Naunyn-Schmiedeberg's Arch Pharmacol DOI 10.1007/s00210-013-0954-0

ORIGINAL ARTICLE

Evaluation of the gastroprotective activity of the extracts, fractions, and pure compounds obtained from aerial parts of Rubus imperialis in different experimental models Priscila Elisabeth Berté & Jhonny da Silva Lopes & Nicole Garbin Comandulli & Daniele Wolff Rangel & Franco Delle Monache & Valdir Cechinel Filho & Rivaldo Niero & Sergio Faloni de Andrade

Received: 17 September 2013 / Accepted: 22 December 2013 # Springer-Verlag Berlin Heidelberg 2014

Abstract Previous phytochemical studies carried out with Rubus imperialis Chum. Schl. (Rosaceae) have demonstrated the presence of triterpenes (niga-ichigoside F1 and 2β,3β,19α-trihydroxyursolic acid) in this species. The literature indicates that triterpenes are closely related to some pharmacological activities, including antiulcer activity. Therefore, in view of the previous promising results with this species, this work extends the phytochemical studies, as well as investigates its gastroprotective action in different models using rodents. The hydroalcoholic extract was tested using the following protocols in mice: ethanol/HCl and nonsteroidal anti-inflammatory drug (NSAID)-induced ulcer, acetic acidinduced chronic ulcer, ligature pylorus model, and free mucus quantification in mucosa. Isolated triterpenes were investigated in the ethanol/HCl-induced ulcer model. The results of this study show that R. imperialis extract (100, 250, or 500 mg) displays gastroprotective activity in the ethanol-induced ulcer model with a percentage of inhibition of gastric lesions of 70, 71, and 86 %, respectively. The extract also significantly reduced the ulcerative lesions in the indomethacin-induced ulcer. In this model, the percentage of inhibition of ulcer was 41, 44, and 70 %, respectively. Regarding the model of gastric secretion, a reduction of gastric juice volume and total acidity was observed, as well as an increase in gastric pH; however, gastric mucus production was not altered by treatment with the extract. It was also observed that the ethyl acetate fraction presented higher activity, leading to the isolation of nigaP. E. Berté : J. da Silva Lopes : N. G. Comandulli : D. W. Rangel : F. D. Monache : V. C. Filho : R. Niero : S. F. de Andrade (*) Programa de Pós-Graduação em Ciências Farmacêuticas e Núcleo de Investigações Químico-Farmacêuticas (NIQFAR), Universidade do Vale do Itajaí – UNIVALI, Rua Uruguai, 458, 88302-202 Itajaí, SC, Brazil e-mail: [email protected]

ichigoside F1 and 2β,3β-19-α-trihydroxyursolic acid, which presented antiulcer activity comparable to that of omeprazole, with an inhibition percentage of 98 and 99 %, respectively. These results demonstrate that R. imperialis extract and isolated compounds (niga-ichigoside F1 and 2β,3β-19-αtrihydroxyursolic acid) produce gastroprotective effects, and this activity seems, at least in part, to be related to antisecretory effects. Keywords Rubus imperialis . Gastroprotective activity . Antiulcer

Introduction The Rosaceae family comprises about 100 genera and approximately 700 species, distributed mainly in tropical and subtropical regions of the world. Some of the species are used in various countries to treat different diseases (Niero and Cechinel 2008). Chemical and pharmacological studies carried out on some species of this family have demonstrated the presence of active principles that exert hypoglycemic, antibacterial, antiallergic, and antiasthmatic effects. In addition, some plants of the species are used in folk medicine as a uterine relaxant, for the treatment of diarrhea and similar enteric disorders, and as an astringent (Novaes et al. 2001; Kim et al. 2005; Emendörfer et al. 2005; Kanegusuku et al. 2007). Moreover, many plants of the Rosaceae family produce edible fruits. One of the species belonging to the Rosaceae family is Rubus imperialis Chum. Schl. (Rosaceae), which is widespread in Southern Brazil, where it is known as amora-branca or amora-do-mato. In Brazil, R. imperialis is used in folk medicine for the treatment of diabetes and pain.

Naunyn-Schmiedeberg's Arch Pharmacol

Previous phytochemical studies carried out by our research group have demonstrated the presence of triterpenes (nigaichigoside F1 and 2β,3β,19α-trihydroxyursolic acid) (Niero et al. 2002; Ardenghi et al. 2006) in this species. Nigaichigoside F1 showed potent antinociceptive activity when analyzed in two models of nociception: the writhing test and the formalin test (Ardenghi et al. 2006). The literature indicates that triterpenes are closely related to some pharmacological activities, including antiulcer activity (Andrade et al. 2006, 2008). Considering the fact that there are many terpenes with gastroprotective activity and that gastroprotective properties of R. imperialis have not yet been properly studied, our research group decided to evaluate the gastroprotective potential of this species in different experimental ulcer models. The gastroprotective effects of the extract, of fractions and of the isolated triterpenes were studied.

