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Article Contribution of Secondary Metabolites to the Gastroprotective Effect of Aqueous Extract of americana L. () Stem Bark in Rats

Ticiana Parente Aragão 1,2, Lady Dayane Kalline Travassos dos Prazeres 1, Samara Alves Brito 1, Pedro José Rolim Neto 3, Larissa Araújo Rolim 4, Jackson Roberto Guedes da Silva Almeida 5 ID , Germana Freire Rocha Caldas 6 and Almir Gonçalves Wanderley 1,7,* ID

1 Department of Pharmaceutical Sciences, Federal University of Pernambuco, Recife 50740-521, PE, Brazil; [email protected] (T.P.A.); [email protected] (L.D.K.T.d.P.); [email protected] (S.A.B.) 2 Department of Nutrition, University of Pernambuco, Petrolina 56328-903, PE, Brazil 3 Laboratory of Medication Technology, Federal University of Pernambuco, Recife 50740-521, PE, Brazil; [email protected] 4 Central of Analysis of Drugs, Medicines and Food, Federal University of San Francisco Valley, Petrolina 56304-205, PE, Brazil; [email protected] 5 Center for Studies and Research of Medicinal , Federal University of San Francisco Valley, Petrolina 56304-205, PE, Brazil; [email protected] 6 Graduate Program in Health Sciences, Biological and Health Sciences Center, Federal University of Maranhão, São Luís 65080-805, MA, Brazil; [email protected] 7 Department of Physiology and Pharmacology, Federal University of Pernambuco, Recife 50670-901, PE, Brazil * Correspondence: [email protected]; Tel.: +55-81-2126-8530; Fax: +55-81-2126-8976

Received: 30 November 2017; Accepted: 4 January 2018; Published: 9 January 2018

Abstract: Ximenia americana L. (Olacaceae) is used in ethnomedicine as cicatrizant and for the treatment of gastric disorders. This study identified the chemical constituents of the aqueous extract of X. americana (XaAE) and evaluated its antiulcerogenic activity. After lyophilization, XaAE was analyzed by liquid chromatography-mass spectrometry (LC-MS) and its antiulcerogenic effect was evaluated in acute gastric lesions induced by ethanol, acidified ethanol, and indomethacin. Antisecretory action, mucus production and the participation of sulfhydryl groups (–SH) and nitric oxide (NO) were also investigated. The chromatographic analysis identified procyanidins B and C and catechin/epicatechin as major compounds. Oral administration of XaAE (100, 200 and 400 mg/kg) inhibited the gastric lesions induced by ethanol (76.1%, 77.5% and 100%, respectively), acidified ethanol (44.9%, 80.6% and 94.9%, respectively) and indomethacin (56.4%, 52.7% and 64.9%, respectively). XaAE reduced gastric contents and acidity (51.4% and 67.7%, respectively) but did not alter the production of gastric mucus. The reduction of the -SH and NO groups promoted by N-ethylmaleimide (NEM) and Nω-nitro-L-arginine-methyl-ester (L-NAME) respectively, reduced the gastroprotective effect of XaAE. In conclusion, XaAE has gastroprotective activity mediated in part by -SH, NO and antisecretory activity. This antiulcer action was initially correlated to its major constituents, procyanidins B and C and catechin/epicatechin.

Keywords: Ximenia; Olacaceae; flavonoids; gastric lesion

1. Introduction Peptic ulcer disease is a chronic, multifactorial disease characterized by lesions that affect the mucosa of the esophagus, stomach and/or duodenum, and may extend to the muscular layer of the mucosa [1].

Molecules 2018, 23, 112; doi:10.3390/molecules23010112 www.mdpi.com/journal/molecules Molecules 2018, 23, 112 2 of 18

Lesions occur due to an imbalance between the defensive factors (mucus, bicarbonate, mucosal blood flow, endogenous prostaglandins) and aggressors (acid secretion, alcohol, nonsteroidal anti-inflammatory drugs, Helicobacter pylori and reactive oxygen species) of the gastric mucosa [2]. These lesions affect about four million people worldwide, more often men than women, with duodenal ulcer more prevalent among young people and gastric ulcer among the elderly [1,3]. Currently, treatment options for peptic ulcer are based on the use of antacids, cytoprotective agents, muscarinic antagonists, H2 antihistamines, proton pump inhibitors, and more recently the use of antimicrobials for the treatment of Helicobacter pylori infection [4,5]. These drugs have led to a reduction in the incidence and prevalence of peptic ulcers. However, the side effects of chronic treatment with these drugs and the increased resistance of H. pylori to antibiotics indicate the need to search new substances that may help in the treatment of this disease. The inhibition of gastric acid secretion with anti-secretory drugs such as proton-pump inhibitors or H2-receptor antagonists has been a viable therapeutic option in the treatment of gastric diseases, despite the recurrence and/or adverse effects [6]. In this context, we used pantoprazole or ranitidine as standard drugs in the experimental models. In addition, carbenoxolone was also used as a standard drug in some experimental models considering its potent cytoprotective gastric action [7]. X. americana L. (Olacaceae), commonly known as prickly plum, was first described and named in 1606, in Australia, by the Spanish naturalist Francisco Ximenes [8]. It is a tropical, uncultivated and uncommon natural cosmopolitan , occurring mainly in and South America, including on the coastal plateaus in the Northeast Brazil [9]. In ethnomedicine, X. americana stem bark is used as an astringent and healing agent, and is also indicated for the treatment of wounds, skin and mucosal ulcerations, gastritis and stomach discomfort [9]. Some studies have explored its ulcer healing, anti-inflammatory [10,11], analgesic [12], antioxidant [13,14], antimicrobial [15], antihelmintic [16] and anticancer [17] action. The lack of literature support for the action of X. americana L. stem bark on the treatment of gastric problems, whose use is based on ethnopharmacological information, motivated us to explore the possible antiulcerogenic action of this species in acute preclinical models of gastric lesions.

2. Results

2.1. Phytochemical Profile The analysis revealed the presence of high levels of phenolic compounds, lignans, monoterpenes, sesquiterpenes, diterpenes, naphthoquinones, triterpenes and steroids, and smaller quantities of alkaloids, and hydrolyzable (Table1).

Table 1. Metabolic compounds identified in the phytochemical characterization of X. americana (XaAE) aqueous extract.

Classes of Secondary Metabolites Standards Revealing Agent Intensity Alkaloids Yohimbine Dragendorff + Quercetin NP/PEG +++ Lignans - Vanillin phosphoric +++ Mono, sesqui and diterpenes - Vanillin sulphuric +++ Naphthoquinones - 10% Ethanolic KOH +++ Saponins Sulfuric vanillin + Hydrolysable tannins Gallic acid Ferric chloride 2% ++ Triterpenes and steroids - Sulfuric vanillin +++ Natural products-polyethylene glycol reagent (NP/PEG) (=NEU-reagent), KOH: potassium hydroxide. (+++) strong; (++) medium; (+) weak. Molecules 2018, 23, 112 3 of 18

