 and M 3 muscarinic acetylcholine receptors.
Based on the information shown in Table 2, M 2 and M 3 muscarinic acetylcholine receptors appeared to be the best protein targets for some VPEO constituents, as shown by their intermolecular docking energy (∆E binding ), score normalization of the binding energy based on the number of non-hydrogen atoms (IE norm.binding ), and Ligand Efficiency (LE) values. Indeed, using the weighted arithmetic mean values (related to % of composition, see supplementary materials) shown in Table 3 obtained for all compounds, binding values were used for the analysis of M 2 muscarinic acetylcholine receptor: ∆E binding , LE, and IE norm.binding values were −7.67, 0.55, −2.06 kcal·mol −1 , and the M 3 muscarinic acetylcholine receptor, the ∆E binding , LE, and IE norm.binding were −8.03, 0.58, and −2.15 kcal·mol −1 , respectively, while, in comparison with the Ca v 1.2 (L-type voltage-gated calcium channel) protein target, the ∆E binding , LE, and IE norm.binding , binding values were −5.11, 0.37, and −1.37 kcal·mol −1 . Therefore, these results show that the M 3 muscarinic acetylcholine receptor, as a target protein, is probably involved in the effects of VPEO, which means that they might be involved in the anti-spasmodic activities of VPEO compounds. Values are listed as a three-colored scheme from red (high energy) to green (low energy).  Table 4 for the top 12 (M 2 Muscarinic Acetylcholine Receptor) and 11 (M 3 Muscarinic Acetylcholine Receptor) compounds, of which the most abundant compounds found in the VPEO are presented. The results showed that approximately 60% of the VPEO compounds acted as potential blockers of M 2 and M 3 Muscarinic Acetylcholine Receptors. In contrast, the others showed higher values and were considered weak blockers with low activity for the other protein target Ca v 1.2 (L-type voltage-gated calcium channel). VPEO compounds obtained from molecular docking (see Table 4) displaying more binding potential to M 2 Muscarinic Acetylcholine Receptor were β-patchoulene, β-caryophyllene, γ-muurolene, and eremophyllene, while for the M 3 Muscarinic Receptor were α-cubebene, β-patchoulene, β-bourbonene, β-caryophyllene, α-guaiene, αhumulene, γ-muurolene, valencene, and δ-cadinene, all obtained ∆E binding values (<−9.0) and IE norm, binding values (<−2.2), representing those with the highest interaction with residues close to the active site. In addition, the binding energies were evaluated for the major VPEO compounds, including natural sesquiterpenes such as α-patchoulene (5.8%), α-humulene (6.1%), seychellene (7.6%), and patchoulol (20.8%).(Detailed values of the interactions between individual compounds of the essential oil and the mentioned targets can be found supplementary materials) Table 5 lists the non-covalent interactions present in the M 2 -ligand complex. The four best compounds, β-patchoulene, β-caryophyllene, γ-muurolene, and eremophyllene, identified by molecular docking presented weak Van der Waals-type and π-alkyl interactions (see Table 5 and Figure 7) with the binding site of M 2 , where the most representative residues were Ala191, Ala194, Cys429, Phe181, Trp155, Trp400, Tyr104, and Tyr403. On the other hand, for the major VPEO compounds (see Table 5 and Figure 7), α-patchoulene, α-humulene, seychellene, and patchoulol had similar interactions of weak Van der Waalstype and π-alkyl interactions with the binding site of M 2 , where the most representative residues of these interactions are Ala191, Ala194, Phe181, Trp155, Trp400, Tyr104, and Tyr403. However, patchoulol has a hydrogen bridge interaction between the hydroxyl group and Asn404, forming a favorable interaction in the binding of this compound by M 2 . Table 5. Amino acid residues of M 2 Muscarinic Acetylcholine Receptor (M 2 R) and hydrogen bonding with the VPEO molecules within a distance of 3.5 Å. To extend the analysis, we focused on all possible protein-ligand interactions in the set of the best eight (identified by molecular docking) and the major VPEO compounds that interact with the M 3 Muscarinic Acetylcholine Receptor binding site (Table 6). These interactions define an interaction framework for the ligands in the analyzed set.  Figure 7. Non-covalent interactions analysis for the best four compounds (β-patchoulene, β-caryophyllene, γ-muurolene, and eremophyllene,), and major constituents of the molecular docking of VPEO (α-patchoulene, α-humulene, seychellene, patchoulol) bound to M2 muscarinic acetylcholine receptor.

