Antiulcerogenic Activity of the Aqueous Fraction of Anacardium Humile St

Full Length Research Paper

Antiulcerogenic activity of the aqueous fraction of Anacardium humile St. Hil (Anacardiaceae)

Anderson Luiz-Ferreira 1,2*, Maira Cola 3, Victor Barbastefano 2, Clélia Akiko Hiruma-Lima 4, Lourdes Campaner Santos 5, Wagner Vilegas 5 and Alba Regina Monteiro Souza Brito 2

1Departamento de Ciências Biológicas, Universidade Federal de Goiás (UFG), Catalão, GO, Brazil. 2Departamento de Biologia Estrutural e Funcional, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brazil. 3Departamento de Farmacologia, Instituto de Biologia, Universidade Federal de Santa Catarina, Santa Catarina, Brazil. 4Departamento de Fisiologia, Instituto de Biociências, Universidade Estadual Paulista (UNESP), Botucatu, SP, Brazil. 5Departamento de Química Orgânica, Instituto de Química, Universidade Estadual Paulista (UNESP), Araraquara, SP, Brazil.

Accepted 19 June, 2012

Anacardium humile St. Hil. is being used in traditional medicine to treat ulcer. The present work evaluated the mechanisms of action involved in the anti-ulcer properties of the aqueous fraction from leaves of A. humile (AHQ). Gastroprotection of A. humile was evaluated in ethanol and piroxicam models. Mechanisms of action such as mucus production, nitric oxide (NO), sulfhydryl compounds (SH) and the anti-secretory action were evaluated. The acetic acid-induced gastric ulcer model was used to evaluate the A. humile healing properties. Results obtained in the ethanol model showed that AHQ provided significant gastroprotection at all the tested doses (50, 100, and 200 mg/kg). Thereby, the following protocols were performed using the dose capable of producing the most effective gastroprotection, which was the AHQ 200 mg/kg (P < 0.001). Moreover, AHQ (200 mg/kg) protected the mucosa from piroxicam-induced damage. Mucus, NO, and SH did not participate on this gastroprotection. AHQ interfere in H + secretion in gastric mucosa, thus exerting an anti-secretory activity. Our results suggest that the gastroprotection and cicatrisation process executed by AHQ occurred due to its anti-secretory activity. This study reinforces its traditional medicinal use. Considering that the current therapies are based on the use of anti-secretory agents, the A. humile arises as a promising alternative antiulcer therapy.

Key words: Anacardium humile St. Hil ., gastric ulcer, gastroprotection.

INTRODUCTION

Medicinal plants are traditionally used to cure various (Morais et al., 2010). diseases and for the production of various drugs Anacardium humile St. Hil. has been used in folk (Mahmood et al., 2011). Natural bioactive compounds medicine as an alternative treatment for ulcers and present in medicinal plants have been investigated for gastritis (de Almeida et al., 1998). Considering this their characteristics and health effects (Ghasemzadeh popular use, our research group demonstrated that this and Ghasemzadeh, 2011). The Anacardiaceae family species possesses antiulcerogenic activity (Luiz-Ferreira has been exploited for its medicinal value. Among these et al., 2008). However, this study did not evaluate the uses are anticonvulsant (Marchetti et al., 2011), anti-asth- mechanisms involved in this activity. Therefore, the aim matic (Hemshekhar et al., 2011), and anti-ulcerogenic of this study was to evaluate the pharmacological role of this species, describing the mechanism involved in the gastroprotective action of the aqueous extract (AHQ) of leaves of A. humile St. Hil. Special attention was paid to *Corresponding author. E-mail: [email protected] . its effects on mucus production and gastric acid 5338 J. Med. Plants Res.

secretion. activity of AHQ was assessed on two experimentally induced gastric ulcer models:

