Study of Antispasmodic and Antidiarrheal Activities of Tagetes lucida (Mexican Tarragon) in Experimental Models and Its Mechanism of Action

Tagetes lucida has been used in traditional medicine as a remedy to alleviate several gastrointestinal disorders that provoke stomachaches, abdominal cramps, and diarrhea. However, there is not enough scientific evidence that supports these effects. Therefore, the purpose of this study was to evaluate antispasmodic and antidiarrheal activities of aqueous extract of T. lucida (AqExt-TL) as well as its mechanism of action in experimental models. Antispasmodic activity and the mechanism of action of AqExt-TL were assessed on segments of the guinea pig ileum precontracted with KCl, acetylcholine (ACh), or electrical field stimulation (EFS). Furthermore, the antispasmodic effect of two coumarins (umbelliferone and herniarin) previously identified in this species was evaluated. Antidiarrheal activity of AqExt-TL was determined using the charcoal meal test in mice. AqExt-TL showed antispasmodic activity in segments of the guinea pig ileum precontracted with KCl (83.7 ± 1.9%) and ACh (77.2 ± 5.3%) at the maximal concentration; however, practically, it did not alter the contractions induced by EFS (10.1 ± 2.2%). Antispasmodic activity of AqExt-TL was not significantly altered by hexamethonium (a ganglionic blocker) or L-NAME (an inhibitor of nitric oxide synthase). However, this extract decreased the maximal contractile response to calcium (82.7 ± 8.5%), serotonin (68.1 ± 8.5%), and histamine (63.9 ± 5.9%) in their concentration-response curves. Umbelliferone and herniarin also induced an antispasmodic effect on tissues precontracted with KCl. In addition, low doses of AqExt-TL reduced to 50% the distance traveled by charcoal meal in the gastrointestinal transit model in mice as loperamide, an antidiarrheal agent, did. These results provided evidence of the antispasmodic and antidiarrheal activity of T. lucida, which supports its use in the folk medicine in relieving symptoms in some gastrointestinal disorders. In the antispasmodic effect, the blockade of histaminergic and serotoninergic pathway as well as the calcium channels seems to be involved. Finally, umbelliferone and herniarin could be partially responsible for the antispasmodic activity induced by T. lucida.


Introduction
Functional gastrointestinal disorders such as dyspepsia or irritable bowel syndrome cause stomachaches, abdominal cramps, and diarrhea. ese symptoms have been traditionally treated with herbal medicines all over the world [1,2]. Tagetes lucida Cav. (T. lucida), also known as "hierba de Santa María," "pericón," "hierbanis," "periquillo," "anicillo" or Mexican tarragon, is an endemic plant in Central America and Mexico that is prepared from fresh flowers or from aerial parts of the plant and orally consumed as infusion to alleviate stomachaches and diarrhea, mental agitation, or symptoms of a hangover, as well as infections caused by parasites [3].
Phytochemical studies of our group of work have identified coumaric components in the aqueous extract of this species using thin layer chromatography and Ultra-High-Performance Liquid Chromatography techniques [4]. Other studies have determined the presence of umbelliferone, herniarin, and scoparone among other coumarins [5,6]; in addition, some flavonoids such as quercetin, patuletin, rutin, isorhamnetin, quercetagetin, and naringenin have been identified [6][7][8].
Despite its use in gastrointestinal complaints in the traditional medicine, the only scientific study that supports its effectiveness in the intestinal smooth muscle was made with the chloroform extract T. lucida leaves. Authors reported that the chloroform extract reduced the contractile activity in the jejunum of rabbits [17]. However, although the aqueous infusion is popularly consumed, there is no evidence on the effect of the aqueous extract of T. lucida on the gastrointestinal system, let alone on the mechanism of action involved. For this, the purpose of this study was to evaluate the antispasmodic and antidiarrheal effects of aqueous extract of aerial parts of T. lucida in two experimental models and to investigate its mechanism of action. We also determined the antispasmodic effect of umbelliferone and herniarin as possible bioactive components of this species.