2.2. High Performance Liquid Chromatography (HPLC-DAD-MS/MS)

MoleculesMoleculesThe 2018 2018 chromatographic, ,23 23, ,112 112 analysis of the aqueous extract of X. americana suggested the presence33 of of 17 17 of procyanidin B, procyanidin C and catechin/epicatechin compounds for the similarity between TheultravioletThe precursorprecursor absorption ionsions andand spectrum, fragmentationfragmentation mass/charge specspectratra ofof ratio thethe major andmajor fragmentation constituentsconstituents inin (MS thethe2) mm as//zz describedextractextract werewere in 577.16Table577.162 . (A),(A), The 866.19866.19 precursor (B)(B) and ionsand 289.03 and289.03 fragmentation (C)(C) andand areare spectrashshownown in ofin theFiguresFigures major 11 constituentsandand 22 toto proveprove in the thethe m identityidentity/z extract ofof procyanidinswereprocyanidins 577.16 (A), B, B, procyanidins procyanidins 866.19 (B) and C, C, 289.03 an andd catechin/epicatechin, catechin/epicatechin, (C) and are shown inrespectively. respectively. Figures1 and 2 to prove the identity of procyanidins B, procyanidins C, and catechin/epicatechin, respectively. TableTable 2. 2. Compounds Compounds detected detected in in X. X. americana americana aqueous aqueous extract extract. . Retention Table 2. Compounds detected in X. americana aqueous extract. Retention 2 2 m/z (+) MS2 (+) m/z (−) MS2 (−) λ Max (nm) Time (min) m/z (+) MS (+) m/z (−) MS (−) λ Max (nm) RetentionTime Time(min) (min) m/z (+) MS2 (+) m/z (−) MS2 (−) λ Max (nm) 7.87.8 355.11355.11 343.87343.87 168.91168.91 124.91124.91 278 278 16.87.8 579.14 355.11 427.11/291.07 343.87 577.16 168.91 425.04/288.95 124.91 278 278 16.816.8 579.14 579.14 427.11/291.07 427.11/291.07 577.16 577.16 425.04/288.95 425.04/288.95 278 278 18.918.9 867.20867.20 867.20 - - - 866.19 866.19 866.19 695.12/577.16/407.04695.12/577.16/407.04695.12/577.16/407.04 278278278 20.620.6 603.10603.10 603.10 313.02313.02 313.02 289.03289.03 289.03 245.02/204.96245.02/204.96 245.02/204.96 278 278 278 32.3 459.11 415.13/353.05/291.09 435.16 329.03/166.91 278 32.332.3 459.11459.11 415.13/353.05/291.09415.13/353.05/291.09 435.16435.16 329.03/166.91329.03/166.91 278 278

CC AA

BB

FigureFigure 1.1. ChromatogramChromatogramChromatogram of ofof X.X.X. americana americanaamericana extractextractextract at atat 270 270 nm. nm. ( (AA))) Procyanidin ProcyanidinProcyanidin B; B;B; ( ((BBB))) Procyanidin ProcyanidinProcyanidin C; C;C; ((CC)) Catechin/epicatechin. Catechin/epicatechin.

OH OH OH OH OH OH

HO O HO O HO O HO O OH HO O OH HO O OH OH OH OH

OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH HO O HO O HO O OH HO O OH C OH OH C

OH OH OH OH OH OH OH OH OH OH HO O HO O OH OH AA OH OH OH OH

BB

FigureFigure 2.2. StructuresStructures ofof thethe major major constituents constituents ofof X.X. americana americana L.L. stem stem bark bark ( ((AAA))) Procyanidin ProcyanidinProcyanidin B; B;B; ((BB)) Procyanidin Procyanidin C; C; ( C(C)) Catechin/epicatechin. Catechin/epicatechin.

TheThe UV UV chromatogram chromatogram at at 270 270 nm, nm, Figure Figure 11, ,1, showsshows shows thatthat that thethe the majormajor major constituentsconstituents constituents inin in thethe the X. X. americana americana extractextract areare procyanidins. procyanidins.procyanidins. Although AlthoughAlthough it itit was waswas not notnot possible possipossibleble to toto identify identifyidentify them, them,them, they theythey are areare procyanidins procyanidinsprocyanidins B1, B1, B2,B1, B2,B3B2, orB3 B3 B4 or or becauseB4 B4 because because they they they have have have the the samethe same same fragmentation fragment fragmentationation profile profile profile as described as as describe describe bydd other by by other other authors authors authors [18]. [18]. [18].

2.3.2.3. Acute Acute Toxicity Toxicity OralOral administration administration of of 2000 2000 mg/kg mg/kg of of XaAE XaAE did did not not induce induce death death nor nor produced produced visible visible signs signs of of behavioralbehavioral changes changes or or toxicity toxicity in in the the treated treated mice mice during during the the 14 14 days days of of observation. observation. There There were were no no significantsignificant differences differences in in water water intake intake (9.24 (9.24 ± ± 0.35 0.35 vs. vs. 8.66 8.66 ± ± 0.29 0.29 mL/day/animal), mL/day/animal), food food consumption consumption

Molecules 2018, 23, 112 4 of 18

2.3. Acute Toxicity Oral administration of 2000 mg/kg of XaAE did not induce death nor produced visible signs of Moleculesbehavioral 2018, changes 23, 112 or toxicity in the treated mice during the 14 days of observation. There were4 ofno 17 significant differences in water intake (9.24 ± 0.35 vs. 8.66 ± 0.29 mL/day/animal), food consumption (5.32 ± 0.13 vs. 5.57 ± 0.09 g/day/animal), and body weight (36.67 ± 0.27 vs. 36.33 ± 0.99 g) between (5.32 ± 0.13 vs. 5.57 ± 0.09 g/day/animal), and body weight (36.67 ± 0.27 vs. 36.33 ± 0.99 g) between the control and treated group, respectively. the control and treated group, respectively.

2.4. Experimental Experimental Protocols

2.4.1. Induction Induction of Gastric Lesions by Absolute Ethanol Administration of of X. americana aqueous extract reduced the formationformation of absolute ethanol-induced ulcerative lesions lesions (Figures (Figures 3 and4 4).). TheThe percentagepercentage inhibitioninhibition of lesionslesions was 76.08%, 77.48%77.48% andand 100%100% in rats pre-treated with the extract at doses of 100,100, 200200 andand 400400 mg/kg,mg/kg, respective respectively,ly, in contrast with the lesioned control control group group (LC, (LC, 116.5 116.5 ±± 19.6719.67 mm mm2).2 ).Rats Rats treated treated with with pantoprazole pantoprazole showed showed a reduction a reduction of 69.86%of 69.86% of the of the lesioned lesioned area. area.

Figure 3. Gastroprotective effect effect of of X. americana (XaAE) aqueous extract on absoluteabsolute ethanol-inducedethanol-induced (99%, 1 mL, orally) gastric lesions in Wistar rats. Animals received 0.9% 0.9% NaCl solution (lesioned (lesioned control, control, LC, 10 mL/kg, orally), orally), pantoprazole pantoprazole (P, (P, 40 mg/kg, orally) orally) or or XaAE XaAE (100, 200 and 400 mg/kg, orally). orally). Results were expressedexpressed asas mean mean± ±SEM SEM (6–7 (6–7 animals/group). animals/group). Statistically Statistically different different when when compared compared to tothe the lesioned lesioned control control group group (analysis (analysis of variance—ANOVA, of variance—ANOVA, followed byfollowed Dunnett’s by multiple Dunnett’s comparisons multiple comparisonstest, ** p < 0.001, test, *** ** p < 0.001, 0.0005, *** **** p

2.4.2. Induction Induction of Gastric Lesions by Acidified Acidified Ethanol X. americana aqueous extract promoted significant significant gastric protection against the acidified acidified ethanol-induced lesions (Figures (Figures 55 andand6 6).). The The results results showed showed that that the the rats rats orally orally pre-treated pre-treated with with the the extract (100, 200 and 400 mg/kg) presented presented a a percen percentagetage of of inhibition inhibition of of 44.86%, 44.86%, 80.56% 80.56% and and 94.93%, 94.93%, 2 respectively, inin contrastcontrast withwith thethe lesionedlesioned controlcontrol groupgroup (LC,(LC, 231.60231.60± ± 29.54 mm2).). Rats Rats treated treated with pantoprazole (P) presented a reduction of 51.99% of the lesioned area.area.

(a) (b) (c) (d) (e)

Figure 4. Typical photograph of the gastroprotective effect of aqueous extract of X. americana (XaAE) stem bark on absolute ethanol-induced (99%, 1 mL, orally) gastric lesions in rats. (a) lesioned control (NaCl 0.9%, 10 mL/kg, orally); (b) pantoprazole (40 mg/kg, orally); (c–e) XaAE (100, 200 and 400 mg/kg, orally, respectively).

Molecules 2018, 23, 112 4 of 17

(5.32 ± 0.13 vs. 5.57 ± 0.09 g/day/animal), and body weight (36.67 ± 0.27 vs. 36.33 ± 0.99 g) between the control and treated group, respectively.

