Ximenia americana L.: Chemical Characterization and Gastroprotective Effect

: Ximenia americana L., popularly known in Brazil as “ameixa do-mato, ameixa-brava, and ameixa-do-sert ã o,” is widely used in folk medicine to treat several intestinal disorders. The present study assessed the potential mechanisms of action underlying the gastroprotective activity of the hydroethanolic extract of Ximenia americana L. (EHXA) stem bark. The acute toxicity of EHXA was estimated, and later, the gastroprotective effect in mice was assessed through acute models of gastric lesions induced by acidiﬁed or absolute ethanol and indomethacin, where the following mechanisms were pharmacologically analyzed: the involvement of prostaglandins (PG), histamine (H 2 ) receptors, ATP-dependent potassium channels, sulfhydryl groups (SH), α 2 adrenergic receptors, nitric oxide (NO), myeloperoxidase (MPO), gastric mucus production, and acetylcholine-mediated intestinal motility. Regarding toxicity, EHXA did not cause deaths or signs of toxicity (LD 50 greater than or equal to 2000 mg/kg/p.o.). When the gastroprotective effect was assessed, EHXA (50, 100, and 200 mg/kg/p.o.) reduced the rate of lesions induced by acidiﬁed ethanol by 65.63; 53.66, and 58.02% in absolute ethanol at 88.91, 78.82, and 74.68%, respectively, when compared to the negative control group. In the indomethacin-induced gastric injury model, the reductions were 84.69, 55.99, 55.99, and 42.50%, respectively. The study revealed that EHXA might stimulate mucus production and reduce intestinal motility through SH groups, NO production, and activation of α 2 adrenergic receptors. The results indicated that EHXA had signiﬁcant gastroprotective activity in the evaluated models. However, further investigation is required to elucidate the cellular and molecular events underlying the action of EHXA components and to correlate them with the modulation of the signaling pathways, as demonstrated by the current pharmacological approach. Therefore, the results demonstrated in the present study, as well as previously reported ﬁndings, support the recommendation of using this species in traditional communities in Brazil.


Results
The chemical composition of the ethanolic extract of X. americana bark (EHXA) was determined using qualitative and quantitative analyses. Qualitative analysis revealed the presence of various metabolite classes, including phenols, anthocyanins, anthocyanidins, flavonols, chalcones, aurones, leucoanthocyanidin, catechins, and steroids [15,31]. In previous research, our group reported the quantitative chemical profile of polyphenols in EHXA using the HPLC-DAD method, which identified significant compounds such as caffeic acid and quercitrin, as well as other phenolic derivatives, including gallic acid, chlorogenic acid, ellagic acid, and flavonoids such as catechin, rutin, quercetin, and kaempferol [31,32].
2.1. Acute Toxicity and Screening for the Gastroprotective Activity of EHXA 2.1.1. Non-Clinical Acute Oral Toxicity-LD 50 The treatment with EHXA (2000 mg/kg) caused no deaths or signs of toxicity in orally treated animals, indicating that the extract has no significant toxicity in vivo.

Acute Gastric Injuries Induced by Acidified Ethanol
The groups treated with EHXA (50, 100, and 200 mg/kg) and omeprazole (30 mg/kg) showed significantly reduced development of ulcerative lesions induced by acidified ethanol (by 65.63%, 53.66%, 58.02%, and 83.82%, respectively) when compared to the negative control group ( Figure 1A), demonstrating the anti-ulcerative potential of the extract.

Results
The chemical composition of the ethanolic extract of X. americana bark (EHXA) was determined using qualitative and quantitative analyses. Qualitative analysis revealed the presence of various metabolite classes, including phenols, anthocyanins, anthocyanidins, flavonols, chalcones, aurones, leucoanthocyanidin, catechins, and steroids [15,31]. In previous research, our group reported the quantitative chemical profile of polyphenols in EHXA using the HPLC-DAD method, which identified significant compounds such as caffeic acid and quercitrin, as well as other phenolic derivatives, including gallic acid, chlorogenic acid, ellagic acid, and flavonoids such as catechin, rutin, quercetin, and kaempferol [31,32].

Non-Clinical Acute Oral Toxicity-LD50
The treatment with EHXA (2000 mg/kg) caused no deaths or signs of toxicity in orally treated animals, indicating that the extract has no significant toxicity in vivo.

Acute Gastric Injuries Induced by Acidified Ethanol
The groups treated with EHXA (50, 100, and 200 mg/kg) and omeprazole (30 mg/kg) showed significantly reduced development of ulcerative lesions induced by acidified ethanol (by 65.63%, 53.66%, 58.02%, and 83.82%, respectively) when compared to the negative control group ( Figure 1A), demonstrating the anti-ulcerative potential of the extract. . The values are the means ± S.E.M (standard error of the mean); n = 6 animals/group. One-way (ANOVA) was used, followed by Tukey's test. ** p < 0.01, **** p < 0.0001, compared to the negative control group.