Plant Material and Preparation of Aqueous Extract of T. lucida (AqExt-TL).
e aerial parts of T. lucida Cav. (Asteraceae) were collected in Amatlán de Quetzalcóatl, State of Morelos, Mexico. e species identification was confirmed by taxonomist M.S. Abigail Aguilar Contreras. A voucher specimen (IMSSM No.15878) was deposited in the herbarium of the "Instituto Mexicano del Seguro Social" in Mexico City. e aqueous extract of Tagetes lucida (AqExt-TL) was obtained using the procedure described by Pérez-Ortega et al. [4]. Briefly, 220 g of dried aerial parts of T. lucida was added to 1000 mL of boiling water for 60 minutes. Infusion was filtered and lyophilized using a Labconco instrument. For the in vitro experiments, the AqExt-TL was prepared with saline solution (0.9%) before adding it to the organ bath to get 31.6, 100, 316, 562, and 1000 μg/mL. For the in vivo experiments, the AqExt-TL was also prepared with saline solution (0.9%) just before its administration.

Animals.
Eight adult male guinea pigs (300 g) and thirty adult male CD1 mice (25 g) were obtained from the Central Bioterium of the Faculty of Medicine of Universidad Nacional Autónoma de Mexico (UNAM) to determine the antispasmodic and antidiarrheal effect, respectively. Five days before the experiments, animals were kept in a room with temperature and humidity controlled (

Isolated Guinea Pig Ileum Preparations (In Vitro Model).
Antispasmodic activity of AqExt-TL was studied using isolated tissues from guinea pig ileum (in vitro experiments). e experiments were performed using ileum sections taken from eight guinea pigs under the procedure described by Ventura-Martinez et al. [21]. Briefly, guinea pigs were sacrificed by cervical dislocation. Approximately 15 cm of ileum was cut of each animal and placed in Krebs-bicarbonate solution (KBS) at 37°C with 95% O 2 and 5% CO 2 . e KBS had the following composition: 1.2 mM MgCl 2 , 1.2 mM NaH 2 PO 4 , 4.7 mM KCl, 2.5 mM CaCl 2 , 25 mM NaHCO 3 , 118 mM NaCl, 11 mM glucose, and 0.3 mM choline chloride. Six segments of 2 cm were obtained of each guinea pig's ileum and placed independently in organ-baths containing KBS at 37°C with 95% O 2 and 5% CO 2 . Segments were equilibrated for 60 minutes under 1 g resting tension. Ileum contractions were measured with force displacement transducers (Grass FT-03C) connected to a polygraph (Grass 7B) with a computerized system of data acquisition (PolyView system, version 2.5).

Antispasmodic Effect of AqExt-TL on the Guinea Pig
Ileum. After the period of stabilization, the antispasmodic activity of AqExt-TL or papaverine (30 μM), used as positive control, was assessed in six ileum segments precontracted with KCl (32 mM). Once the maximal contraction induced by KCl was reached, the corresponding concentration of AqExt-TL was added to the organ bath and remained in contact with the ileum segments for at least 5 minutes or until the relaxing effect obtained was maintained for at least 10 seconds. is procedure was repeated for each concentration of AqExt-TL (from 31.6-1000 μg/mL) and papaverine (30 μM). Between each concentration, the ileum segments were rinsed with 30-60 mL of warm KBS and were allowed to stabilize for 20 minutes, sufficient time to recover their spontaneous activity [22]. Concentrations of AqExt-TL were selected following logarithmic increments to cover the (CRC).
In a second set of experiments, other six ileum segments were contracted with electrical field stimulation (EFS) through two nickel electrodes parallelly positioned to the preparation and connected to a stimulator (Grass S88). e following parameters were used to produce the maximal contractile response: 0.3 Hz of frequency, 3 ms of duration, and 14 V of intensity. e ileum segments electrically stimulated were exposed by separation of cumulative concentrations of the AqExt-TL (from 31.6-1000 μg/mL). is procedure was repeated for papaverine (30 μM) [23]. Between AqExt-TL and papaverine, the ileum segments were rinsed with 30-60 mL of warm KBS and were allowed to stabilize for 20 minutes. e diminution of the contraction induced by KCl or electrical field stimulation (EFS) with each treatment was considered as antispasmodic activity and was compared with the effect induced by saline solution (VEH). e half maximal effective concentration (EC 50 ) of AqExt-TL on each experimental protocol was obtained.
In the other six ileum segments, cumulative (CRCs) from 1 × 10 −9 -1 × 10 −5 M to ACh were obtained in the presence of AqExt-TL (from 31.6-1000 μg/mL) or papaverine (30 μM), incubated for 20 minutes beforehand. Seven CDRs were obtained for each preparation. e ileum segments were rinsed with 30-60 mL of warm KBS and equilibrated by 20 minutes between each CRC. e effect of AqExt-TL or papaverine on contractile response induced by ACh was calculated with the maximal contractile response (E max ) obtained with ACh without treatment, which was considered 100% [24]. Also, the EC 50 values of ACh with VEH or with each treatment were compared.