2.4. Experimental Protocols

2.4.1. Induction of Gastric Lesions by Absolute Ethanol Administration of X. americana aqueous extract reduced the formation of absolute ethanol-induced ulcerative lesions (Figures 3 and 4). The percentage inhibition of lesions was 76.08%, 77.48% and 100% in rats pre-treated with the extract at doses of 100, 200 and 400 mg/kg, respectively, in contrast with the lesioned control group (LC, 116.5 ± 19.67 mm2). Rats treated with pantoprazole showed a reduction of 69.86% of the lesioned area.

Figure 3. Gastroprotective effect of X. americana (XaAE) aqueous extract on absolute ethanol-induced (99%, 1 mL, orally) gastric lesions in Wistar rats. Animals received 0.9% NaCl solution (lesioned control, LC, 10 mL/kg, orally), pantoprazole (P, 40 mg/kg, orally) or XaAE (100, 200 and 400 mg/kg, orally). Results were expressed as mean ± SEM (6–7 animals/group). Statistically different when compared to the lesioned control group (analysis of variance—ANOVA, followed by Dunnett’s multiple comparisons test, ** p < 0.001, *** p < 0.0005, **** p < 0.0001).

2.4.2. Induction of Gastric Lesions by Acidified Ethanol X. americana aqueous extract promoted significant gastric protection against the acidified ethanol-induced lesions (Figures 5 and 6). The results showed that the rats orally pre-treated with the extract (100, 200 and 400 mg/kg) presented a percentage of inhibition of 44.86%, 80.56% and 94.93%, Moleculesrespectively,2018, 23 in, 112 contrast with the lesioned control group (LC, 231.60 ± 29.54 mm2). Rats treated 5with of 18 pantoprazole (P) presented a reduction of 51.99% of the lesioned area.

(a) (b) (c) (d) (e)

FigureFigure 4.4. Typical photograph of the gastroprot gastroprotectiveective effect effect of of aqueous aqueous extract extract of of X.X. americana americana (XaAE)(XaAE) stemstem barkbark onon absoluteabsolute ethanol-induced (99%,(99%, 1 mL, orally) gastric lesions in rats. ( (aa)) lesioned lesioned control control (NaCl(NaCl 0.9%,0.9%, 1010 mL/kg,mL/kg, orally); (b) pantoprazole (40 mg/kg, orally); orally); ( (cc––ee)) XaAE XaAE (100, (100, 200 200 and and 400 400 mg/kg, mg/kg, orally, respectively). Moleculesorally, 2018 respectively)., 23, 112 5 of 17 Molecules 2018, 23, 112 5 of 17

Figure 5. Gastroprotective effect of the aqueous extract of X. americana (XaAE) stem bark on acidified Figure 5. Gastroprotective effect of the aqueous extract of X. americana (XaAE) stem bark on acidified ethanol-inducedFigure 5. Gastroprotective (0.3 M HCl/60% effect ethaof thenol, aqueous 1 mL, orally) extract gastric of X. americanalesions in (XaAEWistar) rats.stem Animalsbark on receivedacidified ethanol-induced (0.3 M HCl/60% ethanol, 1 mL, orally) gastric lesions in Wistar rats. Animals received 0.9% 0.9%ethanol-induced NaCl solution (0.3 (lesionedM HCl/60% control, ethanol, LC, 1 10mL, mL/k orally)g, orally),gastric lesionspantoprazole in Wistar (P, rats. 40 mg/kg,Animals orally) received or NaCl0.9% NaCl solution solution (lesioned (lesioned control, control, LC, LC, 10 mL/kg,10 mL/k orally),g, orally), pantoprazole pantoprazole (P, (P, 40 40 mg/kg, mg/kg, orally)orally) oror XaAE (100, 200 and 400 mg/kg, orally). Results were expressed as mean ±± SEM (6–7 animals/group). XaAEXaAE (100,(100, 200200 andand 400400 mg/kg,mg/kg, orally).orally). ResultsResults werewere expressedexpressed asas meanmean ± SEMSEM (6–7(6–7 animals/group).animals/group). Statistically different when compared to the lesi lesionedoned control group (ANOVA, followed by Dunnett’s Statistically different when compared˂ to the lesioned control group (ANOVA, followed by Dunnett’s multiple comparisons test,test, * *p p< 0.02,0.02, ** **p p< < 0.005, 0.005, **** ****p p< < 0.0001). 0.0001). multiple comparisons test, * p ˂ 0.02, ** p < 0.005, **** p < 0.0001).

(a) (b) (c)(d)( e) (a) (b) (c)(d)(e) Figure 6. Typical photograph of the gastroprotective effect of aqueous extract of X. americana (XaAE) stemFigureFigure bark 6.6. Typical on ethanol/HCl-induced photographphotograph ofof thethe gastroprotectivegastroprot(0.3 M HCl/60%ective effect ethanol, of aqueous 1 mL, orally) extract gastric of X. americana lesions in(XaAE) rats. (stemstema) Lesioned barkbark onon control ethanol/HCl-inducedethanol/HCl-induced (NaCl 0.9%, 10 mL/kg,(0.3 (0.3 M orally);HCl/60% (b )ethanol, ethanol,pantoprazole 1 1 mL, mL, (40orally) orally) mg/kg, gastric gastric orally); lesions lesions (c–e )in XaAE rats. (100,((aa)) LesionedLesioned 200 and controlcontrol400 mg/kg, (NaCl(NaCl orally, 0.9%,0.9%, respectively). 1010 mL/kg, mL/kg, orally);orally); ((bb)) pantoprazolepantoprazole (40(40 mg/kg,mg/kg, orally); ((c–e)) XaAEXaAE (100,(100, 200200 andand 400400 mg/kg,mg/kg, orally, orally, respectively). 2.4.3. Induction of Gastric Lesions by Indomethacin 2.4.3. Induction of Gastric Lesions by Indomethacin X. americana aqueous extract promoted significant gastric protection against indomethacin-induced lesionsX. (Figuresamericana 7 aqueousand 8). The extract doses promoted of 100, 200 significant and 400 gastric mg/kg protection promoted against inhibitions indomethacin-induced of 56.43%, 52.67% andlesions 64.91%, (Figures respectively, 7 and 8). Thein contrast doses of with 100, rats200 anof dthe 400 lesioned mg/kg promoted control group inhibitions (LC, 12.56 of 56.43%, ± 2.91 52.67%mm2). 2 Ratsand treated64.91%, with respectively, pantoprazole in contrast (P, 40 mg/kg)with rats show of edthe a lesioned reduction control of 98.70% group of the(LC, lesioned 12.56 ± area.2.91 mm ). Rats treated with pantoprazole (P, 40 mg/kg) showed a reduction of 98.70% of the lesioned area. 2.4.4. Evaluation of Antisecretory Activity 2.4.4. Evaluation of Antisecretory Activity After four hours of pylorus ligation in rats, it was observed that the intraduodenally administered aqueousAfter extract four hours of X. ofamericana pylorus stemligation bark in rats,(100 itmg/kg) was observed reduced that the thecontent intraduodenally by 48.65% andadministered the total acidityaqueous of extract the gastric of X. americanasecretion stemby 32.29% bark (100 when mg/kg) compared reduced to the the content lesioned by 48.65%control andgroup the (LC,total 0.74acidity ± 0.08of theg and gastric 17.90 secretion ± 1.53 Mequivby 32.29% [H+ ]/mL/4when comparedh, respectively). to the Ranitidine-treatedlesioned control grouprats had (LC, a + 45.95%0.74 ± 0.08reduction g and in 17.90 secreted ± 1.53 gastric Mequiv conten [Hts ]/mL/4and 50.34% h, respectively). in total acidity Ranitidine-treated (Table 3). rats had a 45.95% reduction in secreted gastric contents and 50.34% in total acidity (Table 3).

Molecules 2018, 23, 112 6 of 18

2.4.3. Induction of Gastric Lesions by Indomethacin X. americana aqueous extract promoted significant gastric protection against indomethacin-induced lesions (Figures7 and8). The doses of 100, 200 and 400 mg/kg promoted inhibitions of 56.43%, 52.67% and 64.91%, respectively, in contrast with rats of the lesioned control group (LC, 12.56 ± 2.91 mm2). Rats treated with pantoprazole (P, 40 mg/kg) showed a reduction of 98.70% of the lesioned area.

