Anti-ulcerogenic and antisecretory effects of Celtis iguanaea ( Jacq . ) Sargent hexane leaf extract

The Celtis iguanaea (Jacq.) Sargent (Cannabaceae) is one of the native species of the Cerrado region of Brazil widely used in folk medicine to treat dyspepsia. The objective of the present study was to evaluate the gastroprotective effect of the Celtis iguanaea (Jacq.) Sargent (HE) hexane leaf extract in the lesion and gastric secretion models.Antiulcerogenic activity of the Celtis iguanaea (HE) hexane leaf extract was observed with the experimental models, such as indomethacin and pyloric ligation-induced gastric ulcers. In order to evaluate the antisecretory activity of this extract, isolated Rana catesbeiana mucosa and pyloric ligation in mice were used. The HE treatment reduced the lesion index of indomethacin and pyloric ligation-induced ulcer. This extract also reduced the gastric acid secretion and total acidity (increasing the gastric pH) in mice. The secretion of H+ was reduced in the basal values (15.58 ± 1.99 μEq H+/g/15 min) when isolated Rana catesbeiana mucosa was incubated with HE. Intraduodenal administration of HE reduced the gastric secretion produced by bethanecol or histamine. The antiulcerogenic and antisecretory efficacy of HE in this study suggest anticholinergic and antihistaminergic mechanism or interruption of intracellular events that are linked to acid secretion.


INTRODUCTION
For over a century, gastric ulcer disease has been a major cause of morbidity and mortality (Higham et al., 2002).Factors such as stress, nutritional deficiencies, smoking and continuous ingestion of nonsteroidal-antiinflammatory drugs (NSAIDs) had led to increase in the cases of gastric ulcer (Belaiche et al., 2002).The etiology of gastroduodenal ulcers is usually caused by disequilibrium between aggressive (hydrochloric acid and pepsin) and defensive mucosal factors (blood flow, mucus, bicarbonate secretion, and epithelial layer) (Wallace & Granger, 1996).
C e l t i s i g u a n a e a ( J a c q .) S a r g e n t (Cannabaceae) is popularly known as esporão-degalo.The genus Celtis has approximately 70 species and widely spread in the temperate region of Cerrado (Cronquist, 1981).This species is a thorny plant, 6-9 feet tall, with rounded crown and branches sticking vertically giving a pyramidal shape to the canopy.It has an erect and cylindrical trunk of 15-30 cm in diameter (Lorenzi & Souza, 1999).Ethnopharmacological studies showed the use of its leaves in the form of infusion for the treatment of asthma and digestive disturbs (Paula, 2009;Silva & Proença, 2008;Tene et al., 2007).Phytochemical analysis of the leaves and stem of Celtis iguanaea showed the presence of flavonoids, coumarins and mucilage (Paula et al., 2010).Previous results showed gastroprotective effect of the hexane fraction obtained from the partitioning of Celtis iguanaea ethanol leaf extract (Sousa et al., 2012).
The objective of the present study was to evaluate gastroprotective effect of hexane leaf extract of Celtis iguanaea (Jacq.)Sargent (HE) in the lesion and gastric secretion models.

Animals
Male albino Swiss mice weighing 25-35 g (supplied by Central Animal Laboratory, Federal University of Goiás) and Rana catesbeiana of both sexes weighing between 80-120 g (provided by Fujioka Ranario) were used in all experiments.The animals were maintained under controlled conditions of temperature with a 12 h dark/light cycle and free access to water and standard food.All experiments were conducted between 8:00 a.m. to 4:00 p.m. and were performed in accordance with the Ethical Principles in Animal Research as adopted by the Brazilian Society of Laboratory Animal Science.The experimental protocols was approved by Research Ethic Council of Federal University of Goiás (Protocol number 106/08).

Plant material
T h e l e a v e s o f C e l t i s i g u a n a e a (Jacq.)Sargent (Cannabaceae), as popularly known esporão-de-galo, were collected in Hidrolândia, GO,Midwest Brazil (16° 53'59,4" W) with an altitude of 786 m., on September 2008.Samples were authenticated by Prof. Dr. José Realino de Paula, and a voucher specimen was deposited at the Herbarium of the Federal University of Goiás (40.110/UFG).

Preparation of extract
The leaves were oven dried with forced ventilation at 40°C and then crushed to a fine powder.The HE was obtained by exhaustive extraction in Soxhlet apparatus and concentrated under reduced pressure in rotary evaporator.The extraction process yielded 4.81%.