Determination of the Mechanism of Action of the Antispasmodic Effect of AqExt-TL on the Guinea Pig Ileum.
e mechanism of the antispasmodic activity of AqExt-TL was investigated by exploring the participation of the nitric oxide (NO) pathway, nicotinic receptors, calcium channels, and serotoninergic or histaminergic pathway. To explore the participation of NO pathway or nicotinic receptors in the antispasmodic activity of T. lucida, six ileum segments were used. First, the ileum segments were incubated for 30 minutes with VEH (saline solution) or L-NAME (100 μM), an inhibitor of the NO synthase after obtaining a maximal contractile response with KCl (32 mM). After, the previously calculated EC 50 of AqExt-TL was added. is procedure was repeated but with the ileum segments incubated for 10 minutes with a ganglion blocker, hexamethonium (0.5 mM). e preparations were rinsed with 30-60 mL of warm KBS and were allowed to stabilize for 20 minutes before hexamethonium [21].
An independent group of six ileum segments were used to determine the involvement of the channel's calcium in the antispasmodic activity of T. lucida. For this, the segments were stabilized in Ca +2 -free KBS, and the CRCs to CaCl 2 (0.05-25 mM) were constructed in the absence or presence of AqExt-TL (31.6-1000 μg/mL) or a calcium channel blocker, verapamil (0.01 μM) [25]. Finally, to explore the participation of serotoninergic or histaminergic pathway, CRCs to serotonin (5-HT) or histamine were constructed in other six ileum segments in the absence or presence of the previously calculated EC 50 of AqExt-TL, ketanserin (2.5 μM, a 5-HT 2A antagonist), or pyrilamine (3 μM, an H 1 antagonist) [26]. As in the previous protocols, preparations were rinsed with 30-60 mL of warm KBS and equilibrated for 20 minutes between each CRC. e maximal response obtained with serotonin, histamine, or CaCl 2 , from their respective cumulative CRCs (in the absence of any treatment, VEH), was taken as 100% of the contractile response.

Determination of Antispasmodic Activity of Umbelliferone and Herniarin on the Guinea Pig Ileum.
Antispasmodic activity of umbelliferone (1-316 μg/mL) and herniarin (1-316 μg/mL) was assessed on independent groups of ileum segments (six per group) precontracted with KCl (32 mM). As in the previous protocols, preparations were rinsed with 30-60 mL of warm KBS and equilibrated for 20 minutes between every concentration of each coumarin. e diminution of the contraction induced by KCl with each treatment was considered as antispasmodic activity. Concentrations of each coumarin were selected following logarithmic increments to cover the concentrationresponse curve (CRC). e EC 50 of each coumarin was obtained.

Effect of AqExt-TL on the Gastrointestinal Transit in Mice (In Vivo Model).
e effect of AqExt-TL on the intestinal motility was determined using the charcoal meal test in mice (in vivo experiment) described by Khan et al. [27]. On day of the experiment, AqExt-TL (31.6, 100, or 316 mg/kg), loperamide (LOP 5 mg/kg, as the positive control), or vehicle (saline solution 0.9%) was orally administered to different groups of mice (6 animals per group). After thirty minutes, 0.3 mL of suspension of charcoal meal (10% charcoal in 5% gum Arabic) was given orally to the animals. After twenty minutes, mice were euthanized by cervical dislocation and Evidence-Based Complementary and Alternative Medicine the whole small intestines of animals were extracted from pylorus to the caecum. e distance between the pylorus region and the front of the charcoal meal as well the length of the small intestine was measured to obtain the percentage of gastrointestinal motility with each treatment. Doses of AqExt-TL were selected following logarithmic increments.