2.4.4. Evaluation of Antisecretory Activity After four hours of pylorus ligation in rats, it was observed that the intraduodenally administered aqueous extract of X. americana stem bark (100 mg/kg) reduced the content by 48.65% and the total acidity of the gastric secretion by 32.29% when compared to the lesioned control group (LC, 0.74 ± 0.08 g and 17.90 ± 1.53 Mequiv [H+]/mL/4 h, respectively). Ranitidine-treated rats Moleculeshad a 45.95% 2018, 23 reduction, 112 in secreted gastric contents and 50.34% in total acidity (Table3). 6 of 17 Molecules 2018, 23, 112 6 of 17

FigureFigure 7. GastroprotectiveGastroprotective effect effect of of the the aqueous aqueous extract extract of of X.X. americana americana (XaAE (XaAE)) stem stem bark bark on on Figure 7. Gastroprotective effect of the aqueous extract of X. americana (XaAE) stem bark on indomethacin-inducedindomethacin-induced (30 (30 mg/kg, mg/kg, s.c.) s.c.) gastric gastric lesions lesions in in Wistar Wistar rats. rats. Animals Animals received received 0.9% 0.9% NaCl NaCl indomethacin-induced (30 mg/kg, s.c.) gastric lesions in Wistar rats. Animals received 0.9% NaCl solutionsolution (lesioned (lesioned control, control, LC, LC, 10 10 mL/kg, mL/kg, orally), orally), pantoprazole pantoprazole (P, 40 (P, mg/kg, 40 mg/kg, orally) orally) or XaAE or XaAE(100, solution (lesioned control, LC, 10 mL/kg, orally), pantoprazole (P, 40 mg/kg, orally) or XaAE (100, 200(100, and 200 400 and mg/kg, 400 mg/kg,orally). Results orally). were Results expressed were expressed as mean ± asSEM mean (6–8± animals/group).SEM (6–8 animals/group). Statistically 200 and 400 mg/kg, orally). Results were expressed as mean ± SEM (6–8 animals/group). Statistically differentStatistically when different compared when to compared the lesioned to the control lesioned group control (ANOVA, group (ANOVA,followed by followed Dunnett’s by Dunnett’smultiple different when compared to the lesioned control group (ANOVA, followed by Dunnett’s multiple comparisonsmultiple comparisons test, * p ˂ test,0.05, ***p p< < 0.05, 0.005, ** p****< 0.005,p < 0.0001). **** p < 0.0001). comparisons test, * p ˂ 0.05, ** p < 0.005, **** p < 0.0001).

(a) (b) (c)(d)( e) (a) (b) (c)(d)(e) Figure 8. Typical photograph of the gastroprotective effect of aqueous extract of X. americana (XaAE) Figure 8. Typical photograph of the gastroprotective effect of aqueous extract of X. americana (XaAE) stemFigure bark 8. onTypical indomethacin-induced photograph of the(30 mg/kg, gastroprotective s.c.) gastric effect lesions of in aqueous rats. (a) extractLesioned of controlX. americana (NaCl stem bark on indomethacin-induced (30 mg/kg, s.c.) gastric lesions in rats. (a) Lesioned control (NaCl 0.9%,(XaAE) 10 stemmL/kg, bark orally); on indomethacin-induced (b) pantoprazole (40 mg/kg, (30 mg/kg, orally); s.c.) (c– gastrice) XaAE lesions (100, in200 rats. and (a400) Lesioned mg/kg, 0.9%, 10 mL/kg, orally); (b) pantoprazole (40 mg/kg, orally); (c–e) XaAE (100, 200 and 400 mg/kg, orally,control respectively). (NaCl 0.9%, 10 mL/kg, orally); (b) pantoprazole (40 mg/kg, orally); (c–e) XaAE (100, 200 andorally, 400 respectively). mg/kg, orally, respectively). Table 3. Effect of X. americana (XaAE) aqueous extract on gastric secretion parameters in Wistar rats Table 3. Effect of X. americana (XaAE) aqueous extract on gastric secretion parameters in Wistar rats subjectedsubjected to to pylorus pylorus ligation. ligation. Dose Gastric pH Total Acidity (Mequiv. Groups Dose Gastric Content (g) Gastric pH Total Acidity (Mequiv. Groups Gastric Content (g) + (mg/kg,(mg/kg, i.d.) i.d.) Value [H[H+]/mL/4]/mL/4 h)h) Non-lesionedNon-lesioned control control - - 0.23 0.23 ±± 0.030.03 ******** 4.39 ± 0.51 * 12.13 12.13 ±± 2.162.16 * * LCLC - - 0.74 0.74 ±± 0.080.08 3.25 ± 0.10 17.90 17.90 ±± 1.531.53 RanitidineRanitidine 60 60 0.40 0.40 ±± 0.080.08 **** 3.83 ± 0.15 8.89 8.89 ±± 0.990.99 ****** XaAEXaAE 100 100 0.38 0.38 ±± 0.070.07 **** 3.40 ± 0.08 12.12 12.12 ±± 0.840.84 * * LC:LC: lesioned lesioned control control group group (NaCl (NaCl 0.9%,0.9%, 2.52.5 mL/kg,mL/kg, intraduodenally).intraduodenally). Values representrepresent meanmean ±± SEMSEM (5–7(5–7 animals/group). animals/group). Statistically Statistically differentdifferent whenwhen comparedcompared to the lesioned controlcontrol groupgroup (ANOVA,(ANOVA, followedfollowed by by Dunnett’s Dunnett’s multiple multiple comparisons comparisons test,test, ** pp ˂˂ 0.05,0.05, ** p < 0.005, *** p << 0.0005,0.0005, ******** pp << 0.0001). 0.0001).

2.4.5.2.4.5. Evaluation Evaluation of of the the Participation Participation of of Sulfhydryl Sulfhydryl GroupsGroups (-SH) and Nitric Oxide (ON)(ON) in in Gastroprotection Gastroprotection AsAs expected, expected, in in the the absence absence of of blockade,blockade, XaAEXaAE aqueousaqueous extract and carbenoxolone reducedreduced the the gastricgastric lesions lesions in in rats rats by by 82.69% 82.69% andand 74.17%,74.17%, resprespectively,ectively, when compared toto thethe lesionedlesioned controlcontrol groupgroup (LC, (LC, 137.40 137.40 ±± 18.9018.90 mm²).mm²). However,However, inin thethe presencepresence of blockade, i.e.,i.e., inin thethe presencepresence ofof sulfhydrylsulfhydryl gr groupoup inhibitor inhibitor NN-ethylmaleimide-ethylmaleimide (NEM,(NEM, 1010 mg/kg, i.p.), the gastroprotectivegastroprotective effecteffect ofof

Molecules 2018, 23, 112 7 of 18

Table 3. Effect of X. americana (XaAE) aqueous extract on gastric secretion parameters in Wistar rats subjected to pylorus ligation.

Groups Dose (mg/kg, i.d.) Gastric Content (g) Gastric pH Value Total Acidity (Mequiv. [H+]/mL/4 h) Non-lesioned control - 0.23 ± 0.03 **** 4.39 ± 0.51 * 12.13 ± 2.16 * LC - 0.74 ± 0.08 3.25 ± 0.10 17.90 ± 1.53 Ranitidine 60 0.40 ± 0.08 ** 3.83 ± 0.15 8.89 ± 0.99 *** XaAE 100 0.38 ± 0.07 ** 3.40 ± 0.08 12.12 ± 0.84 * LC: lesioned control group (NaCl 0.9%, 2.5 mL/kg, intraduodenally). Values represent mean ± SEM (5–7 animals/group). Statistically different when compared to the lesioned control group (ANOVA, followed by Dunnett’s multiple comparisons test, * p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.0001).