MATERIALS AND METHODS Ethanol-induced gastric lesion in rats

Animals After a total of 24 h fasting, three groups of rats (n = 5) received an oral administration of AHQ (50, 100, and 200 mg/kg), lansoprazole Male Unib: WH rats (n = 5 to 7, 150 to 250 g) and male Unib: SW (30 mg/kg) or vehicle (10 ml/kg). One hour after treatment, all rats mice (n = 6, 30 to 35 g) from Central Animal House of the received, orally, 1 ml of 99.5% ethanol to induce gastric ulcers Universidade Estadual de Campinas (CEMIBUNICAMP; São Paulo, (Morimoto et al., 1991). The animals were killed by CO 2 gas 1 h Brazil) were used. The animals were fed a certified Nuvilab® after treatment with the ulcerogenic agent and their stomachs were (Nuvital) diet with free access to tap water under standard removed to determine the gastric damage as described previously. conditions of 12 h dark-12 h light, humidity (60 ± 1.0%) and temperature (21 ± 1°C). Fasting was used prior to all assays because standard drugs or essential oil treatment were always Non-steroidal anti-inflammatory drug (NSAID)-induced ulcer administered orally (by gavage) or intraduodenally. Moreover, the animals were kept in cages with raised floors of wide mesh to Animals (n = 6) were fasted for 24 h. The gastric injuries were prevent coprophagy. The experimental protocols were approved by induced by subcutaneous administration of piroxicam 30 mg/kg in the Institutional Animal Care and Use Committee (no. 538-1, male mice. Treatments (p.o.) with vehicle, cimetidine (100 mg/kg), CEEA/IB/UNICAMP). and AHQ (200 mg/kg) were carried out 30 min before administration of the NSAID. Four hours after the NSAID administration, the

animals were killed by CO 2 gas and their stomachs were removed Plant for lesion quantification (Puscas et al., 1997).

A. humile St. Hil. was collected along Monte do Carmo road, in Porto Nacional in Tocantins state, Brazil in November, 2002. The Evaluation of mucosal protective factors plants were identified by Marcos Alves and Eduardo Ribeiro of the University of Tocantins and a voucher specimen (accession number Determination of mucus in gastric content 1922) was deposited in the University of Tocantins Herbarium. Rats (n = 6 to 7) had fasted for 24 h, under anesthesia, the abdomen was incised and the pylorus ligated. The vehicle, Aqueous fraction preparation carbenoxolone (200 mg/kg) or AHQ (200 mg/kg) was administered orally after the pylorus ligature. The animals were killed by CO 2 gas Air-dried and powdered leaves (650 g) of A. humile St. Hil. were 4 h after the drug treatments. The stomach content was immersed exhaustively extracted by successive maceration at room in 10 ml 0.02% Alcian blue in 0.16 M sucrose/0.05 M sodium temperature with dichloromethane (DCM, 5 L) and methanol acetate, pH 5.8, and was incubated for 24 h at 20°C. The Alcian (MeOH, 5 L) (130:1, w/v, one week for each solvent). Solvents were blue binding extract was centrifuged at 2000 ×g for 10 min. The evaporated at 60°C under reduced pressure to yield the DCM (6 g) absorbance of supernatant was measured at 615 nm using a light and MeOH (193 g) extracts. A portion of the MeOH extract (80 g) spectrophotometer U/2000 (Hitachi, Japan). The free mucus in the was partitioned between a mixture of EtOAc/water (5 L, 1:1, v/v) to gastric content was calculated from the amount of Alcian blue yield 74 g of aqueous fraction (AHQ) utilized in this study. binding [mg/wet tissue (g)] (Rafatullah et al., 1990).

Evaluation of the gastric juice parameters Drugs and chemical

Animals (n = 5) were fasted for 24 h with free access to water. One Lansoprazole (proton-pump inhibitors; Medley, Campinas, Brazil), hour after oral treatment or immediately after intraduodenal piroxicam (non-selective COX inhibitor; Hexal, São Paulo, Brazil), administration of AHQ (200 mg/kg), cimetidine (100 mg/kg) or cimetidine (histamine H 2-receptor antagonist; Sigma Chemical Co., vehicle, pylorus ligature was performed (Shay et al., 1945). Four St. Louis, MO, USA), carbenoxolone (drug that enhance mucus hours later, the animals were sacrificed by CO 2 gas, their stomachs production; Sigma Chemical Co., St. Louis, MO, USA), N- were removed, inspected internally, and their contents were drained ethylmaleimide (a sulfhydryl blocker; Sigma Chemical Co., St. into a graduated centrifuge tube and were centrifuged at 2000 ×g Louis, MO, USA), and N (G)-nitro-L- arginine methyl ester (nitric for 10 min. The supernatant volume and pH were recorded with a oxide synthase blocker; Sigma Chemical Co., St. Louis, MO, USA) digital pH meter (PA 200, Marconi S.A., Piracicaba, Brazil). were used in this study. The reagents for buffers and other solutions were all of analytical grade. All the other chemicals and reagents used in this study were of analytical grade. Ethanol-induced gastric lesion in N-ethylmaleimide (NEM) and N (G)-nitro-L- arginine methyl ester (L-NAME)-pretreatment rats