Statistical Analysis.
For the in vitro experiments, the effects of each treatment were analyzed using six ileum segments in each experimental protocol, and the results are expressed as the mean ± standard error of the mean (SEM). Statistical differences between treatments were determined using analysis of variance (ANOVA) by repeated measures followed by Dunnett's post hoc test in comparison to the effect of VEH in the corresponding protocol. Statistical differences between E max and EC 50 of umbelliferone and herniarin in their CRC were determined using Student's test. For the in vivo experiments, the effects of each treatment on the gastrointestinal motility were analyzed using six mice for each treatment, and the results are expressed as the mean ± SEM. Statistical differences between treatments were determined using one-way ANOVA followed by Dunnett's post hoc test in comparison to the effect of VEH. Statistical analysis was done with GraphPad Prism version 6 for Windows (GraphPad Software, San Diego, CA, USA). In all cases, data follow a normal distribution, and post hoc test was used only if F achieved P < 0.05 and there was no significant variance in homogeneity. Differences between means with a P < 0.05 were considered significant at the 95% confidence level.

Results
Regarding the preparation of AqExt-TL, the dry weight yield of the final crude aqueous extract was 8.94 g, which represented 4.08% of the vegetal material.

Antispasmodic Effect of AqExt-TL on the Guinea Pig Ileum and the Analysis of Its Mechanism of Action.
AqExt-TL diminished the contractile response obtained with KCl on the guinea pig ileum in a concentration-dependent manner from 100 μg/mL (at 79.7 ± 2.4%) in comparison to vehicle, reaching its maximal diminution at 1000 μg/mL (at 16.3 ± 1.9%). Under these circumstances, the EC 50 of the antispasmodic effect of AqExt-TL was 300 ± 27.8 μg/mL with an E max of 83.7 ± 1.9% of relaxation. e contractile response induced by EFS on the guinea pig ileum was significantly reduced only with 562 μg/mL of AqExt-TL (at 89.9 ± 3.3%). In both cases, papaverine (30 μM), the positive control, decreased almost completely the contractile effect induced by KCl or EFS on the guinea pig ileum (Figure 1(a)). Moreover, AqExt-TL shifted the CRC of ACh to the right in a concentration-dependent manner and increased the EC 50 of ACh at 30.0 ± 2.4 × 10 −6 M, 64.5 ± 2.8 × 10 −5 , and 55.2 ± 4.5 × 10 −5 M with 100, 316, and 562 μg/mL of the extract, respectively, in comparison with VEH (7.7 ± 0.1 × 10 −7 M). e maximal contraction to 1 × 10 −5 M of ACh on the guinea pig ileum was reduced in presence of 316, 562, and 1000 μg/mL of the AqExt-TL at 65.1 ± 5.2, 61.5 ± 7.4, and 22.8 ± 5.3%, respectively. In fact, the highest concentration of AqExt-TL (1000 μg/mL) produced the higher inhibitory effect on ACh-induced contraction, as papaverine did (Figure 1(b)).
In the experiments to explore the mechanism of action, the results showed that neither L-NAME nor hexamethonium significantly modified the antispasmodic effect induced by 300 μg/mL of AqExt-TL on tissues contracted with KCl (Figure 2(a)). However, the same concentration of AqExt-TL reduced the contractile response to CaCl 2 at 82.7 ± 8.5% at 25 mM, as verapamil did (at 22.6 ± 1.9%), but with much less intensity (Figure 2(b)); even though the extract did not displace the CRC to calcium, it did not change its EC 50 either in comparison with the VEH group (4.5 ± 1.4 vs 2.8 ± 1.05 mM). Finally, the AqExt-TL caused a rightward shift of both histamine (Figure 3(a)) and 5-HT (Figure 3(b)) CRCs with diminution of the maximum response to each agonist, like that caused by their antagonists (pyrilamine and ketanserin, respectively).

Antispasmodic Effect of Umbelliferone and Herniarin on the Guinea Pig Ileum.
Antispasmodic effects of umbelliferone and herniarin, two coumarins, were also determined in this study. Results showed that umbelliferone and herniarin induced an antispasmodic effect on the guinea pig ileum precontracted with KCl from 31.6 to 316.2 μg/mL with an E max of 95.2 ± 3.0% and 55.7 ± 10.1% and an EC 50 of 75.2 ± 1.1 and 160.1 ± 1.2 μg/mL, respectively (Figure 4). e E max and EC 50 values of both coumarins showed significant difference and, according to these, umbelliferone was more effective and potent than herniarin in inducing an antispasmodic effect.

Effect of AqExt-TL on Gastrointestinal Transit in Mice.
Finally, oral administration of low doses of AqExt-TL (31.6 and 100 mg/kg) in mice diminished the distance traveled of the charcoal through the small intestine in comparison to that of the VEH group (47.4 ± 7.3, 33.4 ± 3.8 vs 67.0 ± 4.8%, respectively), as loperamide did (18.7 ± 1.5%), an opioid agonist used as antidiarrheal ( Figure 5).