2.4.5. Evaluation of the Participation of Sulfhydryl Groups (-SH) and Nitric Oxide (ON) in Gastroprotection As expected, in the absence of blockade, XaAE aqueous extract and carbenoxolone reduced the gastric lesions in rats by 82.69% and 74.17%, respectively, when compared to the lesioned control group (LC, 137.40 ± 18.90 mm2). However, in the presence of blockade, i.e., in the presence of sulfhydryl group inhibitor N-ethylmaleimide (NEM, 10 mg/kg, i.p.), the gastroprotective effect of X. americana aqueous extract was reversed (9.92%) when compared to the lesioned control group (LC, 311.60 ± 40.82 mm2). In turn, carbenoxolone continued to be active, promoting a significant reductionMolecules of 74.48% 2018, 23 of, 112 the gastric lesions. The comparisons between the groups before7 of 17 and after blockade areX. americana also shown aqueous in extract Figure was9. reversed (9.92%) when compared to the lesioned control group (LC, Administrations311.60 ± 40.82 mm²). of X. In americana turn, carbenoxoloneaqueous continue extractd to and be active, carbenoxolone promoting a reducedsignificant thereduction gastric lesions area in ratsof 74.48% by 64.00% of the gastric and lesions. 45.10%, The respectively, comparisons between when the compared groups before to and the after lesioned blockade control are group (LC, 109.43also± shown16.08 in mm Figure2) in9. the non-blocked condition. However, after blockade with the nitric Administrations of X. americana aqueous extract and carbenoxolone reduced the gastric lesions oxide synthasearea in inhibitorrats by 64.00%Nω and-nitro- 45.10%,L-arginine-methyl-ester respectively, when compared (L-NAME, to the lesioned 70 mg/kg, control group i.p.), (LC, there was no inhibitory109.43 effect ± of 16.08 aqueous mm²) in extract the non-blocked of X. americana condition.and However, carbenoxolone after blockade on the with gastric the nitric lesions oxide in relation to the lesionedsynthase control inhibitor group Nω-nitro- (199.20L-arginine-methyl-ester± 31.86 mm (2L).-NAME, The comparisons70 mg/kg, i.p.), there between was no theinhibitory groups before and after blockadeeffect of aqueous are also extract shown of X. americana in Figure and 10 carbenoxolone. Depletion on of the sulfhydryl gastric lesions and in nitric relation oxide to the groups by lesioned control group (199.20 ± 31.86 mm²). The comparisons between the groups before and after pre-treatmentblockade with are NEM also shown and L-NAME,in Figure 10. respectively,Depletion of sulfhydryl were ableand nitric to eliminate oxide groups the by gastroprotective pre-treatment effect of XaAE. with NEM and L-NAME, respectively, were able to eliminate the gastroprotective effect of XaAE.

Figure 9. Gastroprotective effect of the X. americana aqueous extract (XaAE) stem bark on absolute Figure 9. Gastroprotectiveethanol-induced (99%, effect 1 mL, oforally) the gastricX. americana lesions inaqueous Wistar rats extract before (non-blocked, (XaAE) stem NaCl bark 0.9%, on absolute ethanol-induced10 mL/kg, (99%, i.p) and 1 after mL, (blocked) orally) gastrictreatment lesions with NEM in (10 Wistar mg/kg, rats i.p.). before The animals (non-blocked, received 0.9% NaCl 0.9%, 10 mL/kg,NaCl i.p) solution and after orally (blocked) (lesioned control, treatment LC, 10 with mL/kg), NEM carbenoxolone (10 mg/kg, (orally) i.p.). or TheXaAE animals(orally). received 0.9% NaClResults solution were orally expressed (lesioned as mean control,± SEM (6–7 LC, animals/group). 10 mL/kg), Statistically carbenoxolone different (orally) when compared or XaAE (orally). to the lesioned control group (ANOVA, followed by Dunnett’s multiple comparisons test, * p ˂ 0.05, ± Results were*** p expressed< 0.0005, **** as p < mean 0.0001). # SEMThe non-blocked (6–7 animals/group). and blocked control Statistically groups were different compared when by an compared to the lesionedunpaired control Student’s group t-test, (ANOVA, ## The non-blocked followed and byblocked Dunnett’s XaAE 100 multiple mg/kg groups comparisons were compared test, * p < 0.05, *** p < 0.0005,by an ****unpairedp < 0.0001).Student’s t#-test.The non-blocked and blocked control groups were compared by an unpaired Student’s t-test, ## The non-blocked and blocked XaAE 100 mg/kg groups were compared by 2.4.6. Determination of Mucus Concentration of the Gastric Mucosa an unpaired Student’s t-test. Ligation of the pylorus in the lesioned control group rats promoted a significant decrease in gastric mucus levels (7.89 ± 0.56 μg Alcian blue/g of tissue) compared to those of the false-operated group (non-lesioned, 13.35 ± 0.96 μg of Alcian blue/g of tissue). Rats treated with XaAE did not have an increased of gastric mucus levels (5.93 ± 0.26 μg Alcian blue/g of tissue) compared to the lesioned control group (LC, 7.89 ± 0.56 μg Alcian blue/g of tissue). However, carbenoxolone (CBX, 200 mg/kg) promoted a significant increase in gastric mucus levels of 148.79% in relation to the lesioned control group (Figure 11).

Molecules 2018, 23, 112 8 of 18

2.4.6. Determination of Mucus Concentration of the Gastric Mucosa Ligation of the pylorus in the lesioned control group rats promoted a significant decrease in gastric mucus levels (7.89 ± 0.56 µg Alcian blue/g of tissue) compared to those of the false-operated group (non-lesioned, 13.35 ± 0.96 µg of Alcian blue/g of tissue). Rats treated with XaAE did not have an increased of gastric mucus levels (5.93 ± 0.26 µg Alcian blue/g of tissue) compared to the lesioned control group (LC, 7.89 ± 0.56 µg Alcian blue/g of tissue). However, carbenoxolone (CBX, 200 mg/kg) promoted a significant increase in gastric mucus levels Moleculesof 148.79% 2018 in, 23 relation, 112 to the lesioned control group (Figure 11). 8 of 17 Molecules 2018, 23, 112 8 of 17

FigureFigure 10. Gastroprotective 10. Gastroprotective effect of effect the X. of americana the X. americana aqueous aqueousextract (XaAE) extract stem (XaAE) bark stemon absolute bark on ethanol-induced absolute (99%,Figure ethanol-induced1 10.mL, Gastroprotective orally) gastric (99%, lesions effect 1 mL, ofin theWistar orally) X. americana rats gastric before lesionsaqueous (non-blocked, in extract Wistar (XaAE)NaCl rats 0.9%, before stem 10 bark (non-blocked, mL/kg, on absolute i.p.) and NaCl ethanol-induced after 0.9%, (blocked) treatment(99%,10 1 mL, mL/kg, with orally) L-NAME i.p.) gastric and (70lesions after mg/kg, (blocked) in Wistar i.p). The treatment rats animals before with (non-blocked, received L-NAME 0.9% NaCl (70 NaCl mg/kg, 0.9%, solution 10 i.p). mL/kg, orally The animalsi.p.) (lesioned and receivedafter control, (blocked) LC, 10treatment mL/kg),0.9% with NaClcarbenoxolone L-NAME solution orally(70(orally) mg/kg, (lesioned or XaAEi.p). The control, (orally). animals LC, Results 10received mL/kg), were 0.9% expressed carbenoxolone NaCl solutionas mean (orally) orally± SEM or(lesioned (6–7 XaAE animals/group). (orally). control, LC, Statistically10 mL/kg),Results carbenoxolone different were expressed when (orally) compared as mean or XaAE ±to SEMthe (orally). lesioned (6–7 animals/group).Results control were group expressed Statistically(ANOVA, as mean followed different ± SEM by when (6–7 Dunnett’s comparedanimals/group). multiple comparisonsStatisticallyto the lesioneddifferent test, * p control˂when 0.05, compared group** p < 0.005). (ANOVA, to the# The lesioned followed non-blocked control by Dunnett’s and group blocked (ANOVA, multiple contro comparisons followedl groups bywere test,Dunnett’s compared * p < 0.05, multiple by an unpairedcomparisons** p

