Journal of Ethnopharmacology 104 (2006) 215224

Antiulcerogenic activity of Alchornea castaneaefolia: Effects on somatostatin, gastrin and prostaglandin

Cl lia Akiko Hiruma-Lima a, , Tamara Roberta Calvo b , Clenilson Martins Rodrigues b , e F bio Donizete Pezzuto Andrade b , Wagner Vilegas b , Alba Regina Monteiro Souza Brito c a
Departamento de Fisiologia, Instituto de Bioci ncias, Universidade Estadual Paulista (UNESP), e Rubi o Junior s/n, CP 510, CEP 18618-000, Botucatu, SP, Brazil a b Departamento de Qumica Org nica, Instituto de Qumica, CP 355, CEP 14800-900, UNESP, Araraquara, SP, Brazil a c Departamento de Fisiologia e Biofsica, Instituto de Biologia, CP 6109, CEP 13083-970, UNICAMP, Campinas, SP, Brazil Received 20 February 2005; received in revised form 20 August 2005; accepted 2 September 2005 Available online 25 October 2005
a

Abstract The hydroethanolic extract of the leaves (HEL) and bark (HEB) obtained from Alchornea castaneaefolia (Euphorbiaceae) were investigated for their ability to prevent ulceration of the gastric mucosa in animal models. HEL (500 and 1000 mg/kg) and HEB (1000 mg/kg) signicantly reduced the gastric injuries induced by the combination of HCl/ethanol and lowered the severity of gastric damage formation induced by indomethacin/bethanechol in mice. Further investigation showed that HEL also inhibited formation of ulcers in mice submitted to stress and pylorus ligature, but HEL did not modify gastric juice parameters in Shay mice. HEL was also effective in promoting the healing process in chronic gastric ulcer induced by acetic acid in rats. An enriched avonoidic fraction (EFF at dose of 100 mg/kg) obtained from HEL reduced gastric lesions induced by HCl/ethanol and indomethacin/bethanechol in mice. Although EFF did not modify the amount of free mucus production by gastric mucosa, it was able to increase prostaglandin production. When administered to rats submitted to ethanol-induced gastric lesions, EFF increased the somatostatin serum levels, while the gastrin serum levels were proportionally decreased. Phytochemical investigation on HEL and EFF led to the isolation of avonoids glycosides as the main compounds, thus suggesting that these substances may be involved in the observed antiulcer activity. 2005 Elsevier Ireland Ltd. All rights reserved.
Keywords: Alchornea castaneaefolia; Antiulcer activity; Flavonoids; Euphorbiaceae

1. Introduction Gastric and duodenal ulcers are illnesses that affect a considerable number of people in the world and some authors consider gastric ulcer as the new plague of the 21st century (OMalley, 2003). Factors such as stress, smoking, nutritional deciencies and frequent ingestion of nonsteroidalantiinammatory drugs (NSAIDs) grow the gastric ulcer incidences (Belaiche et al., 2002). Although there is evidence to implicate Helicobacter pylori in the development of peptic ulcer, the proportion of ulcer not related to Helicobacter pylori or NSAIDs has increased, which will affect the management of peptic ulcer (Chan and Leung, 2002).

Corresponding author. Tel.: +55 1438116077; fax: +55 1438116251. E-mail address: hiruma@ibb.unesp.br (C.A. Hiruma-Lima).

Diverse chemical compounds have been isolated from medicinal plants with antiulcer activity (Lewis and Hanson, 1991). This is an important reason to investigate antiulcer effects in medicinal plants with traditional use in gastric diseases. Central Brazil area is one of the major biogeographic regions of the world and also one of the most threatened (Myers et al., 2000). A recent list of the central Brazil ora registers 6253 native species of vascular plants included in 150160 families (Mendonca et al., 1998). Many of these plants are used as natural medicines by people living in the Cerrado area (a savannah-like vegetation) to treat various diseases (Almeida et al., 1998). An ethnopharmacological inventory made in the Cerrado formation of the central region of Brazil showed a high number of medicinal plants used to treat gastric pain and gastritis (Silva et al., 2000). Based on this inventory, we selected the species Alchornea castaneaefolia to study its antiulcer property indicated by traditional use. Alchornea castaneaefolia A. Juss.