Indomethacin-Induced Acute Gastric Lesions
Considering the importance of non-steroidal anti-inflammatory drugs (NSAIDs) in the development of gastric lesions, we evaluated the effects of EHXA on indomethacin-induced acute gastric lesions. The treatment with EHXA (50, 100, and 200 mg/kg) and omeprazole (30 mg/kg) significantly inhibited the development of ulcerative lesions (by 55.99%, 55.99%, 42.50%, and 84.69%, respectively) when compared to the negative control group ( Figure 1C), confirming the gastroprotective activity of the extract in different models.

The Physical Barrier Test
The oral and intraperitoneal administration of EHXA (50 mg/kg) significantly reduced the area of ulcerative lesions of the stomachs by 74.35% and 60.68%, respectively, compared to the negative control group. These results demonstrate that the gastroprotective effect of EHXA occurs both due to the modulation of gastroprotective signaling pathways and through the formation of a physical barrier ( Figure 2).

Indomethacin-Induced Acute Gastric Lesions
Considering the importance of non-steroidal anti-inflammatory drugs (NSAIDs) in the development of gastric lesions, we evaluated the effects of EHXA on indomethacin-induced acute gastric lesions. The treatment with EHXA (50, 100, and 200 mg/kg) and omeprazole (30 mg/kg) significantly inhibited the development of ulcerative lesions (by 55.99%, 55.99%, 42.50%, and 84.69%, respectively) when compared to the negative control group ( Figure 1C), confirming the gastroprotective activity of the extract in different models.

The Physical Barrier Test
The oral and intraperitoneal administration of EHXA (50 mg/kg) significantly reduced the area of ulcerative lesions of the stomachs by 74.35% and 60.68%, respectively, compared to the negative control group. These results demonstrate that the gastroprotective effect of EHXA occurs both due to the modulation of gastroprotective signaling pathways and through the formation of a physical barrier ( Figure 2). ; n = 6 animals/group. One-way (ANOVA) was used, followed by Tukey's test (**** p < 0.0001, when compared to the negative control group).

Involvement of Prostaglandins E2 (PGE2)
After administering EHXA (50 mg/kg) and misoprostol (0.016 mg/kg), a PGE2 analog, the area of ulcerative lesions decreased by 65.28% and 73.42%, respectively, compared to the negative control group. However, with the administration of indomethacin (a specific inhibitor of the PGE2 synthesis pathway), the gastroprotective effects of EHXA and misoprostol were reversed, indicating that the gastroprotective effect of EHXA depends, at least partially, on the synthesis of PGE2 ( Figure 3A).
Since PGE2-mediated gastroprotection significantly involves mucus production by the gastric mucosa, we analyzed the effects of EHXA (50 mg/kg) and misoprostol (0.016 **** **** Figure 2. Gastroprotective effect of EHXA (50 mg/kg) in acute injuries induced by absolute ethanol in the physical barrier test. The values are the means ± S.E.M (standard error of the mean); n = 6 animals/group. One-way (ANOVA) was used, followed by Tukey's test (**** p < 0.0001, when compared to the negative control group). After administering EHXA (50 mg/kg) and misoprostol (0.016 mg/kg), a PGE 2 analog, the area of ulcerative lesions decreased by 65.28% and 73.42%, respectively, compared to the negative control group. However, with the administration of indomethacin (a specific inhibitor of the PGE 2 synthesis pathway), the gastroprotective effects of EHXA and misoprostol were reversed, indicating that the gastroprotective effect of EHXA depends, at least partially, on the synthesis of PGE 2 ( Figure 3A).

Involvement of Gastroprotective
Since PGE 2 -mediated gastroprotection significantly involves mucus production by the gastric mucosa, we analyzed the effects of EHXA (50 mg/kg) and misoprostol (0.016 mg/kg) on mucus production. These treatments significantly increased the production of adherent mucus by 79.92% and 40.40%, respectively, compared to the negative control group, corroborating the evidence that EHXA might be acting through PGE 2 -dependent mechanisms ( Figure 3B).

Involvement of H 2 Receptors
The administration of EHXA (50 mg/kg) and ranitidine (40 mg/kg), an H 2 receptor antagonist, significantly decreased the occurrence of ulcerative lesions in the stomach by 80.71% and 59.92%, respectively, when compared with the negative control group. However, the pretreatment with histamine (H 2 receptor agonist) only reversed the protective effect of ranitidine, indicating that the gastroprotective effect of EHXA does not occur via the inhibition of H 2 receptors ( Figure 4A).

