gastroprotective activity of the ethanol extract from the inner bark of caesalpinia pyramidalis in...

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Gastroprotective activity of the ethanol extract from the innerbark of Caesalpinia pyramidalis in rats

Ana Roseli S. Ribeiro a, Polyana B.F. Diniz a, Charles S. Estevam a, Malone S. Pinheiro b,Ricardo L.C. Albuquerque-Júnior c, Sara M. Thomazzi a,n

a Departamento de Fisiologia, Universidade Federal de Sergipe, Av. Marechal Rondon, Cidade Universitária, CEP 49100-000 São Cristóvão, Sergipe, Brazilb Laboratório de Biomedicina, Universidade Tiradentes, Av. Murilo Dantas, 300, CEP 49032-490 Aracaju, Sergipe, Brazilc Instituto de Tecnologia e Pesquisa—ITP, Universidade Tiradentes, Av. Murilo Dantas, 300, CEP 49032-490 Aracaju, Sergipe, Brazil

a r t i c l e i n f o

Article history:Received 20 December 2012Received in revised form31 January 2013Accepted 10 March 2013

Keywords:AntiulcerCaesalpinia pyramidalisFabaceaeGastroprotectiveHelicobacter pylori

41/$ - see front matter & 2013 Elsevier Irelanx.doi.org/10.1016/j.jep.2013.03.023

espondence to: Department of Physiology, Ces, Federal University of Sergipe, 49100-0005 79 21056640; fax: þ55 79 21056474.ail addresses: [email protected], sarathom

e cite this article as: Ribeiro, A.R.S.idalis in rats. Journal of Ethnopharm

a b s t r a c t

Ethnopharmacological relevance: Caesalpinia pyramidalis Tul. (Fabaceae), known as “catingueira”, hasbeen used in folk medicine in the treatment of various disorders such as gastritis, heartburn, indigestion,and stomach ache. However, the gastroprotective properties of this species have not yet been studied.Materials and methods: The ethanol extract of Caesalpinia pyramidalis inner bark was used in rats via oralroute, at the doses of 30, 100, and 300 mg/kg. The antiulcer assays were performed using the ethanol-and nonsteroidal anti-inflammatory drug-induced ulcer models. Gastric secretion parameters (volume,pH, and total acidity) were also evaluated by the pylorus ligated model, and the mucus in the gastriccontent was determined. The anti-Helicobacter pylori activity of the ethanol extract of Caesalpiniapyramidalis was performed using the agar-well diffusion and broth microdilution methods.Results: The ethanol extract (30, 100, and 300 mg/kg) produced dose dependent inhibition (Po0.01) onthe ulcer lesion index, the total lesion area, and the percentage of lesion area in the ethanol-inducedulcer model. The ethanol extract (30, 100, and 300 mg/kg) also reduced (Po0.001) the ulcer index in theindomethacin-induced ulcer model. In the model ligature pylorus, the treatment with Caesalpiniapyramidalis ethanol extract failed to significantly change the gastric secretion parameters. However, aftertreatment with the ethanol extract of Caesalpinia pyramidalis (30, 100, and 300 mg/kg), there was asignificant increase (Po0.05) in mucus production. The ethanol extract showed anti-Helicobacter pyloriactivity, with inhibition halos of 12.071.7 mm at 10,000 μg/mL. The minimum inhibitory concentration(MIC) and minimal bactericidal concentration (MBC) values of the ethanol extract were of 625 and10,000 μg/mL, respectively.Conclusions: Collectively, the present results suggest that the ethanol extract of Caesalpinia pyramidalisdisplays gastroprotective actions, supporting the folkloric usage of the plant to treat various gastro-intestinal disturbances.

& 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Peptic ulcer is an integrity disturbance of the gastric and/orduodenum mucosa, which causes local defect or excavation due toactive inflammation (Syam et al., 2009). It is a recurrent chronicillness that affects approximately 10% of the world population(Zapata-Colindres et al., 2006). However, current therapy of pepticulcers may fail due to hypersensitivity, gynecomastia, impotence,arrhythmia, and hematopoietic changes of drugs such as antacids,

d Ltd. All rights reserved.

nter of Biological and HealthSão Cristóvão (SE), Brazil.

[email protected] (S.M. Thomazzi).