Material and methods Plant material The plant material was collected in August 2008 in Treze de Maio (latitude 28° 33′ 28″S and longitude 049° 08′ 48″W) in the state of Santa Catarina and was identified by Dr. Ademir Reis. A voucher specimen was deposited at the Barbosa Rodrigues Herbarium under code VC-Filho 012.

Preparation of extracts and fractions and isolation of compounds Dried and powdered aerial parts of R. imperialis (1.10 kg) were exhaustively extracted at r.t. with methanol for 12 days. The resulting macerate were filtered and concentrated under reduced pressure, using a rotatory evaporator, yielding crude hydroalcoholic extract (CME). This extract (127.5 g; 12.7 %) was then suspended in a MeOH–water mixture (9:1) and successively partitioned with n-hexane, chloroform, and ethyl acetate, furnishing 5.32, 8.39, and 9.25 g, respectively. Each fraction was chromatographed separately on a silica gel column (0.04–0.063 mm, 2.5×50 cm) eluted with a gradient of CHCl3–MeOH or hexane–acetone (100→0) to give several fractions. Similar fractions were combined, and the purity was examined by thin layer chromatography (TLC) using Merck silica gel precoated aluminum plates, with a layer thickness of 200 μm and several solvent systems of different polarities. These procedures led to the isolation of four compounds. The isolated compounds were identified by NMR spectral data, in comparison with authentic samples and the literature data, as niga-ichigoside F1 (1) (265 mg) and 2β,3β-19-αtrihydroxyursolic acid (2) (340 mg) (Fig. 1).

Drugs, reagents, and solvents Indomethacin, cimetidine, and omeprazole were purchased from Sigma-Aldrich (St. Louis, MO, USA). All the other reagents and solvents used were of analytical grade. Evaluation of gastroprotective activity Male Swiss mice (20–25 g) and male Wistar rats (180–200 g) were provided by the Central Animal House of the Universidade do Vale do Itajaí (UNIVALI) (Itajaí, SC, Brazil). Rats were used in assays with the extract whereas mice were used for the evaluation of fractions and isolated compounds, due to low yields of fractions and compounds in the isolation process. The animals were housed in groups of five, in standard cages, at room temperature (25±3 °C) with 12 h dark/12 h light cycles, and received food and water ad libitum. Twelve hours prior to the experiments, they were transferred to the laboratory and given only water ad libitum. In all the experiments, the animals were kept in cages with wide-mesh raised floors to prevent coprophagy. The animals used in the present study were housed and cared for in accordance with the Federal Government legislation on animal care. Also, the experiments were authorized by the Ethical Committee for Animal Care of the Universidade do Vale do Itajaí (Itajaí, SC, Brazil) (process number 023/11p2). Ethanol/HCl-induced ulcer The experiment was performed according to the method described by Schmeda-Hirschmann et al. (2005). After 12 h of fasting, the rats were randomly divided into five groups of six animals each. The first group was given 1.0 mL of vehicle (1 % Tween-80 aqueous solution), and the second group was treated with omeprazole (30 mg/kg). The remaining groups received 100, 250, or 500 mg/kg of hydroalcoholic extract of R. imperialis. All the treatments were administered by gavage. One hour after treatment, all the rats received 0.5 mL of a 0.3 mol/L HCl/60 % ethanol solution (ethanol/HCl) to induce gastric ulcer. Another hour later, the animals were sacrificed by cervical dislocation, and the stomachs removed and opened along the greater curvature. The stomachs were gently rinsed with water to remove the gastric contents and blood clots, for subsequent scanning. The images obtained were analyzed using the parameters described in the section on stomach analysis. In another assay, the gastroprotective activity of hexanic, chloroformic, and ethyl acetate fractions was evaluated. In this experiment, mice were divided into five groups with six animals each, which were submitted to fasting for 12 h prior to receiving 0.2 mL of the vehicle (1 % Tween-80 aqueous solution), 30 mg/kg of omeprazole (standard drug), or 250 mg/kg of the fractions, respectively. One hour after

Naunyn-Schmiedeberg's Arch Pharmacol Fig. 1 Molecular structures of compounds isolated from R. imperialis. 1 niga-ichigoside F1 and 2 2β,3β-19-αtrihydroxyursolic acid