Involvement of H2 Receptors
The administration of EHXA (50 mg/kg) and ranitidine (40 mg/kg), an H2 receptor antagonist, significantly decreased the occurrence of ulcerative lesions in the stomach by 80.71% and 59.92%, respectively, when compared with the negative control group. However, the pretreatment with histamine (H2 receptor agonist) only reversed the protective effect of ranitidine, indicating that the gastroprotective effect of EHXA does not occur via the inhibition of H2 receptors ( Figure 4A).  ; n = 6 animals/group. One-way (ANOVA) was used, followed by Tukey's test (** p < 0.01 and **** p < 0.0001 when compared to the negative control group). ### p < 0.001 when compared to the ranitidine group and ## p < 0.01 when compared to the diazoxide group The values are the means ± S.E.M (standard error of the mean); n = 6 animals/group. One-way (ANOVA) was used, followed by Tukey's test (*** p < 0.001, **** p < 0.0001, when compared to the negative control group). ## p < 0.01, when comparing antagonist + agonist vs. agonist; EHXA alone vs. antagonist + EHXA.

Involvement of H2 Receptors
The administration of EHXA (50 mg/kg) and ranitidine (40 mg/kg), an H2 receptor antagonist, significantly decreased the occurrence of ulcerative lesions in the stomach by 80.71% and 59.92%, respectively, when compared with the negative control group. However, the pretreatment with histamine (H2 receptor agonist) only reversed the protective effect of ranitidine, indicating that the gastroprotective effect of EHXA does not occur via the inhibition of H2 receptors ( Figure 4A).  ; n = 6 animals/group. One-way (ANOVA) was used, followed by Tukey's test (** p < 0.01 and **** p < 0.0001 when compared to the negative control group). ### p < 0.001 when compared to the ranitidine group and ## p < 0.01 when compared to the diazoxide group One-way (ANOVA) was used, followed by Tukey's test (** p < 0.01 and **** p < 0.0001 when compared to the negative control group). ### p < 0.001 when compared to the ranitidine group and ## p < 0.01 when compared to the diazoxide group.

Involvement of ATP-Dependent Potassium Channels
The treatment of animals with EHXA (50 mg/kg) and diazoxide (3 mg/kg), a potassium channel opener, led to a significant reduction in the occurrence of ulcerative lesions in the stomach, with percentual reductions of 76.28% and 71.54%, respectively, compared to the negative control group. However, the pretreatment with glibenclamide, a specific blocker of ATP-dependent potassium channels, had no significant impact on the therapeutic effect of EHXA, indicating that this effect does not result from an interference with potassium-dependent channels ( Figure 4B).

Involvement of Sulfhydryl Groups (-SH Groups)
The treatment with EHXA (50 mg/kg) and carbenoxolone (100 mg/kg) significantly reduced ulcerative lesions, with percentual inhibitions of 79.57% and 73%, respectively, compared with the negative control group. However, when the animals in the EHXA group were pretreated with N-ethylmaleimide (NEM), the gastroprotective effect of EHXA and carbenoxolone was reversed, indicating the participation of sulfhydryl groups ( Figure 5A) in the gastroprotective action of EHXA ( Figure 5A). sium channel opener, led to a significant reduction in the occurrence of ulcerative lesions in the stomach, with percentual reductions of 76.28% and 71.54%, respectively, compared to the negative control group. However, the pretreatment with glibenclamide, a specific blocker of ATP-dependent potassium channels, had no significant impact on the therapeutic effect of EHXA, indicating that this effect does not result from an interference with potassium-dependent channels ( Figure 4B).

Involvement of α2 Adrenergic Receptors
The treatments with EHXA (50 mg/kg) and clonidine (0.05 mg/kg), an α2 adrenergic receptor agonist, significantly decreased the occurrence of ulcerative lesions in the stomachs of ethanol-challenged animals (inhibitions of 79.76% and 73.68%, respectively) compared to the negative control group. However, in the animals pretreated with yohimbine, a specific inhibitor of α2 adrenergic receptors, the gastroprotective effect of EHXA (50 mg/kg) was reversed, suggesting that α2 adrenergic receptors can, at least partially, mediate the gastroprotective effect of EHXA ( Figure 5B).

Involvement of the Nitric Oxide (NO) Pathway
The treatment with EHXA (50 mg/kg) and L-arginine (600 mg/kg) significantly reduced the generation of ulcerative lesions by 73.61% and 71.60%, respectively, compared to the negative control group. However, when the animals in the EHXA group received pretreatment with L-NAME (nonspecific inhibitor of nitric oxide synthase enzymes), a reversal of the gastroprotective effect of EHXA and L-arginine was observed, thus suggesting the potential involvement of NO-dependent mechanisms ( Figure 6A). ; n = 6 animals/group. One-way (ANOVA) was used, followed by Tukey's test (**** p < 0.0001, when compared to the negative control group). #### p < 0.0001, when comparing antagonist + agonist vs. agonist; ### p < 0.001, when comparing antagonist + agonist vs. agonist; EHXA alone vs. antagonist + EHXA.