, et al., Gastroprotective acacology (2013), http://dx.d

anticholinergics, proton pump inhibitors, and H2-receptor antago-nists (Bighetti et al., 2005; Lakshimi et al., 2010).

In recent years, considering the prominence of the disease,interest in the use of natural products as sources of new drugs forthe treatment of peptic ulcer has significantly increased (Falcãoet al., 2008). Plants belonging to the genus Caesalpinia (Fabaceae)have been used in folk medicine and its antiulcer activity has beendemonstrated (Bacchi et al., 1995; Sharma and Rajani, 2011).

Caesalpinia pyramidalis Tul., known as “catingueira”, is anendemic medicinal plant species of the Northeast region of Brazil,mainly from the caatinga (Agra et al., 2007; Albuquerque et al.,2007). Bark, flower, leaf, root, and stem are traditionally used inthe form of drink and to wash the affected site in the treatment ofgastritis, heartburn, indigestion, stomach ache, stomach problems,cough, bronchitis, asthma, respiratory infection, influenza, colic,

tivity of the ethanol extract from the inner bark of Caesalpiniaoi.org/10.1016/j.jep.2013.03.023i

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A.R.S. Ribeiro et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎2

fever, flatulence, diarrhea, collision, injury, diabetes, and as expec-torant and aphrodisiac (Albuquerque et al., 2007; Cartaxo et al.,2010).

Pharmacological studies of Caesalpinia pyramidalis have shownit to be useful in the treatment of diarrhea, inflammation, and pain(Braga, 1960; Santana et al., 2012; Santos et al., 2011).

Therefore, the present study was carried out in order toevaluate the gastroprotective potential of the ethanol extract ofCaesalpinia pyramidalis inner bark.

2. Materials and methods

2.1. Plant collection and extraction of the ethanol extract

The inner bark of Caesalpinia pyramidalis was collected in themunicipality of Canindé de São Francisco-SE, Brazil, in September2008 (09166′00′′S, 37178′94′′W). The plant was authenticated byProfessor Ana Paula Prata, Department of Biology, FederalUniversity of Sergipe. A voucher specimen has been deposited atthe Herbarium of the Federal University of Sergipe (number ASE13,164). The inner bark was dried at 40 1C in a forced air oven fortwo days and subsequently powdered (2,840 g) and extracted bymaceration at room temperature with 90% ethanol for five days.The extract was filtered in vacuum, and the solvent was removedusing a rotary evaporator (45 1C). The percentage of yield of theethanol extract (EE) was 2.6% (73.8 g).

The EE of Caesalpinia pyramidalis was analyzed using highperformance liquid chromatography (Shimadzu, Prominencemodel, Kyoto, Japan) consisting of a vacuum degasser DGU-20A3model, SIL-10A autosampler, two high pressure pumps LC-6A, andan SPDM20Avp photodiode array detector system coupled with aCBM 20A interface. Analysis was performed in an analyticalPhenomenex LUNA® C18 column (250�4.6 mm i.d., 5 mm ofparticle diameter, Torrance, CA, USA). Separation of compoundswas done by reverse mode gradient elution as previouslydescribed (Santana et al., 2012).

2.2. Drugs

The following substances were used: Alcin blue, carbenoxolone,cimetidine, indomethacin, and omeprazole (Sigma Chemical Co.,St. Louis, MO). All the other reagents used were of analytical grade.All substances used were dissolved in 0.2% Tween 80 in 0.9% NaClsolution, with the exception of indomethacin which was dissolvedin 2% sodium bicarbonate.

2.3. Microorganism

Helicobacter pylori strain ATCC 43504 was obtained from theInstituto Nacional de Controle de Qualidade em Saúde—INCQS—Fundação Oswaldo Cruz—Fiocruz/RJ, Brazil. Stock cultures weremaintained in Tryptic Soy broth at −20 1C.

2.4. Animals

Young adult Wistar rats (180–300 g) of both sexes wereobtained from the Central Biotery of the Federal University ofSergipe (São Cristóvão, Brazil). Animals were maintained at con-trolled room temperature (2172 1C) with free access to food(Purina®) and water, under a 12 h light/dark cycle. Twenty-fourhours before the experiments, they were transferred to thelaboratory and given only water, ad libitum. The experimentswere performed after approval of the protocol by the InstitutionalEthics Committee (CEPA/UFS 63/11) and were carried out in

Please cite this article as: Ribeiro, A.R.S., et al., Gastroprotective acpyramidalis in rats. Journal of Ethnopharmacology (2013), http://dx.d

accordance with the current guidelines for the care of laboratoryanimals.