Discussion
Tagetes lucida Cav. is used in traditional medicine to treat several gastrointestinal affections in Mexico, Guatemala [28], and Egypt [29]. Even though it is consumed as aqueous infusion, there is low evidence on the effect of the aqueous extract of T. lucida on the gastrointestinal system. In this study, the antispasmodic and antidiarrheal effects of AqExt-TL were demonstrated using in vitro (guinea pig ileum) and in vivo (charcoal meal gut travel distance in mice) models, respectively. e maximum antispasmodic effect obtained with the AqExt-TL on the guinea pig ileum was independent of tissue relaxation capacity since papaverine (a positive control) completely relaxed tissues precontracted. Papaverine causes relaxation of smooth muscle by inhibition of the cyclic adenosine monophosphate (cAMP) phosphodiesterase, which results in an increase in intracellular cAMP. is event leads to a decrease in the myosin light chain phosphorylation and to an increase in actin depolymerization, thus preventing contraction. On the other hand, papaverine increases Ca 2+ uptake by the sarcoplasmic reticulum membrane of smooth muscle cells and consequently diminishes the intracellular calcium [30]. It has been traditionally used as reference smooth muscle relaxant to compare spasmolytic or antispasmodic efficacy of several treatments [21,22]. e antispasmodic effect of AqExt-TL obtained in this study is in agreement with the spasmolytic effect previously reported on the uterine contractility in rats [18]. Also, this effect could be related to the antinociceptive effect recently reported in a visceral pain model in mice, where a hydroalcoholic extract of T. lucida reduced contortions produced by acetic acid in mice [19]. Altogether, these results support its use in traditional medicine, where an infusion prepared with this species is used for stomach pain and diarrhea [2].
It is known that contractions induced by high concentrations of KCl are due to an increase in intracellular calcium by opening of L-type Ca 2+ voltage-dependent channels, which leads to the activation of the myosin light chain on the smooth muscle [30,31]. Meanwhile, ACh induces contraction by activation of muscarinic receptors (M 3 ) on the membrane of smooth muscle and the activation of phospholipase C. is generates inositol triphosphate (IP 3 ) that stimulates calcium release from the sarcoplasmic reticulum and induces an increase in the cytosolic levels of calcium [32]. Finally, the contractile effect of EFS is due to a release of ACh from myenteric neurons and its consequent bounding to M 3 receptors on the membrane of smooth muscle [33]. Our results suggest that the antispasmodic effect of T. lucida does not involve the release of ACh from myenteric neurons rather than a direct effect on smooth muscle cells. It was confirmed when the antispasmodic effect of T. lucida was not altered by hexamethonium, a ganglion blocker that inhibits release of ACh of the cholinergic terminals in enteric neurons [33]; this effect is not related to neurotransmitter release. Furthermore, the possible participation of nitric oxide (NO) pathway in the antispasmodic effect of T. lucida was investigated; for example, NO is considered a major inhibitory nonadrenergic and noncholinergic neurotransmitter in the gastrointestinal tract that induces relaxation of smooth muscle cells [34,35]. NO is synthesized from L-arginine by activation of NO synthase, and L-NAME, an inhibitor of NO synthase, inhibits the synthesis of this neurotransmitter. In this study, the antispasmodic effect induced by AqExt-TL was not significantly modified by L-NAME, suggesting that the NO pathway is not involved in this effect.
In addition, antispasmodic activity of T. lucida could partially involve a blocking of voltage-dependent calcium channels, since the AqExt-TL decreased the contractile response to calcium, like verapamil did (a calcium channel blocker), but with much less intensity.
is protocol has been used in several studies to determine the participation of calcium channels in the relaxing activity of several extracts [22,36]. Other neurotransmitters, as histamine and serotonin, could be involved in contractions of smooth muscle cells, and compounds that blocked their receptors could inhibit the contractile effect of these endogenous agonists [37]. In this study, the AqExt-TL caused a rightward shift of both histamine and 5-HT CRCs with diminution of the maximum response to each agonist, like that caused by their respective antagonists (pyrilamine for H 1 receptors and ketanserin for 5-TH 2A receptors) suggesting that part of the antispasmodic effect induced by AqExt-TL involves an antagonism on these receptors or a diminution in the release of these neurotransmitters from myenteric neurons.