Involvement of α 2 Adrenergic Receptors
The treatments with EHXA (50 mg/kg) and clonidine (0.05 mg/kg), an α 2 adrenergic receptor agonist, significantly decreased the occurrence of ulcerative lesions in the stomachs of ethanol-challenged animals (inhibitions of 79.76% and 73.68%, respectively) compared to the negative control group. However, in the animals pretreated with yohimbine, a specific inhibitor of α 2 adrenergic receptors, the gastroprotective effect of EHXA (50 mg/kg) was reversed, suggesting that α 2 adrenergic receptors can, at least partially, mediate the gastroprotective effect of EHXA ( Figure 5B).

Involvement of the Nitric Oxide (NO) Pathway
The treatment with EHXA (50 mg/kg) and L-arginine (600 mg/kg) significantly reduced the generation of ulcerative lesions by 73.61% and 71.60%, respectively, compared to the negative control group. However, when the animals in the EHXA group received pretreatment with L-NAME (nonspecific inhibitor of nitric oxide synthase enzymes), a reversal of the gastroprotective effect of EHXA and L-arginine was observed, thus suggesting the potential involvement of NO-dependent mechanisms ( Figure 6A).

Nitrate/Nitrite Quantification
The treatment with EHXA (50 mg/kg) and L-arginine (600 mg/kg) significantly increased nitrate and nitrite production by 61.62% and 75.32%, respectively, in tissue homogenates when compared to the negative control group. This result corroborates the previous result of the nitric oxide test ( Figure 6B).

Myeloperoxidase (MPO) Activity
The administration of EHXA (50 mg/kg) and indomethacin (10 mg/kg) resulted in a significant reduction in the myeloperoxidase (MPO) activity produced in the inflamed tissue of animals challenged with acidified ethanol, with inhibitions of 69.93% and 65.07%, respectively, compared to the negative control group (Figure 7), indicating that the activity of EHXA is associated with a decrease in the inflammatory response in the gastric mucosa. One-way (ANOVA) was used, followed by Tukey's test (**** p < 0.0001, when compared to the negative control group). #### p < 0.0001, when comparing antagonist + agonist vs. agonist; EHXA alone vs. antagonist + EHXA.

Nitrate/Nitrite Quantification
The treatment with EHXA (50 mg/kg) and L-arginine (600 mg/kg) significantly increased nitrate and nitrite production by 61.62% and 75.32%, respectively, in tissue homogenates when compared to the negative control group. This result corroborates the previous result of the nitric oxide test ( Figure 6B).

Myeloperoxidase (MPO) Activity
The administration of EHXA (50 mg/kg) and indomethacin (10 mg/kg) resulted in a significant reduction in the myeloperoxidase (MPO) activity produced in the inflamed tissue of animals challenged with acidified ethanol, with inhibitions of 69.93% and 65.07%, respectively, compared to the negative control group (Figure 7), indicating that the activity of EHXA is associated with a decrease in the inflammatory response in the gastric mucosa. One-way (ANOVA) was used, followed by Tukey's test (**** p < 0.0001, when compared to the negative control group). #### p < 0.0001, when comparing antagonist + agonist vs. agonist; EHXA alone vs. antagonist + EHXA.

Nitrate/Nitrite Quantification
The treatment with EHXA (50 mg/kg) and L-arginine (600 mg/kg) significantly increased nitrate and nitrite production by 61.62% and 75.32%, respectively, in tissue homogenates when compared to the negative control group. This result corroborates the previous result of the nitric oxide test ( Figure 6B).

Myeloperoxidase (MPO) Activity
The administration of EHXA (50 mg/kg) and indomethacin (10 mg/kg) resulted in a significant reduction in the myeloperoxidase (MPO) activity produced in the inflamed tissue of animals challenged with acidified ethanol, with inhibitions of 69.93% and 65.07%, respectively, compared to the negative control group (Figure 7), indicating that the activity of EHXA is associated with a decrease in the inflammatory response in the gastric mucosa. One-way (ANOVA) was used, followed by Tukey's test (**** p < 0.0001, when compared to the negative control group). #### p < 0.0001 and ## p < 0.01, when comparing blank group.

Inhibition of Gastrointestinal Motility by EHXA and ATROPINE
The treatment with EHXA (50 mg/kg) and the muscarinic receptor antagonist atropine (0.01 mg/kg) partially inhibited the intestinal motility of animals by 23.47% and 32.50%, respectively, when compared to the negative control group. This finding suggests that EHXA may inhibit gastrointestinal motility by interfering with the cholinergic system ( Figure 8).