2.5. Antiulcerogenic activity

2.5.1. Ethanol-induced ulcerThe experiment was carried out according to the method of

Robert et al. (1979). After 24 h of fasting, the rats (n¼8/group)were pre-treated orally (p.o.) with the EE (30–300 mg/kg),omeprazole (30 mg/kg), or vehicle (0.2% Tween 80, 10 mL/kg).One hour after treatment, all the rats received 4 mL/kg of absoluteethanol to induce gastric ulcer. One hour later, the animals wereanesthetized and euthanized by cervical dislocation, and theirstomachs were removed and opened along the greater curvature.The stomachs were gently rinsed with water to remove the gastriccontents and blood clots, for subsequent scanning. The imagesobtained were analyzed using a specific “EARP” software (devel-oped by Dr. Eros Comunello, Universidade do Vale do Itajaí,São José, SC, Brazil) measuring each lesion point. The ulcers wereclassified as level I, ulcer areao1 mm2; level II, ulcer area1–3 mm2; and level III, ulcer area43 mm2. The following para-meters were determined: (i) Ulcerative Lesion Index (ULI) as 1�(number of ulcers level I)þ2� (number of ulcers level II)þ3�(number of ulcers level III); (ii) curative ratio, which was deter-mined as follows: %C¼100 (ULI treated � 100/ULI vehicle); (iii)total area of lesion (mm2); and (iv) percentage of lesion area inrelation to total stomach area (Andrade et al., 2006).

For the histological analysis, the absolute ethanol-inducedulcerative response in the stomachs was carried out after 1 h.The stomach samples were fixed in 10% formalin and embedded inparaffin. The samples (EE at 100 and 300 mg/kg, omeprazole, andvehicle) were cut in serial 5 μm thick sections and stained inhematoxylin–eosin. A microscopic score was determined for thefollowing parameters: (i) disruption of the superficial region of thegastric gland with epithelial cell loss, (ii) interstitial edema, and(iii) submucosal leukocytes infiltration, using a scale ranging from0 to 3 (0: none; 1: mild; 2: moderate; and 3: severe) for eachcriterion. The sections were assessed by an experienced patholo-gist without the knowledge of the treatments (four histologicalsections/animal, n¼8/group).

2.5.2. Nonsteroidal anti-inflammatory drug (NSAID)-induced ulcerThe experiment was carried out according to the method of

Djahanguiri (1969) with a few modifications. After 24 h of fasting,the rats (n¼8/group) were pre-treated orally with the EE (30–300 mg/kg), cimetidine (100 mg/kg), or vehicle (0.2% Tween 80).One hour after treatment, all the rats received indomethacin(100 mg/kg, p.o.) to induce gastric ulcer. Six hours after treatmentwith indomethacin, the animals were anesthetized and eutha-nized by cervical dislocation. The stomachs were removed, andopened along the greater curvature. The stomachs were gentlyrinsed with water to remove the gastric contents and blood clots.For determination of the ulcer index, scores were attributed asfollows: (i) score 1 each, loss of mucosal folding, mucosal dis-coloration, edema, or hemorrhage; (ii) score 2, less than 10petechiae; and (iii) score 3, more than 10 petechiae (Gamberiniet al., 1991).

2.6. Evaluation of mucosal protective factors

2.6.1. Determination of the gastric juice parameters—pyloric ligatureThe assay was performed using the method of Shay et al. (1945)

with a few modifications. After 24 h of fasting, the animals wereanesthetized with tyopental sodium (10 mg/kg, i.p.), the abdomenwas incised and the pylorus ligated. Immediately after pylorus





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ligature, the rats (n¼8/group) were pre-treated intraduodenallywith the EE (30–300 mg/kg), cimetidine (100 mg/kg), or vehicle(0.2% Tween 80). Four hours later, the animals were anesthetizedand euthanized by cervical dislocation; the abdomen was opened,and another ligature placed at the esophageal end. The stomachswere removed and the gastric contents collected and centrifugedat 3000 rpm (8000� g, 25 1C, 10 min). The amount of gastric-juiceacid (mL) and the pH values were determined. The total acidsecretion in the gastric juice in the supernatant volume wasdetermined by titration as pH 7.0, using a 0.01 N NaOH solution,and phenolphthalein as indicator.