0378-8741/$ see front matter 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.09.007

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belongs to the Euphorbiaceae family and is popularly known as sar , sar o and gurupi . This species is a mediuma a a sized tree (16 m), very common in the margins of Tocantins River, Cerrado regions, alongside riverbanks, both in sandy and clay soils. It can also be found in the regions from the State of Amazons to the State of Bahia and Mato Grosso. Alchornea castaneaefolia is important for reconstituting forest and river margins due to its xing properties (Pott and Pott, 1994). In folk medicine, leaves and barks of Alchornea castaneaefolia are used as an infusion for the treatment of ulcer, gastritis, rheumatism, arthritis, u and muscular pain (Duke and Vasquez, 1994). Despite the popular use of Alchornea castaneaefolia as a medicinal plant, there are no data available about its pharmacological effect on the gastrointestinal system or its possible toxic properties. A few references regarding the genus Alchornea were found. Alchornea cordifolia has antiinammatory effect, and also antimicrobial and antiplasmodial activities (Ajali, 2000; Ebi, 2001; Banzouzi et al., 2002; Osadebe and Okoye, 2003). This plant presented alchornein, alchorneinone, isoalchornein, yohimbine and other alkaloids as well as anthranilinic and gentisic acids (Ogungbamila and Samuelsson, 1990). Flavonoids, tannins, steroids, terpenes, saponins and alkaloids were isolated from Alchornea cordifolia (Tona et al., 1998). Triterpenoids such as taraxerone, friedeline, epifriedelinol, taraxerol, seco3,4-friedeline and seco-3,4-taraxerone were also isolated from Alchornea latifolia. The last two triterpenoids present an open A ring and showed cytotoxicity in vitro assays in human cancer cells as well as potent inhibition of topoisomerase II (Setzer et al., 2000). The present work was carried out to investigate the possible antiulcerogenic effect of the hydroalcoholic extract of leaves (HEL) and bark (HEB) of Alchornea castaneaefolia against acute and chronic experimental models of gastric ulcer in rodents. The possible mechanism of action of enriched avonoidic fraction (EFF) isolated from HEL was also studied. Finally, we also performed a phytochemical investigation on the extracts obtained from Alchornea castaneaefolia. 2. Methods and materials 2.1. Animals Male Swiss albino mice (2535 g) and male Wistar rats (150250 g) from the Central Animal House of the UNICAMP were used. The animals were fed a certied Nuvilab (Nuvital) diet with free access to tap water under standard conditions of 12 h dark12 h light, humidity (60 1.0%) and temperature (21 1%). Fasting was used prior to all assays because standard drugs and infusion and extract were always administered orally (by gavage) or by intraduodenal route using a saline solution (10 ml/kg) as the vehicle. Moreover, the animals were kept in cages with raised oors of wide mesh to prevent coprophagy. The UNICAMP Institutional Animal Care and Use Committee, following the recommendations of the Canadian Council on Animal Care (Olfert et al., 1993), approved all the employed protocols.