Inhibition of Gastrointestinal Motility by EHXA and ATROPINE
The treatment with EHXA (50 mg/kg) and the muscarinic receptor antagonist atropine (0.01 mg/kg) partially inhibited the intestinal motility of animals by 23.47% and 32.50%, respectively, when compared to the negative control group. This finding suggests that EHXA may inhibit gastrointestinal motility by interfering with the cholinergic system ( Figure 8).  [31,32]. A similar chemical composition was observed in other studies, indicating low chemical variation among the samples [14,15].
According to a previously published study, the HPLC analysis of Ximenia americana fruit pulp extract identified various phenolic compounds, including rutin, gallic acid, catechin, epicatechin, p-coumaric acid, and ferulic acid [29]. The ethanolic extract obtained from the stems of Ximenia americana was found to contain steroids such as stigmasterol and sitosterol, as well as triterpenoids such as betulinic acid, oleanolic acid, 28-O-(-D-glucopyranosyl) oleanolic acid, 3-oxo-oleanolic acid, 3βhydroxycicloart-24(E)-ene-26-oic acid, and furanoic and widdrane sesquiterpenoids [15,33,34]. Another study also reported the identification of several flavonoids, such as quercetin, kaempferol, and isorhamnetin, in the leaves and fruits. Analysis of the fatty acid profile of this species by HPLC identified the presence of palmitic acid, stearic acid, oleic acid, linoleic acid, and alpha-linolenic acid [35].