Figure 11. Effect of the aqueous extract from X. americana (XaAE) stem bark on the gastric mucus contentFigure 11. 11.after EffectEffect pylorus of of the the aqueousligation aqueous extractin extract Wistar from from X.rats. americana X. Th americanae animals(XaAE) (XaAE) stemreceived barkstem 0.9% onbark the NaClon gastric the solution mucusgastric contentmucusorally (lesionedaftercontent pylorus after control, ligationpylorus LC, in 10ligation Wistar mL/kg), rats.in carbenoxolone Wistar The animals rats. received Th(oerally) animals 0.9%or XaAE NaClreceived (orally). solution 0.9% The orally NaCl non-lesioned (lesioned solution control, grouporally receivedLC,(lesioned 10 mL/kg), nocontrol, treatment. carbenoxolone LC, 10 Results mL/kg), (orally) were carbenoxolone orexpressed XaAE (orally). as(o rally)mean The or ± non-lesioned XaAESEM (6–7(orally). animals/group). group The received non-lesioned noStatistically treatment. group differentResultsreceived were whenno treatment. expressed compared asResults meanto the ± werelesionedSEM expressed (6–7 control animals/group). as group mean (ANOVA, ± StatisticallySEM (6–7 followed animals/group). different by Dunnett’s when comparedStatistically multiple to comparisonsthedifferent lesioned when control test, compared **** group p < 0.0001). (ANOVA,to the lesioned followed control by Dunnett’s group (ANOVA, multiple comparisonsfollowed by test,Dunnett’s **** p < multiple 0.0001). comparisons test, **** p < 0.0001). 3. Discussion 3. Discussion This study investigated the antiulcerogenic pharmacological activity of the aqueous extract of X. americanaThis study stem investigated bark in acute the gastricantiulcerogenic lesions induced pharmacological by ethanol, activity acidified of theethanol aqueous (HCl/ethanol) extract of andX. americana indomethacin, stem bark as well in acute as its gastricinfluence lesions on gastric induced acid by secretion ethanol, parameters,acidified ethanol sulfhydryl (HCl/ethanol) groups, nitricand indomethacin, oxide, and mucus. as well as its influence on gastric acid secretion parameters, sulfhydryl groups, nitricThe oxide, results and obtainedmucus. demonstrate for the first time that the aqueous extract of the X. americana has aThe gastroprotective results obtained effect demonstrate in the acute for modelsthe first tested. time that These the modelsaqueous are extract the most of the used X. americana because theyhas a represent gastroprotective the etiological effect inagents the acute most modelscommonl tested.y involved These inmodels gastric are ulcers the most[19]. Hydrochloricused because acid,they representfree radicals, the ethanoletiological and agents non-steroi mostdal commonl anti-inflammatoryy involved in drugs gastric (NSAIDs) ulcers [19]. and Hydrochloric H. pylori are someacid, freeof the radicals, aggressive ethanol factors and [4,20,21]. non-steroi dal anti-inflammatory drugs (NSAIDs) and H. pylori are some of the aggressive factors [4,20,21].

Molecules 2018, 23, 112 9 of 18

3. Discussion This study investigated the antiulcerogenic pharmacological activity of the aqueous extract of X. americana stem bark in acute gastric lesions induced by ethanol, acidified ethanol (HCl/ethanol) and indomethacin, as well as its influence on gastric acid secretion parameters, sulfhydryl groups, nitric oxide, and mucus. The results obtained demonstrate for the first time that the aqueous extract of the X. americana has a gastroprotective effect in the acute models tested. These models are the most used because they represent the etiological agents most commonly involved in gastric ulcers [19]. Hydrochloric acid, free radicals, ethanol and non-steroidal anti-inflammatory drugs (NSAIDs) and H. pylori are some of the aggressive factors [4,20,21]. In the acute toxicity test, X. americana aqueous extract, when given orally at the dose of 2000 mg/kg, did not produce signs of toxicity or death. These data indicate that its LD50 is above 2000 mg/kg and, therefore, can be considered non-toxic under the present experimental conditions. Although Maikai et al. [22] did not register death loss (DL50 > 5000 mg/kg) in mice and rats by oral and intraperitoneal administration of the aqueous extract of X. americana stem bark, clinical signs of toxicity such as excitement, restlessness, difficulty breathing, loss of appetite, general weakness and depression were observed at high doses, as well as body mass loss. Similar results to ours were obtained by Agyigra et al. [23] who estimated a DL50 by oral administration of the methanolic extract of X. americana stem bark of more than 5000 mg/kg in rats and mice. Ethanol is considered to be one of the most intense agents inducing gastric lesions because it promotes serious mucosal disorders due to its direct action, while gastric acidity has little effect on the formation of ulcers [24,25]. Lesions formed by ethanol result from a series of factors such as decreased gastric mucus production, prostaglandins, and sulfhydryl groups, decreased gastric motility, altered transmucosal potential and gastric mucosal blood flow, as the increased generation of free radicals, the release of histamine, ischemia and gastric vascular permeability [20]. Its administration produces hemorrhagic, and necrotic lesions in the mucosa consisting of elongated bands generally parallel to the longitudinal axis of the stomach [26]. The presence of HCl accelerates the process [24]. Oral administration of XaAE reduced the ethanol- and HCl/ethanol-induced gastric lesions at all doses tested, indicating a gastroprotective action. Indomethacin has been reported to decrease antioxidant enzyme activity [27], increase oxidative stress and inhibit cyclooxygenase (COX), inducing gastric ulceration in the stomach [28,29]. This ulcerogenic mechanism of indomethacin is attributed to a reduced secretion of bicarbonate, mucus and blood flow and inhibition of mucosal repair [30]. In the indomethacin-induced acute ulcer model, in which prostaglandins are suppressed, the extract was able to reduce gastric lesions initially suggesting an inhibitory action on the cyclooxygenase pathway, reducing the production of cytoprotective prostaglandins. However, other mechanisms may contribute to this activity, since Soro [12] showed that the aqueous extract of the X. americana stem bark (25, 50 and 100 mg/kg, i.p.) presented analgesic activity in abdominal writhing tests by acetic acid and formalin only in the 2nd phase, which characterizes the possible anti-inflammatory action of the extract. Proton pump inhibitors are “prodrugs” considered to be very effective and relatively equivalent, reducing by up to 95% the daily production of gastric acid [31]. Pantoprazole has a linear (and therefore more predictable) pharmacokinetic profile, less variable bioavailability and a rapid onset of action when compared to other class representatives such as omeprazole and lansoprazole [32]. In all models of induction of acute gastric lesions, rats treated with pantoprazole showed a reduction of the lesioned area. The phytochemical study determined the presence of alkaloids, flavonoids, lignans, monoterpenes, sesquiterpenes and diterpenes, naphthoquinones, saponins, hydrolysable tannins, triterpenes, and steroids. The HPLC-DAD-MS/MS identified procyanidins B, procyanidin C, and catechin/epicatechin as the most abundant compounds. Molecules 2018, 23, 112 10 of 18