2.6.2. Determination of mucus in gastric contentThis assay was performed according to the methodology

described previously by Sun et al. (1991) with a few modifications.After 24 h of fasting, the animals were anesthetized with tyopentalsodium (10 mg/kg, i.p.), the abdomen was incised and the pylorusligated. Immediately after pylorus ligature, the rats (n¼8/group)were pre-treated intraduodenally with the EE (30–300 mg/kg),carbenoxolone (200 mg/kg), indomethacin (100 mg/kg), or vehicle(0.2% Tween 80). The animals were anesthetized and euthanizedby cervical dislocation 4 h after the drug treatments. The stomachcontent was immersed in 10 mL of 0.02% Alcian blue, 0.16 Msucrose, 0.05 M sodium acetate solution, pH 5.8, and incubatedfor 24 h at 20 1C. The Alcian blue binding extract was centrifugedat 3000� g for 10 min. The absorbancy of the supernatant wasmeasured by spectrophotometry at 620 nm. The free mucus in thegastric content was calculated from the amount of Alcian bluebinding (mg/g tissue).

2.7. Anti-Helicobacter pylori activity

2.7.1. Agar-well diffusion methodAntibacterial activity was evaluated by modified agar-well

diffusion method (Okunji et al., 1990). Equidistant wells of 8 mmin diameter were bored into the blood agar (Mueller-Hinton agarwith 5% sheep blood) plate inoculated with Helicobacter pylori, in asuspension of 6�108 CFU/mL (McFarland turbidity standard 0.5).Each well was filled with 60 μL of the EE at 10,000 μg/mL.Tetracycline (30 μg/mL) was used as the standard drug. The plateswere left at room temperature for 30 min and then incubated for24 h at 37 1C under microaerophilic conditions. After this period,the growth inhibition halos were measured with a digital pachy-meter. The diameters of inhibitory zones were measured intriplicate and mean values≥08 mm were considered active.

2.7.2. Broth microdilution methodThe minimum inhibitory concentration (MIC) and minimal

bactericidal concentration (MBC) of the EE were determined bybroth microdilution method, according to the Clinical and Labora-tory Standards Institute (CLSI) recommendations (NationalCommittee for Clinical Laboratory Standards, 1993).

Table 1Effect of the ethanol extract of Caesalpinia pyramidalis (EE) on ethanol-induced ulcers i

Treatment (p.o.) Dose (mg/kg) Total area of lesion (mm2)

Vehicle – 111.84713.37Omeprazole 30 0.2770.27 nnn

EE 30 45.4776.38 nnn

100 36.4174.37 nnn

300 0.9370.46 nnn

Results as mean7SEM (n¼8/group). Statistical comparison was performed using ANOVnn Po0.01.nnn Po0.001.


Tryptic Soy broth inoculated with 6�108 CFU/mL of Helicobacterpylori (100 μL) was added to each well in the microplate. Serialdilutions of the EE ranging from 10,000 to 0.305 μg/mL were alsoadded (100 μL). The microplate was incubated at 37 1C undermicroaerophilia for 3 days. After incubation, the plates were visuallyexamined and each well was replicated in blood agar (Mueller-Hinton agar with 5% sheep blood), to determine whether growthhad occurred. The MIC defined as the lowest concentration to causecomplete bacterial growth inhibition and the MBC defined as thelowest concentration that causes bacterial death were observedafter 24 h. The experiments were performed in triplicate.

2.8. Statistical analysis

The results are presented as the means7SEM of n animals pergroup. Statistical evaluation of the data was performed using one-wayanalysis of variance (ANOVA) followed by Tukey's test. The analysis ofthe microscopic alterations and indomethacin-induced ulcers werecarried out by the Kruskal–Wallis test, followed by Dunn's test. Pvalues lower than 0.05 were considered significant.

3. Results

In the ethanol-induced ulcer model, the treatment withCaesalpinia pyramidalis EE (30, 100, and 300 mg/kg) produceddose dependent inhibition on the ulcer lesion index, the totallesion area, and the percentage of lesion, compared with thevehicle group (Po0.01, Table 1). Omeprazole (30 mg/kg) signifi-cantly also reduced all the gastric ulcer parameters (Po0.001,Table 1).