Discussion
Essential oils are gaining interest due to their intricate chemical composition and diverse pharmacological effects. Among these actions, the antispasmodic effect is well known, although further research is required to better understand the cellular and molecular mechanisms of action [41]. Therefore, we studied the antispasmodic effect of Valeriana pilosa root essential oil (VPEO) and its underlying mechanisms of action using the isolated rat ileum ex vivo model. Our findings reveal that VPEO displays spasmolytic and antispasmodic effects in a dose-dependent manner at concentrations ranging from 100 to 1000 µg/mL. The relaxant activity of VPEO in the isolated rat ileum was reversible after washing, indicating that the inhibition observed was not attributable to intestinal damage caused by the oil's interaction with cell lipid bilayers [42]. . Non-covalent interactions analysis for the best nine compounds (α-cubebene, βpatchoulene, β-bourbonene, β-caryophyllene, α-guaiene, α-humulene, γ-muurolene, valencene, and δ-cadinene), and major (α-patchoulene, α-humulene, seychellene, patchoulol) constituents of the molecular docking of VPEO bound to M 3 muscarinic acetylcholine receptor.

Discussion
Essential oils are gaining interest due to their intricate chemical composition and diverse pharmacological effects. Among these actions, the antispasmodic effect is well known, although further research is required to better understand the cellular and molecular mechanisms of action [41]. Therefore, we studied the antispasmodic effect of Valeriana pilosa root essential oil (VPEO) and its underlying mechanisms of action using the isolated rat ileum ex vivo model. Our findings reveal that VPEO displays spasmolytic and antispasmodic effects in a dose-dependent manner at concentrations ranging from 100 to 1000 µg/mL. The relaxant activity of VPEO in the isolated rat ileum was reversible after washing, indicating that the inhibition observed was not attributable to intestinal damage caused by the oil's interaction with cell lipid bilayers [42].
Smooth muscle excitation and contraction in response to acetylcholine (ACh) release from autonomic nerves primarily involve the activation of muscarinic acetylcholine receptors (mAChRs) in the gastrointestinal tract and many other visceral organs [43]. The mAChR family comprises five molecularly distinct subtypes: M 1 -M 5 [44]. Smooth muscle mAChRs consist of M 2 and M 3 subtypes, with M 2 being predominant (M 2 :M 3 = 3-5:1) [43,45], although all five mAChR subtypes have been detected in the gastrointestinal smooth muscle at the mRNA level [46].
Activation of mAChRs triggers multiple biochemical and electrical signaling events that result in muscle contraction [47]. Our results demonstrated that VPEO, at a concentration of 250 µg/mL, reduced tonic contraction in ACh-precontracted tissues. Additionally, preincubation of intestinal tissues with VPEO (250 µg/mL) significantly reduced the pEC 50 of the tissue to ACh, as evidenced by a rightward shift in the dose-response curve. These initial findings suggest that VPEO blocks muscarinic receptors involved in rat ileum contraction. To explore this hypothesis, we evaluated whether the presence of muscarinic antagonists such as atropine, hyoscine butylbromide, and solifenacin would attenuate the relaxing effect of VPEO. Our results showed that solifenacin, a selective blocker of M 3 muscarinic receptors, slightly reduced the relaxation induced by VPEO, but not significantly. In contrast, hyoscine butylbromide, an M 2 -M 3 muscarinic blocker, significantly reduced the relaxing effect of VPEO. Moreover, when the non-selective muscarinic blocker atropine was employed, the relaxing effect of VPEO on muscle tone in the rat ileum was further reduced. Traditional studies using various mAChR antagonists have suggested that the M 3 subtype primarily mediates contraction in visceral smooth muscles, while the contribution of the M 2 subtype remains less clear [48,49]. However, other investigations have demonstrated that the M 2 subtype modulates contraction, at least in part, by inhibiting cyclic AMP (cAMP)-dependent relaxation [43] and regulating smooth muscle ion channel activity [50][51][52]. Nevertheless, the precise mAChR subtype that mediates the contractile response remains largely unknown. Our findings indicate that, in addition to blocking M 3 receptors, the antispasmodic effect of VPEO also involves modulation of M 2 receptors. Consequently, both experimental and theoretical outcomes reveal the impact of VPEO on M 2 and M 3 muscarinic receptors.