At present, there is no way to attribute the gastroprotective activity to one of the identified major compounds and consequently to estimate their potency. We intend to try to correlate the effect of the extract with those obtained from the isolated compounds. However, the possibility of synergistic interactions between these compounds in the crude extract may make it difficult to establish some correlation. Shettar [13] reported the presence of flavonoids in phytochemical analysis and their high antioxidant potential of the aqueous extract of X. americana leaves. His studies suggest that leaves can be used as natural antioxidants to prevent the incidence and progression of many diseases. In recent years, flavonoids have been the most widely studied natural compounds with potential for antiulcer activity [33–35]. In this group, the catechins are the ones with the most well-defined mechanism of action, which include histidine decarboxylase inhibition [19,36] and proton pump inhibition [37]. The gastroprotective activity for epicatechin was also reported by Rozza et al. [38] that demonstrated that this protection of mucosa occurs by stimulation of the increase of the mucus barrier and neutralizing the gastric juice, as well as through the involvement of SH compounds, adrenergic activation, NO, SOD and HSP-70. Catechin and its derivatives act as gastroprotective agents by inhibition of gastric H+,K+-ATPase [37]. Catechin also demonstrated a gastroprotective mechanism of endocrine action by reducing levels of gastrin and histamine [39]. Lewis [40] also was shown the gastroprotective activity of proanthocyanidin B-2 in mice against stress-induced gastric lesions. The obstruction promoted by the pyloric ligature causes accumulation of acid in the gastric content and exaggerated distension of the mucosa, consequently leading to gastric hypersecretion and the formation of lesions [41]. In addition, the formation of endogenous histamine and its release from mast cells also is related to the pathogenesis of gastric ulcers produced by pyloric ligation, suggesting that H2 antihistamines and histidine decarboxylase inhibitors may be useful in the prevention of such lesions [36]. In this model, treatment with the aqueous extract of X. americana stem bark reduced both the volume and total acidity of gastric juice, the major parameters of basal gastric secretion. Considering that inhibition of the proton pump is one of the mechanisms of action associated with catechins [37], it is possible to suggest that the inhibitory action of gastric acidity of XaAE may be related to the presence of these constituents. In order to understand the mechanisms involved in the gastroprotective action promoted by the XaAE, experimental models have been performed in order to investigate the participation of sulfhydryl groups (-SH), nitric oxide and mucus. Endogenous sulfhydryl compounds are important for maintaining the integrity of the gastric mucosa, by participating in the production of mucus and binding to free radicals formed during inflammation or exposure to harmful agents, promoting neutralization [42,43]. Ethanol decreases the activity of -SH groups, causing oxidative stress, and confirming the involvement of these groups with the gastroprotective effect [43]. According to El-Ashmawy et al. [44] the garlic extract, which is rich in sulfhydryl compounds, demonstrated antioxidant and anti-inflammatory activity required for protection of gastric mucosa. Changes in intracellular redox status including the increase in intracellular levels of reactive − oxygen species, such as superoxide anion (O2 ), hydroxyl radical (OH) and hydrogen peroxide (H2O2), decreased levels of sulfhydryl compounds and mainly peroxidation, are important markers of oxidative stress involved in the pathogenesis of various inflammatory or tumoral diseases [45,46]. However, multiple studies suggest the protective effect of medicinal plants and extracts in the treatment and prevention of the burden of tissue oxidative stress, with a significant improvement in lipid peroxidation biomarker levels [47–49]. In this study, data demonstrated that gastric exposure to ethanol-associated to depletion of the sulfhydryl compounds promoted by NEM (-SH group inhibitor) decreased the gastroprotection of mucosa. Our results also showed that treatment with NEM eliminated the gastroprotective effect of the extract previously observed, suggesting a possible participation of sulfhydryl groups in its action. Molecules 2018, 23, 112 11 of 18

NO also plays a mucosal defensive role by regulating the blood flow and stimulating gastric mucus secretion by the activation of the enzyme guanylyl cyclase [50]. Inhibition of nitric oxide synthase (NOS) by L-NAME results in a decrease in this endogenous mediator and, consequently, an increase of the gastric lesion [51]. In our experiment, the blockade on NOS by L-NAME promoted a significant increase in gastric lesions and also reversed the gastroprotective action of X. americana aqueous extract, also indicating a possible contribution of this mediator. It should be noted that although the 70 mg/kg dose of L-NAME with a focus on gastroprotection mechanisms involving the role of nitric oxide as a mediator of gastroprotection has been obtained from studies in the literature [51–54], this same dose is also capable of increasing the blood pressure of rats [55]. The primary function of the mucus layer is structural, creating a stable layer that supports surface contact with acid and acting as a protective physical barrier against luminal pepsin. Secretion of − bicarbonate (HCO3 ) adhered to the mucus layer (stable and adherent gel) creates a nearly neutral pH gradient on the epithelial surface of the stomach and duodenum, providing the first line of defense against luminal acid [56]. Carbenoxolone, the cytoprotective agent used as the standard control in the model of determination of gastric mucus concentration, is a semi-synthetic derivative of the glycyrrhizinic acid, the active ingredient of Glycyrrhiza glabra, which has been extensively studied and reported to have anti-ulcer effects in experimental animals and humans [7]. Carbenoxolone maintains the prostaglandin content of the gastric mucosa at high levels due to its inhibitory action on the catabolic enzymes 15-hydroxy-prostaglandin-dehydrogenase and ∆13-prostaglandin-reductase, besides increasing cyclic AMP levels by inhibition of mucosal phosphodiesterases. High levels of prostaglandins promote the action of mucosal defense against ulceration [40]. Our data show that carbenoxolone increased the concentration of Alcian blue complexed to mucus, but this effect was not verified with the extract of X. americana, thus initially rejecting the hypothesis of the contribution of mucus in the gastroprotective effect of the extract.

4. Materials and Methods

4.1. Collection of Plant Material X. americana L. stem bark was collected in the Letras rach (Coordinates: 08◦14005.69” S, 039◦09031.39” W, 425 m altitude), in the municipality of Salgueiro, state of Pernambuco, Brazil, during the flowering period in November 2015. Plenty of material of the sampled species was deposited in the Semi-Arid Tropic Herbarium (HTSA), of Embrapa, in the municipality of Petrolina, state of Pernambuco, Brazil, under registry number HTSA 6633.

4.2. Extract Preparation and Phytochemical Profile (TLC) To prepare the extract, stem barks were powdered in a knife mill (Wyllie Macro—TE 650), and then immersed in distilled water, using the proportion of 20% of their dry weight, and reserved for 24 h. The solution was then filtered and lyophilized. The yield of X. americana L. (XaAE) stem bark aqueous extract was 15.76% (w/w), which was stored under refrigeration (6–10 ◦C) until pharmacological tests and phytochemical analyses. The extract was previously analyzed by thin layer chromatography (TLC) to identify the phytochemical classes [57].

4.3. High Performance Liquid Chromatography (HPLC-DAD-MS/MS) The extract was subjected to Liquid Chromatography coupled to Mass Spectrometry (LC-MS), using an octadecylsilane column (250 × 4.6 mm, 5 µm, Luna C18, Phenomenex-Shimadzu, Kyoto, Japan) as stationary phase, and the mobile phase consisted of 2 solvents: solvent A-0.1% formic acid in ultrapure water and solvent B-0.1% formic acid in methanol (HPLC grade) with flow rate of 1.0 mL/min. Molecules 2018, 23, 112 12 of 18

The stationary phase was maintained at 30 ◦C and the volume injected was 20 µL for the samples (1 mg/mL) in the high performance liquid chromatography coupled to the diode array detector and the mass spectrometer (HPLC-DAD-MS/MS) for monitored analyses of 190 to 400 nm and 50 to 1000 m/z. The analyses used an LC-20 (Shimadzu, Kyoto, Japan) equipped with a quaternary pump system model LC-20ADVP, degasser model DGU-20A, PDA detector model SPD-20AVP, oven model CTO-20ASVP, automatic injector model SIL-20ADVP and controller model SCL-20AVP coupled to a mass spectrometer from AmaZonL (Bruker Daltonics, Billerica, MA, USA) equipped with an electrospray ionization source operating in the analyzer mode and by capture of positive and negative ions to divide the HPLC eluent and a flow rate of 0.2 mL/min was introduced at the source. The following parameters of the mass spectrometer were used: capillary voltage of 3.5 kV; desolvation temperature of 320 ◦C; gas flow of 10 L/min; and pressure of 60 psi, using nitrogen as drying and nebulization gas.

4.4. Animals Three-month-old Wistar rats (Rattus norvegicus) and Swiss mice (Mus musculus) of both sexes were provided by the Vivarium of the Department of Physiology and Pharmacology of the Federal University of Pernambuco (DFF/UFPE) and the Aggeu Magalhães/Fiocruz Research Center (CPqAM/Fiocruz), respectively. The animals were kept under controlled lighting, temperature (22 ± 3 ◦C) and humidity (55–60%) conditions, and received standard water and diet ad libitum. The experimental protocols were submitted and approved by the UFPE Ethics Committee on Animal Use (CEUA) of the UFPE (Process 0021/2016).