Results of histopathological analyses of the gastric mucosa areshown in Fig. 1 and Table 2. Animals pre-treated with the vehicleshowed severe lesions which were characterized by disruption ofthe superficial region of the gastric gland with loss of epithelialcells, intense edema, and infiltration of inflammatory cells.Animals pre-treated with the EE (100 and 300 mg/kg, p.o.) showedless mucosal damage when compared with the vehicle group(Fig. 1 and Table 2). Pre-treatment with omeprazole also inhibitedlesions promoted by ethanol (vehicle group; Fig. 1 and Table 2).

The treatment with Caesalpinia pyramidalis EE (30, 100, and300 mg/kg) and cimetidine (100 mg/kg) significantly reduced theulcer index, compared with the vehicle group (Po0.001) in theindomethacin-induced ulcer (Table 3).

In the model of evaluation of gastric secretion, using ligaturepylorus, the treatment with Caesalpinia pyramidalis EE failed tosignificantly reduce the secretion volume and total acid secretion(Table 4). The EE also failed to significantly increase the pH value,compared with the vehicle group. However, cimetidine signifi-cantly changed all the gastric juice parameters (Po0.01, Table 4).

On the other hand, in the mucus determination model, aftertreatment with the EE of Caesalpinia pyramidalis (30, 100, and300 mg/kg), there was a significant increase (Po0.05) in mucus

n rats.

Lesion area (%) Ulcer lesion index Curative ratio (%)

11.3371.56 25.5071.84 –

0.0470.04 nnn 0.3370.33 nnn 98.7071.314.3870.63 nnn 18.2071.52 nn 28.6274.094.0870.83 nnn 11.9071.23 nnn 53.3373.320.1670.08 nnn 0.9270.40 nnn 96.3971.56

A followed by Tukey's test vs. vehicle group.





Fig. 1. Photomicrographs of gastric mucosa (HE; magnification 100� for a, d, g, and j; 400� for b, c, e, f, h, i, k, and l). (a–c) Vehicle group showing disruption of the epithelialstructure (des), pronounced interstitial edema of the submucosa (ed3), and inflammatory infiltrate (arrows). (d–f) Omeprazole group showing gastric epithelium (ge) and submucosawith normal characteristics. The EE of Caesalpinia pyramidalis groups, at 100 (g–i) and 300 mg/kg (j–l), showing the gastric epithelium (ge) preserved and discreet interstitial edema ofthe submucosa (ed1). get: gastric epithelial tissue; sct: submucosal connective tissue; pml: peripheral muscle layer (four histological sections of each animal, n¼8/group).

Table 2Effect of the ethanol extract of Caesalpinia pyramidalis (EE) on ethanol-inducedmicroscopic damage in gastric mucosa.

Treatment(p.o.)

Dose(mg/kg)

Epithelial cell loss(score 0–3)

Edema(score 0–3)

Inflammatorycells (score 0–3)

Vehicle – 3.0 (2.0–3.0) 3.0 (2.0–3.0) 1.0 (0.0–1.0)Omeprazole 30 1.0 (1.0–2.0) nnn 1.0 (0.0–2.0) nnn 0.5 (0.0–1.0)EE 100 1.0 (0.0–3.0) nnn 1.0 (0.0–2.0) nnn 0.0 (0.0–2.0) nnn

300 1.0 (0.0–2.0) nnn 1.0 (0.0–2.0) nnn 0.0 (0.0–0.0)

Data shown are medians with minimum and maximal scores in brackets. Kruskal–Wallis followed by Dunn's test vs. vehicle group (four histological sections of eachanimal, n¼8/group).

nnn Po0.001.

Table 3Effect of the ethanol extract of Caesalpinia pyramidalis (EE) on indomethacin-induced ulcers in rats.

Treatment (p.o.) Dose (mg/kg) Ulcer index Inhibition (%)

Vehicle – 3.0770.25 –

Cimetidine 100 0.4070.21 nnn 86.97EE 30 0.6770.28 nnn 78.18

100 0.4070.27 nnn 86.97300 0.7370.30 nnn 76.22

Results as mean7SEM (n¼8/group). Statistical comparison was performed usingKruskal-Wallis followed by Dunn's test vs. vehicle group.

nnn Po0.001.


production, compared with the group treated with vehicle (Table 5).Carbenoxolone (200 mg/kg) also increased the mucus production(Po0.05, Table 5).