Pharmacological assays
Quantification of H + secreted Rana catesbeiana isolated gastric mucosa was incubated in Ussing chamber (Villegas 1968;Ferreira 2002), for the study of the gastric secretion.
After the destruction of central nervous system (CNS) of a Rana catesbeiana, the abdomen was opened longitudinally and the stomach removed and the mucosa was washed in 15 mL of Ringer solution without buffer.The mucosa was then mounted in the Ussing chamber with the luminal side filled with Ringer solution while serous side was filled with buffered ringer solution.Oxygenation was performed by oxygen bubbling.HE concentrations (10, 30, 100, 300 the 1000 µg/mL) were achieved in Ringer solution of serous side after a stabilization period of 60 minutes.The pH of the solution in contact with the luminal side was monitored by a pH adjusted to 6.5 and (initial pH) at 0. 001 N NaOH throughout the experiment.

Nonsteroidal anti-inflammatory drug (NSAID)-induced ulcer
Mice Swiss (n=8) fasted for 16 hr were randomly divided into six groups.Animals were orally treated with vehicle (2% Tween-80 10 mL/kg), ranitidine (50 mg/kg), or HE (25, 50, 100 and 200 mg/kg).One hour after treatment, all the animals received indomethacin (30 mg/kg s.c.) to induce gastric ulcer.Six hours after indomethacin treatment, the animals were sacrificed by cervical dislocation.The stomachs were removed, and opened along the minor curvature.The gastric mucosa was examined for the presence and severity of ulcerative lesions.According to Macaúbas et al. (1988), the degree of lesion score were measured with a ruler and a magnifying glass (10x amplification) and expressed as the mean rate.Parameters for the ulceration index (UI) area; color of the mucosa, loss of mucosal folds, petechial hemorrhages, edema, hemorrhage and number of ulcer.

Pyloric ligation in mice
The pyloric sphincter of mice was ligated surgically, as described by Vissher et al. (1954).HE (100 mg/kg) or vehicle (2% Tween 80 10 mL/ kg) were immediately administered intraduodenally (i.d.) and the incision was closed.Four hours later the animals were killed, the stomachs were excised, opened along the smaller curvature and the UI was determined.
The luminal contents were collected, centrifuged for 30 min at 1500 rpm.The volume (mL) and the value of gastric pH were measured.Each sample was titrated against 0.01 N NaOH using the phenolphthalein reagent as an indicator, and the total acid was expressed as microequivalents of H + per litre per 4 h (mEq[H + ]/L/4h).This model was also used to establish the effect of HE on secretion induced bethanechol (20 mg/kg s.c.) and histamine (30 mg/kg s.c.).

Statistical analysis
Data were showed as mean ± standard error of mean (SEM) and analyzed statistically by oneway ANOVA followed by Student-Newman-Keuls' test as the post hoc test, or Student "t" test unpaired where necessary.Differences between experimental groups were considered statistically significant when P ≤ 0.05 (Sokal & Rohlf, 1981).