4.5. Acute Toxicity Acute toxicity was evaluated in Swiss mice of both sexes (n = 3/group) [58]. The animals were deprived of food for 12 h and then given a single oral dose of XaAE (2000 mg/kg) or vehicle (0.9% NaCl solution, 10 mL/kg). After administration, the animals were observed individually during 30, 60, 120, 180, 240, 300 and 360 min intervals and, daily for 14 days. The following parameters were evaluated: behavioral changes, clinical signs of toxicity, consumption of water and feed (non-cumulative), and variation of body mass [59].

4.6. Experimental Protocols

4.6.1. Induction of Gastric Lesions by Absolute Ethanol After 16 h of fasting, the animals (n = 6–7/group) were pre-treated orally with 0.9% NaCl (lesioned control, LC), pantoprazole (P, 40 mg/kg) or XaAE (100, 200 and 400 mg/kg) one hour before administration of the ulcerogenic agent (absolute ethanol, 1 mL, orally) according to the method described [60], with minor modifications. The animals were euthanized in a CO2 chamber one hour after the induction of the lesions. The stomachs were removed, photographed and the gastric ulcer area was determined with the aid of Image J Software version 1.4 (Bethesda, MD, USA). The results were expressed as ulcerative lesion area (mm2).

4.6.2. Induction of Gastric Lesions by Acidified Ethanol (HCl/Ethanol) After 16 h of fasting, the animals (n = 6–7/group) were pre-treated orally with 0.9% NaCl solution (lesioned control, LC), pantoprazole (P, 40 mg/kg) or XaAE (100, 200 and 400 mg/kg) one hour before administration of the ulcerogenic agent (solution 0.3 M HCl/60% ethanol, 1 mL orally) according to the method described [61], with minor modifications. The animals were euthanized in a CO2 chamber and the gastric ulcer areas were determined as previously described. Molecules 2018, 23, 112 13 of 18

4.6.3. Induction of Gastric Lesions by Indomethacin After 16 h of fasting, the animals (n = 6–8/group) were pre-treated orally with 0.9% NaCl (lesioned control, LC), pantoprazole (P, 40 mg/kg) or XaAE (100, 200 and 400 mg/kg). After 30 min of treatment, gastric lesions were induced with indomethacin (30 mg/kg, s.c.) according to the method described [62], with minor modifications. The animals were euthanized in a CO2 chamber 6 h after the induction of the lesions and the gastric ulcer areas were determined as previously described.

4.6.4. Evaluation of Antisecretory Activity This methodology was performed according to the protocol previously described [63], with some adaptations. The animals were divided into 4 groups (n = 5–7) and fasted for 16 h with free access to 5% water solution. For pylorus ligation, the animals were anesthetized (xylazine 6 mg/kg associated with ketamine 60 mg/kg, i.p.), the abdomen was opened for exposure of the stomach and the pylorus was tied with a suture. Immediately after pylorus ligation, NaCl solution 0.9% (lesioned control), ranitidine (60 mg/kg) or XaAE (100 mg/kg) were administered intraduodenally and the abdominal wall was sutured. Four hours later, the animals were euthanized by CO2 asphyxiation, the esophagus was pinched, the stomach removed, and the gastric contents collected and centrifuged at 176× g/30 min. Weight and pH of the gastric contents were determined. Total acidity of the gastric contents was determined by titration, using 1% phenolphthalein as indicator. The total concentration of the acid was expressed as Mequiv. [H+]/mL/4 h. For this experiment, a non-lesioned control group was used, in which the animals did not receive intraduodenal treatment, but underwent surgery stress.

4.6.5. Evaluation of the Participation of Sulfhydryl Groups (-SH) and Nitric oxide (NO) in Gastroprotection For determination of the role of sulfhydryl groups and nitric oxide, after 24 h of fasting the animals were separated into 12 groups (n = 6–7). Three groups received pre-treatment with 0.9% NaCl (lesioned control, i.p.) and three groups with N-ethylmaleimide (NEM, 10 mg/kg, i.p.). To evaluate the participation of NO, three groups were pre-treated with 0.9% NaCl (lesioned control, i.p.) and three groups treated with Nω-nitro-L-arginine-methyl-ester (L-NAME, 70 mg/kg, i.p.). After 30 min of pre-treatment, each group received an oral dose of NaCl 0.9% carbenoxolone (CBX, 100 mg/kg) and XaAE (100 mg/kg). After 1 h, all the animals received 1 mL of absolute ethanol (orally) for induction of gastric lesions. After 1 h of ethanol administration, the rats were euthanized in a CO2 chamber, the stomachs removed and opened along the greater curvature. The stomachs were removed, photographed and the gastric lesion area was determined with the aid of Image J Software. The results were expressed as ulcerative lesion area (mm2)[51,64].

4.6.6. Determination of Mucus Concentration of the Gastric Mucosa The content of soluble glycosaminoglycans in the gastric mucosa was investigated using the Alcian blue dye, specific for acid mucins. The animals were divided into four groups (n = 6–7): NaCl 0.9% (lesioned control), carbenoxolone (CBX, 200 mg/kg), XaAE (100 mg/kg) and Sham-operated (non-lesioned control). After 1 h of treatment, the animals were anesthetized (xylazine, 6 mg/kg associated with ketamine, 60 mg/kg, i.p.) and the pylorus was tied. After 4 h of ligation, the animals were euthanized, and the esophagus pinched so that stomach contents were not lost. The stomachs were removed, washed out with distilled water, dried, and opened along the great curvature to remove the gastric material to be weighed. The non-glandular portion was discarded, and the glandular portion was weighed and reserved in an individual container with 10 mL of 0.1% Alcian blue solution for 2 h. Excess of dye was removed by washing the stomach with 7 mL of 0.25 mol/L sucrose solution; a first wash was performed for 15 min and a second for 45 min. The dye complexed to the mucus of the stomach wall and it was removed with 10 mL of 0.5 mol/L magnesium chloride solution, mixing by vortexing, for 1 min in intervals of 30 min, for 2 h. The stomachs were removed and Falcon tubes Molecules 2018, 23, 112 14 of 18 with the solution were stored in the refrigerator for approximately 8 h. After this time, the tubes were shaken for 1 min, aliquots of 4 mL were collected and added to 4 mL of ethyl ether and vortexed for 2 min. The obtained emulsion was centrifuged for 10 min at 1016.6× g/25 ◦C. The tubes were placed in an ice bath, the film formed above the supernatant was discarded and the aqueous phase separated. The results were obtained by spectrophotometric reading at 595 nm and interpolated in a standard curve of Alcian blue (100, 50, 25, 12.5 and 6.25 µg/mL), to calculate the concentration (µg) of the dye retained in the stomach in relation to the control group [65].

4.6.7. Statistical Analysis Values are expressed as mean ± standard error of the mean (SEM). Differences between groups were determined by analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. The level of significance for rejection of the null hypothesis was 5% (p < 0.05). In acute toxicity and influence of sulfhydryl groups and nitric oxide on gastroprotection, an unpaired Student’s t-test was used to check statistical differences between the two groups. Statistical analyses were performed using the GraphPad Prism version 7.0 (GraphPad Software, Inc., La Jolla, CA, USA).

5. Conclusions The set of results indicates that X. americana aqueous extract has a gastroprotective action whose initial mechanism of action involves the participation of sulfhydryl groups, nitric oxide, and antisecretory activity. The data also indicate that flavonoids, procyanidins B, procyanidins C, and catechin/epicatechin, identified as major constituents, are related to this activity.

Acknowledgments: To Norberto Peporine Lopes and José Carlos Tomaz from the Center for Mass Spectrometry of Organic Micromolecules (CEMMO) of the Faculty of Pharmaceutical Sciences of the University of São Paulo (Ribeirão Preto) for the allowing us to use the LC-MS. Author Contributions: T.P.A., L.D.K.T.d.P. and S.A.B. were responsible for the development of experimental protocols. J.R.G.d.S.A. contributed to the preparation of the aqueous extract. P.J.R.N. and L.A.R. were responsible for the chromatographic analysis. A.G.W., G.F.R.C. and T.P.A. conducted the analyses, interpreted the data, and drafted the manuscript. All authors read and approved the final manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the XaAE (aqueous extract of X. americana stem bark) are available from the authors.

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