Antiulcer action Rats were divided into groups of animals that fasted for 24 h. They had previously been treated intraperitoneally with NEM at a dose of Based on their respective specifications, the groups under each 10 mg/kg (n = 5), L-NAME at a dose of 70 mg/kg (n = 6) or vehicle experimental model included positive (lansoprazole, (n = 6). Thirty minutes later, the groups received an oral dose of the carbenoxolone, or cimetidine) and negative (Saline 0.9%) controls. vehicle (n = 6), carbenoxolone (n = 6; 100 mg/kg) or AHQ (n = 6; After each experiment, the animals were killed; the stomachs were 200 mg/kg). After 60 min, all groups were treated orally with 1 ml of opened along the greater curvature and pressed onto a glass plate. absolute ethanol for gastric-ulcer induction (Arrieta et al., 2003). Ulcerative lesion (U.I) was calculated according to the methodology Animals were killed by CO 2 gas 1 h after ethanol administration described by Szelenyi and Thiemer (1978). The antiulcerogenic and the stomachs excised and gastric damage determined as Lima-Saraiva et al. 5339

Table 1. Effects of AHQ from A. humile on two models of acute gastric lesion induced in rats and mice.

Gastric lesions models Treatment (p.o.) Dose ULI (mm) Inhibition (%) Saline 10 ml/kg 65.4 ± 17.7 - Lansoprazole 30 mg/kg 2.2 ± 0.8*** 97

Ethanol 50 mg/kg 42.4 ± 17.7* 35 AHQ – Anacardium humile 100 mg/kg 39.9 ± 9.9** 39 200 mg/kg 5.8 ± 3.6*** 91

Saline 10 ml/kg 13.7 ± 2.5 - Piroxicam Cimetidine 100 mg/kg 2.9 ± 1.6*** 79 AHQ – Anacardium humile 200 mg/kg 8.9 ± 3.1** 35

The results are the mean ± SD ANOVA, followed by Dunnett's test. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the vehicle.

described earlier. lesion formation when compared with vehicle group.

Healing action Evaluation of mucosal protective factors Acetic acid-induced gastric ulcer Determination of mucus in gastric content On this gastric ulcer induction, a cicatrisation model, rats were not fasted. Anesthesia (50 mg/kg of ketamine and 10 mg/kg of We observed the effect of AHQ (200 mg/kg) on adherent xylazine) was administered for the application of 100 µl of absolute acetic acid into the subserosal stomach layer of each animal. Two mucus production by the gastric mucosa (Figure 1). days after surgery, treatments (p.o.) with vehicle (n = 5), cimetidine Pretreatment with carbenoxolone (200 mg/kg) induced (n = 5; 100 mg/kg), and AHQ (n = 5; 200 mg/kg) were administered significant increase in mucoprotective effect in animals once daily for 14 consecutive days. The animals were sacrificed on submitted to pylorus ligature. On the other hand, AHQ the 15th day by CO 2 gas and then their stomachs were removed for (200 mg/kg) was not able to increase mucus production. lesion quantification (Okabe and Amagase, 2005).