High concentrations of potassium ions (hypermolar KCl) cause tonic contraction of smooth intestinal muscles by a mechanism that depends on extracellular calcium influx [56,57]. Our study demonstrated that increasing concentrations of VPEO reduced contractions induced by 60 mM KCl. Additionally, when the tissue was pre-incubated for 20 min with the essential oil, the dose-response curves in response to KCl were significantly reduced. The action of VPEO can be attributed to its major component, patchouli alcohol (20.8%), a tricyclic sesquiterpene that has displayed similar results in other types of smooth muscle, such as the aorta [58] and rat corpus cavernosum [59]. However, the synergistic effects of other VPEO components, such as α-patchoulene (5.8%), α-humulene (6.1%), and seychellene (7.6%), at lower concentrations cannot be rejected.
Furthermore, it was confirmed that the relaxation induced by VPEO was reduced in the presence of verapamil, a voltage-gated calcium channel blocker. These results are consistent with similar studies conducted on the rhizome of Valeriana hardwickii Wall, which caused a rightward shift in calcium concentration-response curves in the rabbit jejunum, similar to that caused by verapamil [60,61]. The findings from our study on VPEO suggest the presence of an antispasmodic effect potentially mediated through calcium channel blockade.
It has been shown that ATP-sensitive potassium (K ATP ) channels regulate intestinal contractility [28]. These channels are heterooctameric proteins composed of pore-forming subunits of the inwardly rectifying K + channel (K IR ) and subunits of the sulfonylurea regulatory receptor (SUR). The data demonstrate decreased intrinsic basal contractility in the small intestine and colon due to increased basal K ATP channel activity, which can be inhibited by glibenclamide, an ATP-sensitive K + channel blocker [62], and BaCl 2 , an inward rectifier K + channel blocker [63,64]. We observed that VPEO reduced BaCl 2 -induced contractions, shifting the dose-response curve to the right, suggesting that VPEO might activate K + channels, similar to the crude extract of Valeriana wallichii, which displays antispasmodic activity mediated by activation of the K ATP channel [65]. To confirm these results, tissues were pre-incubated with glibenclamide and BaCl 2 , and the relaxing effect of VPEO on basal tone was evaluated. Our results showed that the activation of K ATP and K IR channels did not produce the relaxation exhibited by VPEO in the rat ileum.
It is important to mention that several studies have shown that phytochemical analysis of essential oils from numerous plants can induce relaxation in smooth muscle across various animal species, which is often attributed to their antioxidant activity and ability to enhance gut and barrier function in animals [6,49,67]. Terpenes have been demonstrated to enhance gut microbiota, such as D-limonene [68,69], suggesting that this could be a possible explanation for how essential oils produce a calming effect on the gut.
In summary, our study provides experimental evidence showing antispasmodic properties of VPEO. Future in vivo experiments evaluating the effect of VPEO on the intestinal microbiota or determining optimal dosage are required to obtain more clues into the potential use of VPEO as a new pharmacological approach for treating muscle spasms.

Conclusions
The findings obtained from this study on Valeriana pilosa essential oil (VPEO) unequivocally demonstrated its spasmolytic and antispasmodic effects in rat ileum. In silico and pharmacological analyses suggest that VPEO exerts its actions through blockade of muscarinic receptors and calcium channels. Collectively, these outcomes provide molecular insights that shed light on the traditional utilization of this plant for the treatment of gastrointestinal disorders.