Discussion
The present study reports, for the first time, the potential of the hydroethanolic extract (by high-performance liquid chromatography-HPLC) of the same extract, revealing the presence of the compounds quercitrin and caffeic acid as major components, in addition to several others, such as quercetin, rutin, gallic acid, catechin, chlorogenic acid, ellagic acid, and kaempferol [31,32]. A similar chemical composition was observed in other studies, indicating low chemical variation among the samples [14,15].
According to a previously published study, the HPLC analysis of Ximenia americana fruit pulp extract identified various phenolic compounds, including rutin, gallic acid, catechin, epicatechin, p-coumaric acid, and ferulic acid [29]. The ethanolic extract obtained from the stems of Ximenia americana was found to contain steroids such as stigmasterol and sitosterol, as well as triterpenoids such as betulinic acid, oleanolic acid, 28-O-(-Dglucopyranosyl) oleanolic acid, 3-oxo-oleanolic acid, 3β-hydroxycicloart-24(E)-ene-26-oic acid, and furanoic and widdrane sesquiterpenoids [15,33,34]. Another study also reported the identification of several flavonoids, such as quercetin, kaempferol, and isorhamnetin, in the leaves and fruits. Analysis of the fatty acid profile of this species by HPLC identified the presence of palmitic acid, stearic acid, oleic acid, linoleic acid, and alpha-linolenic acid [35].
Notably, compounds present in EHXA have already been proven to present significant gastroprotective effects. Quercitrin has a broad spectrum of bioactivities, including antioxidant, anti-inflammatory, antimicrobial, immunomodulatory, analgesic, wound-healing, vasorelaxant, and gastroprotective [36]. This compound effectively reduced acute colitis in a study by Sanches et al. (2002) [37]. In addition, the gastroprotective activity of gallic acid, which is also present in EHXA, has been evidenced in several in vivo models [38]. Evidence suggests the involvement of the NF-κb pathway in its mechanism of action [39]. Caffeic acid also demonstrated biological activities related to our study, such as antimicrobial, anticancer [40], antioxidant [41], anti-inflammatory, and antinociceptive [42]. Romana-Souza et al., in 2018, showed that caffeic acid administration promotes wound healing by inhibiting the activation of the NO/NOS2 pathway via NF-κB and preventing oxidative stress through reducing peroxynitrite production and nitrite synthesis, thus corroborating our results [43]. A study by Kolgazi et al. (2021) also demonstrated that the reduction in macroscopic and microscopic lesions in the ethanol model was associated with decreased MPO activity and modulation of the NO pathway [44].
It is worth mentioning that the gastroprotective potential of the EHXA constituent epicatechin has been associated with [45] adrenergic receptor activation and modulation of SH, NO, SOD, and HSP-70. Given the proven biological activities of the constituents present in EHXA, we can suggest that its gastroprotective and anti-ulcerative activity may be attributed to these bioactive compounds in the extract.
Regarding the assessment of acute toxicity, the present study demonstrated that EHXA did not cause mortality nor visible signs of toxicity after treatment with the extract, corroborating the result obtained by [14], which also describes the low toxicity of Ximenia americana L.
Regarding the screening models assessed in the present study, treatments with EHXA (50, 100, and 200 mg/kg) reduced gastric lesions in the acidified ethanol, absolute ethanol, and indomethacin models, thus demonstrating their effectiveness in gastric protection.
The gastric ulcer model induced by acidified ethanol is widely used to assess new substances with gastroprotective potential in mice. Acidified ethanol acts by promoting gastric lesions with areas of intense hemorrhage, reductions in the concentrations of glutathione and antioxidant enzymes, increased vascular permeability and infiltration of neutrophils, the subsequent release of reactive oxygen species, and the loss of gastric epithelial cells [14,[46][47][48].
Regarding the administration of absolute ethanol, it generates severe lesions in the gastric mucosa, reducing gastric blood flow and causing infiltration of inflammatory cells, tissue destruction, extensive hemorrhages, and production of free radicals [49][50][51]. On the other hand, gastric ulcers induced by indomethacin cause an increase in the levels of inflammatory cytokines such as IL-1β and IL-6. It decreases the mucosa's anti-inflammatory cytokines, such as IL-4 and TGF-β [52].
Based on the relevant data supporting the gastroprotective effects of EHXA, we evaluated whether the extract acts by modulating systemic pathways or by forming a protective barrier. In the present study, EHXA significantly reduced the lesions when the animals were treated both orally and intraperitoneally, demonstrating that EHXA was acting both at the protective barrier level and on the signaling pathways of the gastroprotective response. It is worth mentioning that the stomach's first line of defense against aggressive agents is the physical barrier formed by mucus, surfactant phospholipids, and bicarbonates that cover the surface layer and increase blood flow and cell renewal, in addition to maintaining the structural integrity of the mucosa [53].
To better characterize the gastroprotective effect observed in the screening test, we investigated the involvement of signaling pathways that could contribute to the protective mechanisms exhibited by the EHXA.
Prostaglandins, especially PGE 2 , have a significant cytoprotective effect, stimulating the secretion of sodium bicarbonate in the stomach and inhibiting the release of histamine, TNF-α, platelet-activating factor (PAF), IL-1, IL-8, and LTB 4 . This mediator also contributes to gastroprotection by inhibiting mast cell activation, gastric motility, gastric acid secretion, and leukocyte adherence to the vascular endothelium [54,55]. Importantly, evidence indicates that these effects involve the activation of EP1, EP3, and EP4 receptors [56,57].
Using pharmacological tools, we demonstrated that PGE 2 was able to significantly mediate the effects of EHXA concerning gastric ulcer generation. This finding was corroborated by evidence that EHXA significantly stimulates mucus production in the gastric mucosa. Ethanol promotes damage to the gastric mucosa by destroying protective factors, such as mucus and the non-protein sulfhydryl group, causing an increase in lipid peroxidation and reactive oxygen species production [51,55,58] and a decrease in glutathione levels [59]. It is worth mentioning that in the study described by Mizui, T.& Doteuchi, M 1983 [60], the crude extract, quercetin, and the isolated compound epicatechin were tested and their antioxidant effects were demonstrated in in vitro tests, thus corroborating our findings as well as the evidence that the antioxidant activity is related to the gastroprotective activity of several substances. Sulfhydryl groups (SH) play an important role in gastric cytoprotective mechanisms [61]. They are found in high concentrations in the stomach, where they are linked to free radicals formed after injury [62]. When adhering to the mucous layer, they form a barrier that supports the surface neutralization of acid [63]. The formation of a complex between glutathione and acetaldehyde, due to ethanol oxidation by the enzyme alcohol dehydrogenase, increases lipid peroxidation and can also cause oxidative stress [64,65]. In this context, this study showed that SH groups participate in the mechanism of action of EHXA. Corroborating the present study [14] showed that an aqueous extract of the species Ximenia americana L. reduced gastric ulcer lesions through sulfhydryl groups, nitric oxide, and antisecretory activity.
Regarding the involvement of α 2 adrenergic receptors, these proteins are formed from deduced amino acid sequences to form the three genes of the α 2 receptor and its subunits α2A, α2B, and α2C. α2 receptors can bind with different G protein subunits and stimulate the G o protein, inhibiting the Ca 2+ channel. The α 2 adrenergic receptor inhibits adenyl cyclase, causing the dissociation of the G i protein and its subunits. That is, the α subunit is loaded with guanosine triphosphate (GTP) and another βγ unit when these α 2 adrenergic receptors are stimulated in the stomach, reducing gastric motility [66].
Nitric oxide is involved in maintaining the integrity of the gastric epithelium. According to Cho (2001), nitric oxide exerts either protective or harmful effects, depending on the type of nitric oxide synthetase [67]. NO also contributes to the regulation of gastric blood flow and directly stimulates gastric mucus secretion through the stimulation of guanylate cyclase in epithelial cells. Obstruction of NO production leads to vascular impairment and subsequent alkaline flow into the lumen [67].
To better understand the role of NO in the effect of EHXA in ethanol-induced ulcers, the concentrations of nitrite (NO 2 ) and nitrate (NO 3 ) in the gastric mucosa were assessed as an estimate of NO production [68]. It was possible to verify that nitric oxide is produced at the lesion site and that it is potentially associated with the protective role of EHXA.
Myeloperoxidase (MPO) is an enzyme found mainly in neutrophils, which release it in the inflammatory site in response to several stimuli [69]. In the present study, EHXA reduced MPO levels in the gastric mucosa, suggesting a reduction in neutrophil recruitment and inhibition of the inflammatory response in the injured area. Studies carried out by Pantoja et al., 2018 [70] with total polysaccharides of the extract (TPL-Xa) or tea (Tea-Xa) of Ximenia americana L. showed that its gastroprotective effect is due to the reduction in neutrophil infiltration and, consequently, the reduction in MPO activity.
Regarding gastrointestinal motility, EHXA and the muscarine receptor antagonist atropine presented significant inhibitory effects. Motility is regulated via the cholinergic system in the circular muscular layer of the gastrointestinal tract through M1 and M3 receptors [71]. The study carried out in [72] with caffeic acid, one of the main constituents of EHXA, demonstrated its effective potential in muscle relaxation, corroborating our results.