Evaluation of the gastric juice parameters Statistical analysis

Parametric data were analyzed using a one-way analysis of The gastric juice parameters of the rats that were treated variance (ANOVA) followed by Dunnett’s test or Tukey’s test and with AHQ (200 mg/kg) administered by intraduodenal were compared to the vehicle group. The results were presented as route was verified. These results demonstrated that (AHQ the mean ± standard deviation (SD). All analyses were performed 200 mg/kg) was able to diminish the H + concentration in using GraphPad Prism software. A value of P < 0.05 was the gastric juice without modifying its volume (Table 2). considered significant. This effect was similar to those of the standard drug cimetidine (100 mg/kg). RESULTS

Ethanol-induced gastric lesion in NEM and L-NAME Ethanol-induced gastric lesion in rats pretreatment rats

In this model, the results showed that AHQ provided The pre-treatment with L-NAME did not affect the significant gastroprotection in all the tested doses (p < gastroprotection exerted by AHQ (200 mg/kg) (Figure 0.05, p < 0.01, and p < 0.001) (Table 1). Therefore, we 2A). In addition, as presented in Figure 2B, NEM did not focused on the following protocols using AHQ (200 affect the gastroprotection exerted by AHQ (200 mg/kg), mg/kg) which was the dose capable of producing the but increased the ulcer area in animals submitted to most effective gastroprotection. ethanol-induced gastric ulcer.

NSAID-induced ulcer Healing action

The antiulcerogenic activity of AHQ (200 mg/kg) in Acetic acid-induced gastric ulcer piroxicam-induced gastric lesion model is reported in Table 1. When AHQ was administered, it inhibited the Oral administration of AHQ (200 mg/kg) for 14 5340 J. Med. Plants Res.

200 ** 150

100

Alcian Blue Alcian 50 [mg/wet tissue (g)] tissue [mg/wet 0 Vehicle Carbenoxolone AHQ

Figure 1. Effects of AHQ (200 mg/kg) from A. humile on quantification of adherent mucus in gastric mucosa of rats. The results are presented as mean ± SD ANOVA followed by Dunnet’s test, **p < 0.01 compared to the vehicle.

Table 2. Effects of AHQ (200 mg/kg) from A. humile administered by intraduodenal route on biochemical parameters of gastric juice obtained from pylorus ligature mice.

Route Treatment Dose (mg/kg) Gastric juice (Content mg) pH (units) Vehicle - 0.6 ± 0.3 2.8 ± 0.2 Intraduodenal Cimetidine 100 0.5 ± 0.1 3.3 ± 0.2* AHQ-A. humile 200 0.5 ± 0.2 3.2 ± 0.3*

The results are the mean ± SD ANOVA followed by Dunnett's test. *p < 0.05 compared to the vehicle.

Figure 2. Effect of oral AHQ (200 mg/kg) from A. humile treatment, under the ethanol-induced gastric lesion model, on rats pretreated with L-NAME (A) or NEM (B). The results are the mean ± SD ANOVA followed by Tukey's test. **p < 0.01, ***p < 0.001 compared to the vehicle.

consecutive days accelerated the healing of gastric between aggressive and defensive factors of gastric mucosa in rats when compared with the control group mucosa. Tight junctions between epithelial cells, mucus treated with vehicle (Figure 3). and bicarbonate secretion are protective factors and gastric acid secretion and proteolytic activity of pepsin are, instead, aggressive factors (Tytgat, 2011). The DISCUSSSION excess of gastric acid secretion and increased production of pepsin have been considered as the main etiological Peptic ulcer (PU) is the consequence of an imbalance factors in the development of PU (Mózsik, 2010; Tytgat, Lima-Saraiva et al. 5341

6 ) 2

* 2 ** Ulcer Area (mm Area Ulcer

0 ** Sham Vehicle Cimetidine AHQ

Figure 3. Effect of oral administration, for 14 consecutive days, of AHQ (200 mg/kg) from A. humile on the healing ulcer in rats with chronic ulcer induced by 0.1 ml of absolute acetic acid. The results are the mean ± SD ANOVA followed by Tukey's test. **p < 0.01, ***p < 0.001 compared to the vehicle.