The EE showed anti-Helicobacter pylori activity, with inhibitionhalos of 12.071.7 mm at 10,000 μg/mL. Tetracycline (30 μg/mL),


used as the standard drug in the assay, presented inhibition halosof 40.070.5 mm.

The MIC and MBC values obtained in the broth microdilutionmethod of the EE were 625 and 10,000 μg/mL, respectively.





Table 4Effect of the ethanol extract of Caesalpinia pyramidalis (EE), administered intra-duodenally, on the biochemical parameters of gastric juice obtained from pylorusligature in rats.

Treatment(p.o.)

Dose (mg/kg) Volume (mL) pH [Hþ]mEq/L/4 h

Vehicle – 1.0470.19 3.6070.18 48.8672.82Cimetidine 100 0.4270.05 nn 4.9670.33 nnn 28.0972.45 nn

EE 30 0.7370.07 3.5070.13 49.1073.76100 0.6870.09 3.4870.18 45.0973.50300 0.7070.07 3.2570.14 51.1273.57

Results as mean7SEM (n¼8/group). Statistical comparison was performed usingANOVA followed by Tukey's test vs. vehicle group.

nn Po0.01.nnn Po0.001.

Table 5Effect of the ethanol extract of Caesalpinia pyramidalis (EE) on Alcian blue bindingto free gastric mucus from pylorus ligature in rats.

Treatment (p.o.) Dose (mg/kg) Alcian blue bound (mg/g tissue)

Vehicle – 1.0070.13Carbenoxolone 200 1.4370.07 n

Indomethacin 100 0.9170.07EE 30 1.4470.05 n

100 1.4470.11 n

300 1.6170.09 nnn

Results as mean7SEM (n¼8/group). Statistical comparison was performed usingANOVA followed by Tukey's test vs. vehicle group.

n Po0.05.nnn Po0.001.

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4. Discussion

The present study demonstrates that the oral administration ofthe EE of Caesalpinia pyramidalis possesses gastroprotective activity,as evidenced by its inhibition of the development of ulcers inducedby chemical agents and increase of mucosal protective factor afterthe intraduodenal administration.

The functional integrity of gastric mucosa depends on a balancebetween protective mechanisms and aggressive factors. Thus, thesuccess of gastric pharmacological treatment relies not only on theblockage of acid secretion, but also on the augmentation of theprotective factors of the gastric mucosa (Dajani and Klamut, 2000).

Ethanol-induced ulcer model was first performed to assess thegastroprotective effects of the EE of Caesalpinia pyramidalis. It iswell established that, ethanol is considered one of the agents thatinduce more intense gastric ulcers because it promotes seriousdisturbances in the gastric mucosa (Hiruma-Lima et al., 2009).Ethanol acts by exerting a direct toxic effect on the epithelium,leading to the formation of characteristic necrotic lesions due to areduction in the mucus production and bicarbonate secretion(Massignani et al., 2009). In addition, it induces reduction ofgastric blood flow, the solubilization of components of the mucusof the stomach, and oxidative stress, increase of xanthine oxidaseactivity and levels of malondialdehyde with reduction in levels ofglutathione (Marrotta et al., 1999).

Here, we clearly demonstrated that the vehicle group, treatedorally with absolute ethanol, produced the expected characteristiczone of necrotizing mucosal lesions with severe macroscopic andmicroscopic gastric mucosal damage (epithelial cell loss, edema,and inflammatory cell infiltrate). On the other hand, Caesalpiniapyramidalis EE-treated groups significantly decreased the ulcerlesion index, the total lesion area, and the percentage of lesion aswell as the epithelial cell loss, edema, and inflammatory cellinfiltrate. These results indicate that Caesalpinia pyramidalis EE


displays an antiulcerogenic effect related to cytoprotective activity,since it significantly reduced the ethanol-induced ulcer.