Legal Requirements: Ethical Aspects of the Research
These in vivo assays were conducted using Swiss mice (Mus musculus) of both sexes, with body masses between 20-30 g. The animals were monitored at the Experimental Animal House at URCA and housed in polypropylene cages under controlled conditions, at a temperature of 24 ± 2 • C under a 12 h light/dark cycle. They had free access to a specific food for rodents (Labina, Presence ® , São Paulo, Brazil) and drinking water, except for during an 8-12 h fasting period before testing. The research followed the animal use rules and was approved by the Commission for Experimentation and Use of Animals of the Regional University of Cariri (CEUA/URCA) under number 00157/2021.2.

Hydroethanolic Extract from the Bark of Ximenia americana L. (EHXA) and Chemical Profile
The collection of botanical materials, the extract preparation, and the phytochemical profile were described by Silva et al., 2018 [31].

In Vivo Assays
In the screening tests of gastric injury induced by acidified ethanol, absolute ethanol, and indomethacin, the animals (n = 6) were divided into groups and treated with either H 2 O (0.1 mL/10 g/p.o.) or different doses of EHXA (50, 100, and 200 mg/kg/p.o.). The lowest effective dose of EHXA (50 mg/kg/p.o.) identified in the screening tests was used for the other tests and the assessment of the signaling pathways of the gastroprotective response. The investigated mechanisms included the determination of gastric mucosal adherent mucus; the involvement of H 2 receptors; ATP-dependent potassium channels; sulfhydryl groups (SH); the cholinergic pathway; α 2 adrenergic, nitric oxide, and nitrite/nitrate production; and myeloperoxidase activity. All procedures followed the rules for the use of animals, and the study was approved and registered under number 00157/2021.2.

Non-Clinical Acute Oral Toxicity-LD 50
The non-clinical oral acute toxicity evaluation followed the Organization for Economic Cooperation and Development [77], with some modifications, and used the Malone and Robichaud table [78]. The animals were divided into two groups (n = 3). The control group received water for injection (0.01 mL/g/p.o.), and the group treated with EHXA received a single dose of 2000 mg/kg/p.o. The animals were observed at 30, 60, 120, 180, and 360 min post-treatment, and then subsequently every 24 h for 14 days.  [79]. After 30 min, the animals were euthanized by cervical dislocation. The stomachs were removed and opened by the greater curvatures, washed with 0.9% saline, and prepared for evaluation and quantification of gastric lesions as described in Section 4.4.1.

The Physical Barrier Test
Mice (n = 6) were divided into 3 groups, receiving either water for injection (0.1 mL/10 g/p.o.), EHXA (50 mg/kg/p.o.), and EHXA (50 mg/kg, i.p.). After 30 min of i.p. or 1 h after administering the p.o., 96% ethanol (0.2 mL/animal) was administered. After 30 min, the animals were euthanized by cervical dislocation. The stomachs were removed and opened along the greater curvature, washed with 0.9% saline, and prepared for evaluation and quantification of gastric lesions as described previously.  [82]. After 30 min, the animals were euthanized, and the stomachs were removed, washed, and prepared for evaluation and quantification of gastric lesions as described previously.