2.2. Vegetal material and extract preparation Bark and leaves of Alchornea castaneaefolia were collected at Tocantins River bed, Porto Nacional city (Tocantins StateTO), Brazil, by Dr. Hiruma-Lima. A owered voucher was identied by Prof. Solange Lolis from UNITINS, Porto Nacional city and deposited under No. 4527 at UNITINS herbarium. Dry leaves (400 g) of Alchornea castaneaefolia were powdered and macerated with 3 l of 70% ethanol for 1 week. The solution was ltered through lter paper and the solvents were evaporated under reduced pressure to obtain 5 g (1.3% from the dry weight) of HEL. A similar procedure with dried barks provided 7 g (1.8%) of HEB. 2.3. Phytochemical analysis An aliquot of HEL (3.0 g) was fractionated by gel permeation chromatography (GPC) on a Sephadex LH-20 column (Pharmacia, 1 m 3 cm) eluted with methanol at a ow rate 0.5 ml/min. One hundred twenty fractions (8 ml) were automatically collected on a Redifrac collector and checked by thin layer chromatography (TLC) on Si gel plates, CHCl3 :MeOH:nPrOH:H2 O (5:6:1:4, v/v/v/v, lower phase) was revealed with UV light (254 nm) and further with anisaldehyde/sulfuric acid or natural products/polyethyleneglycol (NP/PEG) reagents (Wagner et al., 1986). Mainly glycolipids and free sugars (detected by their NMR spectra) were obtained from Fr. 1-55 and were not further investigated. Fr. 55-120 (1 g, 33% from HEL) gave mainly avonoids, gallic acid and methyl gallate. An aliquot (200 mg) of Fr. 55-120 was further puried by high performance liquid chromatography (HPLC) Knauer Chance equipped with a RP18 column (Phenomenex Luna 250 mm 10 mm, 10 m) eluted with a mixture of methanol:water 7:3 (v/v) and monitored with a Knauer Ultraviolet (UV) detector (254 nm), giving compounds 17. Substances 17 were analysed using one-dimensional (1 H and 13 C NMR, DEPT and TOCSY) and bi-dimensional experiments (COSY 1 H1 H, HMQC, HMBC) on a 500 MHz Varian Inova equipment. Samples were dissolved in hexadeuterated dimethylsulphoxide DMSO-d6 . Their structures were identied on the basis of their 1 H and 13 C NMR data compared to those reported in the literature (Agrawal, 1989; Harborne, 1996). For the biological assays, the fractionation of HEL was repeated and Fr. 55-120 was added and named enriched avonoidic fractionEFF (3 g). 2.4. HPLC-UV-PDA chromatographic prole of the EFF from Alchornea castaneaefolia Analyses were performed on a Varian HPLC ProStar 210 apparatus equipped with an RP18 column (Phenomenex Luna 250 mm 4.6 mm, 5 m), a guard column (Phenomenex 4 mm 3 mm), a Varian ProStar 330 Ultraviolet photo-diode array (UV-PDA) detector and a Rheodyne injector (loop of 20 l). Elution was performed with a linear gradient of acetonitrile/water 2535% for 30 min, ow rate of 1.0 ml/min, and monitored at 254 nm.

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2.5. Drugs and chemicals The following drugs were used: cimetidine (Tagamet SmithKline, Brazil), lansoprazole (Ilzatec Boehringer Ingelheim, Brazil), acetyl- -methylcholine chloride (bethanechol), Alcian Blue, carbenoxolone and indomethacin (Sigma Chemical Co., U.S.A.). The chemicals used and other solutions were all of analytical grade. All drugs and reagents were prepared immediately before use. 2.6. Acute toxicity The acute toxicity studies of Alchornea castaneaefolia were performed in mice. In this assay, increasing doses of HEL were orally administered to groups of 10 animals for each dose after a 12 h fast. Animals receiving the vehicle (saline) served as control. The signals and symptoms associated with the HEL administration (5 g/kg, p.o.) were observed after 0, 30, 60, 120, 180 and 240 min and then once a day for the next 14 days. At the end of the period, the number of survivors was recorded. The acute toxicological effect was estimated by the deaths occured, and it was expressed as LD50 (Litcheld and Wilcoxon, 1949; Souza Brito, 1995). 2.7. Antiulcerogenic activity 2.7.1. HCl/ethanol-induced ulcer The antiulcerogenic activities of HEL (250, 500 and 1000 mg/kg), HEB (1000 mg/kg) and EFF (100 mg/kg) were studied in HCl/ethanol-induced gastric ulcer (Mizui and Doteuchi, 1983). Mice were divided into groups with 511 animals that were fasted 24 h prior to receiving an oral dose of the vehicle, saline (10 ml/kg), lansoprazole (30 mg/kg), HEL (250, 500 and 1000 mg/kg), HEB (1000 mg/kg) and EFF (100 mg/kg). After 50 min, all groups were orally treated with 0.2 ml of a 0.3 M HCl/60% ethanol solution (HCl/ethanol) for gastric ulcer induction. Animals were killed 1 h after the administration of HCl/ethanol, and the stomachs excised and inated by saline injection (2 ml). The extent of the lesions was measured and the lesion index expressed as the sum of all lesions (Szelenyi and Thiemer, 1978). 2.7.2. Indomethacin-induced gastric ulcers in cholinomimetic-treated mice In this model, mice were divided into groups with 511 animals and gastric lesions were induced with indomethacin (30 mg/kg, s.c.) and bethanechol (5 mg/kg, i.p.) administered to mice after a 24 h fast. HEL (250, 500 and 1000 mg/kg), HEB (1000 mg/kg) and EFF (100 mg/kg), cimetidine (100 mg/kg) and saline were administered orally 30 min before the induction of gastric lesions. The animals were killed 4 h after treatment with the ulcerogenic agent (Rainsford, 1978). The stomachs were removed and gastric damage determined as described above. 2.7.3. Hypothermic-restraint stress ulcer The antiulcerogenic activity of HEL in the hypothermicrestraint stress-induced gastric ulcer model was assessed in mice.