In order to provide more evidence concerning the gastroprotec-tive effect of Caesalpinia pyramidalis EE, we investigated its action onindomethacin-induced ulcer model. NSAID are considered anotherwell established and common cause of peptic ulcers in humans(Bjarnason et al., 2007). These drugs are known to induce ulcersduring the course of anti-inflammatory therapy, by inhibiting pros-taglandin synthetase through the inhibition of cyclo-oxygenase(COX)-1 and COX-2 activities, and direct cytotoxic effects on theepithelium (Moleiro et al., 2009; Rainsford, 1987). Prostaglandinsplay an important protective role by stimulating the secretion ofbicarbonate and mucus, maintaining the blood flow of the mucosa,and they are responsible for regulating mucosal cell renewal. Ourresults demonstrated that the EE of Caesalpinia pyramidalis presentsgastroprotective potential in NSAID-induced ulcer model. This actiondenotes that the EE of Caesalpinia pyramidalis could be administeredtogether with NSAID with the aim of minimizing this side effect onthe gastric mucosa.

Previous study suggests that natural compound may provide acytoprotective effect by the induction of COX-2 expression ingastric mucosa and maintenance of basal prostaglandin E2 levelindependent of action of NSAID in gastric mucosa (Moleiro et al.,2009). However, prostaglandin inhibition seems not to be theexclusive mechanism of NSAID-induced gastric lesion. Othermechanisms such as induction of oxidative damage throughincreased lipid peroxidation, thiol depletion, and inactivation ofgastric mucosal peroxidase are also involved (Pérez et al., 2013;Rao et al., 2004).

The next step of this study was the evaluation of the effect ofthe EE of Caesalpinia pyramidalis on gastric juice parametersthrough the pylorus ligature model. In this model, the digestiveeffect of accumulated gastric juices and interference in gastricblood flow are responsible for inducing ulceration (Patel et al.,2000). The intraduodenal administration of the EE of Caesalpiniapyramidalis was not able to change the volume, acidity, and pH ofthe gastric juice. Therefore, the EE of Caesalpinia pyramidalis didnot act as an antacid or as buffer solution in contact with gastricmucosa, as indicated by the lack of antisecretory effects in thegastric juice.

Another parameter measured in this study was the amount ofgastric mucus in pylorus-ligated rats. Gastric mucus is the first lineof defense against acid and adheres together with bicarbonatesecreted by the epithelium to serve as a barrier against self-digestion (Allen and Flemströn, 2005). We observed that theamount of adhered gastric mucus was augmented by treatmentwith the EE of Caesalpinia pyramidalis when compared to theanimals treated with vehicle. These results indicate an increase inthe amount of mucus as a potential mechanism of action for thegastroprotective effect of the EE of Caesalpinia pyramidalis.

Considering that the EE of Caesalpinia pyramidalis has gastro-protective actions, additional assays were carried out in order toevaluate its anti-Helicobacter pylori activity. Our findings show thatthe EE of Caesalpinia pyramidalis presents a MIC value of 625 μg/mL.Natural products with concentrations above 500 μg/mL have weakantimicrobial activity, their use being difficult to treat bacterial orfungal infections (Holetz et al., 2002).

Generally, the major components are found to reflect quite wellthe biophysical and biological features of the extracts from whichthey were isolated. Our previous study confirmed the presence offlavonoids in the EE of Caesalpinia pyramidalis inner bark, anddemonstrated the presence of rutin (Santana et al., 2012). Theliterature shows that some potent antiulcer plants containphenolic compounds, namely, baicalein, cinnamic acid, oleuropein,rutin, quercetin, and tephrosin, as active constituents (Sumbulet al., 2011).






5. Conclusion

In summary, the present results provide convincing evidencethat the EE from Caesalpinia pyramidalis displays pronouncedgastroprotective activity, as evidenced by the significant inhibitionof the formation of ulcers induced by different agents and increasein the amount of mucus, supporting the folkloric usage of the plantto treat various gastrointestinal disorders. This ethanol extract is ofgreat interest as a source of novel molecules for developingstrategies more appropriate in treating ulcer. In order to identifythe active compounds present in the EE of Caesalpinia pyramidalis,pharmacological and chemical studies are in progress.

Acknowledgments

The authors are grateful to Coordenação de Aperfeiçoamentode Pessoal de Nível Superior (CAPES) and Conselho Nacional deDesenvolvimento Científico e Tecnológico (CNPq). SMT is recipientof CNPq productivity grants.

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Glossary

COX: cyclo-oxygenase;EE: ethanol extract;MBC: minimal bactericidal concentration;MIC: minimum inhibitory concentration;NSAID: nonsteroidal anti-inflammatory drugs.


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