COOGly COOH

OH

treatment, all the mice received 0.5 mL of a 0.3 mol/L HCl/ 60 % ethanol solution (ethanol/HCl) to induce gastric ulcer. Another hour later, the animals were sacrificed by cervical dislocation, and the stomachs were further processed as described above. Nonsteroidal anti-inflammatory drug-induced ulcer Experiments were carried out according to the method described in Nwafor et al. (2000), with a few modifications. After 12 h of fasting, the rats were randomly divided into five groups of six animals each. The first group was given 1.0 mL of vehicle (1 % Tween-80 aqueous solution), and the second group was treated with cimetidine (100 mg/kg). The three remaining groups received 100, 250 or 500 mg/kg of hydroalcoholic extract of R. imperialis. All the treatments were administered orally. One hour after treatment, all the mice received a combination of indomethacin (100 mg/kg, p.o.) and bethanechol (5 mg/kg, i.p.) to induce gastric ulcer. Four hours after treatment with indomethacin and bethanechol, the animals were sacrificed and their stomachs further processed as described in “Stomach analysis” section. Additionally, hexanic, chloroformic, and ethyl acetate fractions were tested at a dose of 250 mg/kg, using mice similar to those used for the extract. Omeprazole was used as a positive control for the ethanol/ HCl-induced ulcer because cimetidine does not display a sufficient degree of gastroprotection in this model, likely due to the fact that ethanol is very aggressive for the mucosa. However, cimetidine, which is more soluble and stable, was used in the other ulcer models. Chronic ulcer The methodology described by Takagi et al. (1969) was used, with some modifications. The rats were divided into three groups (n=6). They were then anesthetized and subjected to a longitudinal incision below the xiphoid apophysis. After exposure of the stomach, 50 μL of 20 % acetic acid solution was injected into the subserosal layer of the outer wall of the organ. The site was held down for 30 s to prevent leakage of the injected fluid. The stomach was carefully washed with

2

1

saline, and the abdominal wall was sutured. Two days after surgery, when the animals had recovered, treatment was carried out lasting 7 days. The animals received oral cimetidine at a dose of 100 mg/kg (positive control), vehicle (negative control), or R. imperialis extract (500 mg/kg). After 7 days of treatment, the animals were sacrificed by cervical dislocation and the stomachs removed and processed as described in “Stomach analysis” section.

Stomach analysis The images obtained were analyzed using specific “EARP” software to measure each lesion point. The total area of lesion and the percentage of lesion area in relation to total stomach area were determined (Santin et al. 2011). In addition, the inhibition (expressed as percent) was calculated as follows: inhibition (%)=−[(total area of lesion (mm2) in treated group/ total area of lesion (mm2) in control group)−1]×100

Determination of gastric secretion The assay was performed using the method of Shay et al. (1945) with a few modifications. The animals (rats) were divided into groups (n=6). After 24 h of fasting, the animals were anesthetized with thiopental sodium (10 mg/kg, i.p.), the abdomen was incised, and the pylorus ligated. Immediately after pylorus ligature, R. imperialis hydroalcoholic extract was administered at doses of 100, 250, or 500 mg/kg. Cimetidine (100 mg/kg) was used as positive control, and 0.5 mL of vehicle (1 % Tween-80 aqueous solution) was administered as negative control. All the samples were administered intraduodenally. Four hours later, the animals were sacrificed by cervical dislocation; the abdomen was opened and another ligature placed at the esophageal end. The stomachs were removed and the gastric contents collected and centrifuged at 3,000 rpm (8,000×g, 25 °C, 10 min). The amount of gastric juice acid (in milliliters) and the pH values were determined. Total acid secretion in the gastric juice was determined in the supernatant volume by titration at pH 7.0, using a 0.01 mol/L NaOH solution, and phenolphthalein as indicator.

Naunyn-Schmiedeberg's Arch Pharmacol

Determination of mucus in the gastric content This assay was performed according to the methodology previously described in Sun et al. (1991), with a few modifications. The rats were divided into groups of six each. After 24 h of fasting, under anesthesia, the abdomen was incised and the pylorus ligated. Immediately after pylorus ligature, R. imperialis extract was administered intraduodenally at doses of 100, 250, and 500 mg/kg, respectively. Carbenoxolone (250 mg/kg) was used as positive control, and 1 mL of vehicle (1 % Tween-80 aqueous solution) was administered as negative control. The animals were killed 4 h after the drug treatments. The stomach content was immersed in 10 mL of 0.02 % Alcian blue 0.16 M sucrose/0.05 M sodium acetate solution at pH 5.8 and incubated for 24 h at 20 °C. The Alcian blue binding extract was centrifuged at 3,000×g for 10 min. The absorbance of the supernatant was measured by spectrophotometry at 615 nm. The free mucus in the gastric content was calculated by the amount of Alcian blue binding (in milligrams per gram of tissue). Evaluation of gastroprotective activity of niga-ichigoside F1 and 2β,3β,19α-trihydroxyursolic acid The gastroprotective activity of the isolated compounds was evaluated using the model of gastric lesions induced by ethanol/HCl, according to the methodology described by Schmeda-Hirschmann et al. (2005). The mice were divided into groups of six animals and fasted for 12 h prior to receiving 0.5 mL of the vehicle (1 % Tween-80 aqueous solution), 30 mg/kg of omeprazole (standard drug), or isolated compounds. All further procedures were carried out as described above. Statistical analysis The data are reported as mean ± standard error of the mean (SEM) and were compared using one-way analysis of variance (ANOVA), followed by Dunnett’s pairwise test, and p values