RESULTS AND DISCUSSION
Herbal extracts have been and remain important sources of biologically active compounds.These extracts are crucial to drug development (Nodari & Guera, 2001).In the present study, we evaluated anti-ulcerogenic and antisecretory effects of HE.Recent studies showed that the hexane fraction of the ethanol leaf extract of Celtis iguanaea is an effective anti-ulcerogenic and antisecretory agent (Sousa et al., 2012).The chemical study realized by Trevisan et al. (2012) of the hexane fraction allowed the isolation and identification of pentacyclic triterpenes series friedelano, friedelin and epifriedelinol when it was previously isolated from the bark of Celtis sinensis the epifriedelinol (Kim et al., 2005).To verify the hypothesis of an activity involving mechanisms of acid secretion, the HE was evaluated in an experimental model of an isolated Rana catesbeiana gastric mucosa.It is known that increased secretion of acid may be regarded as an important factor in the formation of ulcer while the reduced secretion of gastric acid would result in decreased ulcerative lesion.We observed that the concentrations of HE (100, 300 or 1000 µg/ mL) decreased significantly the basal gastric acid secretion (P≤0,01) (Figure 1).
The anti-ulcer activity of HE was studied using in vivo acute gastric lesions induced experimentally by indomethacin (Djahanguiri, 1969) or by ligation of the pylorus (Vissher et al., 1954).
Nonsteroidal anti-inflammatory drugs ( N S A I D s ) s u c h a s i n d o m e t h a c i n c a u s e s gastroduodenal ulceration by suppression of prostaglandin synthesis (Wallace, 2001).Prostaglandins E 2 and I 2 are known for their ability to inhibit gastric acid secretion and stimulate the secretion of mucus and bicarbonate besides the maintenance of mucosal blood flow.The oral treatment of HE showed a reduction UI proportional to the dose of 50 mg/kg when compared with the control group (Table 1).The ranitidine 50 mg/ kg (positive control) decreased the UI by 55.5%.These results suggest possible involvement of prostaglandins and/or mucus in the anti-ulcerative effect of the extract (Table 1).As we did not observe a perfect dose-effect relationship within the range of 50 to 200 mg/kg, we assumed that the doses being tested produce the maximum effect of HE at this range, hence, we opted for 100 mg/kg p.o. that showed similar results with ranitidine to further our study.
In continuation with the study of antiulcerogenic activity, we used the pyloric ligation model.Among the various mechanisms involved in the pathogenesis of peptic ulcer induced by pyloric ligation, secretion and accumulation of gastric acid are possibly the most relevant factors (Muthuraman & Soo, 2010).In this model, the HE reduced the UI by 37. 4 %, when compared with the control group (Table 1).
Since the HE showed antisecretory activity in gastric secretion model, we proceeded with the evaluation of possible mechanisms involved.In the model of gastric acid secretion induced by pyloric ligation, treatment with HE (100 mg/kg) reduced the volume of gastric juice and the total acidity while increased pH significantly (p<0,001) when compared to the control group (Table 2).
Acetylcholine released by the postganglionic cholinergic neurons binds to M 3 muscarinic subtype located in the parietal cells, thereby leading to gastric acid secretion (Aihara et al.,2005).The M 3 receptor when stimulated activates phospholipase C by Gq protein that induces generation of IP 3 and diacylglycerol, followed by an increase in intracelular [Ca 2+ ] (Caulfield & Birdsall, 1998).Bethanecol is a selective agonist of muscarinic receptor that increases gastric acid secretion (Schubert, 2000).The pretreatment with HE (100 mg/kg i.d.) or atropine (50 mg/kg i.d.) reduced significantly the volume (mL), total acid secretion and raise the pH in mice after administration of bethanecol (20 mg/kg s.c.) (P≤0,001) (Table3).
Histamine stimulates gastric acid secretion by binding directly to H 2 receptors on parietal cell.When stimulated adenylate cyclase increases the cAMP, which in turn activates protein kinase A. The activation of this pathway stimulates the proton pump in the parietal cell, and results in acid secretion (Jain et al., 2007).In the same way, the pretreatment with HE (100 mg/kg i.d.) or ranitidine (50 mg/kg i.d.) reduced the volume (mL), total acid and raise the pH in mice after administration of histamine (30 mg/ kg s.c.) (Table 3).The ligation of the pyloric end of the stomach causes accumulation of gastric acid, that could result in peptic ulcers.The drugs capable of reducing the gastric acid secretion are effective in reducing ulcers in this model.The inhibition of secretion stimulated by histamine or bethanechol, may suggest the blockade or inhibition of a common target of the cascade of events that leads to gastric acid secretion.

CONCLUSION
In conclusion, our results suggest that the efficacy of hexane leaf extract of Celtis iguanaea in preventing ulcers is based on its anti-ulcerogenic and an antisecretory effect.The blockade of bethanecol and histamine by HE suggested an anticholinergic and antihistaminergic mechanism or an interruption of intracelular events that are linked to acid secretion.

FIGURE 1 .
FIGURE 1. Determination of H + (µEq/g/15 min.) in the presence of Rana catesbeiana stomach mucosa incubated with HE in Ussing chamber.The values are represented with mean ± SEM. ** P ≤ 0.01 compared to control Tween.

TABLE 1 .
Effect of HE on indomethacin-induced gastric ulcers or pyloric ligation in mice.The values represent the mean ± SEM of the ulceration index (UI) of each group.* P ≤ 0.05 and *** P ≤ 0.001 compared with the vehicle treated group.N = number of animals in each group.

TABLE 2 .
Effects of HE intraduodenally administered on gastric secretion in mice.The values represent the mean ± SEM. 7 animals were used in each group.* P ≤ 0.05 and *** P ≤ 0.001 compared with the vehicle treated group.

TABLE 3 .
Effects of intraduodenally administration of the HE, atropine or ranitidine in the pyloric ligation model in mice.The values represent the mean ± SEM. 9 animals were used in each group.* P ≤ 0.05 and *** P ≤ 0.001 compared with the vehicle treated group and # P ≤ 0.05 ## P ≤ 0.01 and ### P ≤ 0.001 compared with the secretagogue.