Determination of Gastric Mucosal Mucus
Six mice were divided into three groups: negative control (water for injection 0.1 mL/10 g/p.o.), misoprostol (0.016 mg/kg/p.o.), and EHXA (50 mg/kg/p.o.). After 1 h, gastric lesions were induced by administering 96% ethanol (0.2 mL/animal, p.o.). Thirty minutes after the ethanol administration, the mice were euthanized, and their stomachs were removed and washed. Subsequently, the gastric mucosa was taken out, weighed, and incubated in 10 mL of 0.1% Alcian blue solution for two hours. The dye, complexed with mucus, was extracted using magnesium chloride by stirring it for 1 min every 30 min for 2 h. After this, the homogenate was centrifuged at 3600 rpm for 10 min. The concentration of Alcian blue was measured using spectrophotometry at 598 nm and expressed as µg Alcian blue/mL/g tissue [83].

Involvement of H 2 Histamine Receptors
Mice (n = 6) were divided into groups as follows: water for injection (0.1 mL/10 g/p.o.), ranitidine (40 mg/kg/p.o.), and EHXA (50 mg/kg/p.o.). The participation of histamine H 2 receptors in the gastroprotective effect of EHXA was evidenced by the previous administration of histamine (3 mg/kg, s.c.) 30 min before the administration of EHXA (50 mg/kg/p.o.) or ranitidine (40 mg/kg/p.o.) [84,85]. After 30 min of ranitidine administration and 1 h after administration of EHXA and negative control, the animals received 96% ethanol (0.2 mL/animal; p.o.). After 30 min, the animals were euthanized and their stomachs were removed, washed, and prepared for evaluation and quantification of gastric lesions as previously described.

Involvement of the Nitric Oxide Pathway
A group of animals was divided into five subgroups of six mice, with the first three groups receiving water for injection (0.1 mL/10 g/p.o.), L-arginine (600 mg/kg, i.p.), and EHXA (50 mg/kg/p.o.), respectively. This study aimed to investigate the role of nitric oxide (NO) in the gastroprotective effect of EHXA. For this, the inhibitor L-NAME (20 mg/kg, i.p.) was administered 30 min prior to the administration of EHXA (50 mg/kg) or L-arginine (600 mg/kg). After 1 h (p.o) or 30 min (i.p) of treatment, 96% ethanol (0.2 mL/animal, p.o) was administered [82]. After 30 min, the mice were euthanized, and their stomachs were removed, washed, and prepared for evaluation and quantification of gastric lesions.

Nitrate/Nitrite Quantification
This method was based on the diazotization reaction and Griess reagent coupling, which reveals the presence of nitrite in the injured tissue sample. The absorbance reading was performed by a spectrophotometer with a 560 nm filter [86].

Myeloperoxidase (MPO) Activity
Six mice were randomly assigned to three groups: negative control (water for injection 0.1 mL/10 g/p.o.), indomethacin (10 mg/kg/p.o.), and EHXA (50 mg/kg/p.o.). The treatments were administered 1 h before inducing lesions with acidified ethanol (0.2 mL of 0.3 M HCl solution in 60% ethanol/animal, p.o.). After 1 h, the mice were euthanized by cervical dislocation, and their stomachs were removed and opened along the greater curvature. The stomachs were then washed with 0.9% saline solution and prepared for the evaluation of myeloperoxidase enzyme activity. MPO levels were determined by measuring the optical density of the mixture of the samples and o-dianisidine solution using a spectrophotometer at 600 nm, with hydrogen peroxide as a substrate for MPO. The results were expressed as UMPO/µL of wash [87].
After one hour, a semi-solid-colored marker (0.05% phenol red in 1.5% carboxymethylcellulose) was orally administered, and the mice were euthanized 30 min later. The small intestines were removed and measured to determine the total length. The percentage of the distance traveled by the marker was calculated and used as a marker of motility [88].

Statistical Analysis
The mean ± standard error of the mean (S.E.M) was used to express the results. To analyze the differences between means, one-way and two-way analyses of variance (ANOVA) were employed, followed by Tukey's multiple comparison test. Statistical analysis was carried out using GraphPad Prism 6.1, with a significance level of 5% (p < 0.05) set for rejecting the null hypothesis.

Conclusions
In conclusion, EHXA showed a significant gastroprotective effect that might be associated with the inhibition of MPO activity and modulation of α 2 adrenergic receptors, NO synthesis, SH groups, stimulation of mucus secretion mediated by PGE 2 , and reduction in intestinal motility.
Based on the obtained data, we can attest to the preclinical efficacy of EHXA and suggest its potential mechanisms of action. However, there is a need for further studies to prove its effectiveness in the treatment of gastric ulcers and to elucidate the molecular mechanisms of action of its constituents.

Institutional Review Board Statement:
The animal study protocol was approved by the Commission for Experimentation and Use of Animals of the Regional University of Cariri (CEUA/URCA), registered under number 00157/2021.2.

Data Availability Statement:
The data supporting reported results can be solicited from the corresponding author.