2011). Therefore, the main therapeutic target for PU is damage in the upper and lower gastrointestinal tract the control of this secretion using antacids, H 2 receptor (Sostres and Lanas, 2011). The systemic effects of blockers or proton pump blockers. Nevertheless, these NSAIDs involve prostaglandin production inhibition that treatments are often associated with adverse side effects leads to an impairment of bicarbonate and mucus and ulcer recurrence after therapy interruption (Tytgat, secretion. In the experimental induction of gastric ulcers 2011). In this study, we used models that resemble the by a NSAID, the AHQ (200 mg/kg) presented ulcer in humans (ethanol and NSAID) and the gastroprotection. Given that the ulcerogenic properties of antiulcerogenic mechanisms involved. The healing NSAIDs are due to the fact that they diminish the capacity of the aqueous fraction obtained from the leaves protective factors of the mucosa such as prostaglandins of A. humile St. Hil. (AHQ) was also verified with acetic and mucus (Konturek et al., 2005; Polat et al., 2010), it acid-induced ulcers model. can be affirmed that the antiulcerogenic activity of AHQ Ethanol is known to be one of many factors that observed in this model must augment these mucosal increase the risk of gastric ulcer formation. Hemorrhagic protective factors. In this sense, aiming this hypothesis, ulceration of the stomach in humans and experimental we evaluated the AHQ action on mucus secretion in the animals is also caused by ingestion of elevated amounts gastric mucosa. Figure 1 shows that AHQ (200 mg/kg) of ethanol. The main features of ethanol-induced gastric was not able to increase mucus production. This result ulcer are epithelial loss, mucosal edema, and sub- shows that factors cytoprotective do not participate in epithelial hemorrhage (de-Faria et al., 2012). Initially, the cytoprotection of AHQ. gastroprotective AHQ was tested in ethanol-induced Mucosal microcirculation is essential for oxygen and gastric lesions at these doses (50, 100, and 200 mg/kg). nutrient delivery and toxic substances removal. The This experimental model provides the screening of doses epithelial cells lining the microvessels generate potent and efficacy of the treatments against ulcer formation. vasodilators such as nitric oxide (NO) that increases The ulcerogenic activity of ethanol is driven by its removal and/or dilution of the back-diffusing acid and/or capacity to dissolve the constituent gastric mucus, while noxious agents when the gastric mucosa is exposed to concomitantly diminishing the transmucosal action an irritant agent (Laine, 2008). Thus, NO endogenous potential, thus, increasing the flow of Na + and H + in the and exogenous, plays a major role in mucosal defense by lumen and stimulating the secretion of histamine, pepsin modulating the mucosal circulation. The administration of and H + ions (Moraes et al., 2009). Considering that the L-NAME, NO synthesis inhibitor, would certainly be AHQ (50, 100, and 200 mg/kg) exerted 35, 40, and 88%, harmful, especially for gastric mucosa. As well as NO, the respectively protection on the gastric mucosa, it is sulfhydryl compounds (SH) are important for the undeniable that this fraction exert substantial protective maintenance of gastric mucosa, because of its protection action on the gastric mucosa. against pro-oxidant agents. Luiz-Ferreira et al. (2010) The increased use of NSAIDs causes gastrointestinal reported that the administration of glutathione depletors toxicity, including the PU (Hampton and Hale, 2011). (NEM) significantly potentiates the effects of ethanol on Topical and systemic effects of NSAIDs in the gastric mucosa injury. AHQ (200 mg/kg) protected the gastrointestinal mucosa are associated with mucosal gastric mucosa from the irritant agent when NEM is co 5342 J. Med. Plants Res.

administered. Thus, our result suggests that the AHQ secretory drugs; A. humile arises as a promising gastroprotective effect does not depend of the presence alternative antiulcer therapy. of SH compounds in the gastric mucosal barrier. Furthermore, the evaluation of NO participation in the gastroprotection promoted by AHQ demonstrated that REFERENCES despite the inhibition of NO by the action of the L-NAME- blocking NOs, AHQ continued exerting its effect. Ajibola ES, Adeleye OE, Okediran BS, Rahman SA (2010). Effect of Intragastric Administration of crude aqueous leaf extract of After noticing that cytoprotector factors are not involved Anacardium occidentale on gastric acid secretion in rats. Niger J. in AHQ gastroprotection, we verified its ability to inhibit Physiol. Sci. 25(1):59-62. acid secretion. 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