On the other hand, our results showed that both umbelliferone and herniarin, two coumarins previously identified from the aerial parts of T. lucida [5,6], induced an antispasmodic effect on the guinea pig ileum; this suggests that both coumarins could be involved in the antispasmodic effect induced by this specie. is is not the first study where coumarins seem to be related to a relaxant effect. Oliveira [38] reported the spasmolytic effect of scopoletin, a coumarin isolated from Brunfelsia hopeana, but this is the first report on the spasmolytic effect of umbelliferone and herniarin. In fact, the antispasmodic effect of these compounds could be related to the antinociceptive effect recently reported on T. lucida in a visceral model pain, where one coumarin seems to be responsible for its antinociceptive effect [39]. Furthermore, other studies have related the biological effects of T. lucida to the presence of several coumarins such as esculetin, umbelliferone, scopoletin, 7-8dihydroxycoumarin, and 6-hydroxy-7-methoxycoumarin [7]. Several flavonoids have been also identified in polar extracts of T. lucida such as patuletin, isorhamnetin, quercetin, rutin, quercetagetin, and naringenin [6][7][8]. In fact, in a previous report of our group of investigation, quercetin and rutin found in another species of Tagetes, Tagetes erecta, showed spasmolytic activity [40]. e presence of quercetin in extracts of T. lucida suggests that it also could be one of the metabolites involved in its antispasmodic effect; previous reports indicate that the spasmolytic effect of quercetin is due to a blockade of voltage-dependent calcium channels of the guinea pig ileum [41,42], a mechanism studied in this work that seems to be involved in the antispasmodic effect of AqExt-TL.
Finally, the relaxation of the gastrointestinal tract shown by AqExt-TL was according to the inhibition of the propulsion of the charcoal meal in mice due to a reduction in gastrointestinal motility, as our results show. is inhibition of charcoal meal gut travel distance in healthy mice induced by AqExt-TL was similar to the effect induced by loperamide, an antidiarrheal drug. Loperamide, an opiate analogue, is a widely used as an antidiarrheal agent that binds to the opioid receptor in the gut wall [43]. is results in inhibition of ACh and prostaglandin release from myenteric neurons, reducing propulsion peristalsis [43]. e reduction in the intestinal motility induced by T. lucida could be related to its antidiarrheal effects reported in traditional medicine [2]. However, it is necessary to do more experiments in other in vivo models to demonstrate this activity as well as the mechanism involved. Data from this study give evidence of some bioactive metabolites of T. lucida as partially responsible for its medicinal properties in gastrointestinal affections. It is known that a standardized herbal extract provides a specific concentration of one or more chemical compounds. is fact is important since a specific bioactive metabolite can be characterized, which sometimes allows also knowing targets involved in a selective mechanism of action. A disadvantage of standardization is that it may concentrate one constituent at the expense of other potentially important ones changing the natural balance of the plant's components. In contrast, a full-spectrum extract, like the one used in this study, includes all the plants' chemical or bioactive compounds that may likely attribute the pharmacological activity by interactions between constituents that might be not fully explored. ey promote phytochemical arrays with internal complexity working together. It is likely the most common condition in the effect of medicinal plants use in folk medicine.

Conclusions
In summary, our study provides evidence that the aqueous extract of T. lucida aerial parts possesses antispasmodic activity mediated through an antagonism on histaminergic and serotoninergic receptors as well as blocking calcium channels, but it does not involve nicotinic receptors or nitric oxide pathway. is, together with the decrease in intestinal motility induced by the extract in mice, supports the use of T. lucida aerial parts in traditional Mexican medicine to treat colic and diarrhea involved in several gastrointestinal disorders. Umbelliferone and herniarin were bioactive compounds partially involved in the antispasmodic effect induced by the aqueous extract of T. lucida.

Abbreviations
ACh: Acetylcholine ANOVA: Analysis of variance AqExt-TL: Aqueous extract of T. lucida CaCl 2 : Calcium chloride CRC: Concentration-response curve EFS: Electrical field stimulation EC 50 : Half maximal effective concentration KBS: Krebs-bicarbonate solution LOP: Loperamide NO: Nitric oxide KCl: Potassium chloride SEM: Standard error of the mean E max : e maximal contractile response VEH: Vehicle.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.