Mice were divided into groups of 910 animals. After 24 h of starvation, the animals received a single oral administration of HEL (500 mg/kg), cimetidine (100 mg/kg) or saline. One hour after pretreatment, gastric ulceration was induced by immobilizing the animals inside a closed cylindrical cage maintained at 4 C (Levine, 1971). After 4 h, the animals were killed and the stomachs removed and examined for ulcers as described previously. 2.7.4. Shay ulcer A total of 25 mice were randomly divided into three groups (n = 79) that were fasted for 24 h with free access to water. Thirty minutes after oral administration of single doses of HEL (500 mg/kg), cimetidine (100 mg/kg) as positive control or vehicle (saline, 10 ml/kg), the pylorus ligature was performed (Shay et al., 1945). Four hours later, the animals were killed, the abdomen opened and another ligature placed around the esophagus close to the diaphragm. The stomach was removed, inspected internally, and its content drained into a graduated centrifuge tube and centrifuged at 3000 rpm for 10 min. The supernatant volume and pH were recorded with a digital pH meter (PA 200, Marconi S.A., Brazil). The total acid content of gastric secretion was also determined by titration to pH 7.0 with 0.01 N NaOH using a digital burette (E.M., Hirschmann Technicolor, Germany). Gastric lesions were evaluated by examining the inner gastric surface with a magnifying glass and the mucosal lesions were counted and scored as previously described. 2.7.5. Determination of gastric secretion by intraduodenal administration of HEL A total of 23 mice were randomly divided into three groups (n = 78), which were fasted for 24 h with free access to water. Immediately after pylorus ligature, a single dose of HEL (500 mg/kg), cimetidine (100 mg/kg) as positive control or vehicle was administered by intraduodenal route. Four hours later, the animals were killed and all of the procedures described below were followed (Shay et al., 1945). 2.7.6. Acetic acid-induced gastric ulcers Male Wistar rats (n = 57) fasted for 24 h were used in this experiment. Under anesthesia, a laparotomy was done on all animals through a midline epigastric incision. After exposing the stomach, 0.05 ml (v/v) of a 30% acetic acid solution was injected into the subserosal layer in the glandular part of the anterior wall. The stomach was bathed with saline in order to avoid adherence to the external surface of the ulcerated region. The abdomen was then closed and the animals were fed normally. HEL (500 mg/kg), cimetidine (100 mg/kg) or vehicle (10 ml/kg) was administered orally once a day for 14 consecutive days beginning 1 day after surgery. Bodyweight was recorded daily throughout the experiments to evaluate the possible chronic toxicity induced by HEL. On the day after the last drug administration, the rats were killed and the stomachs were removed and pH of the gastric juice secretions was recorded with a pH meter (Marconi, Brazil). The gastric lesions were evaluated by examining the inner gastric surface with a dissecting magnify-

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ing glass. The ulcer area (mm2 ) and curative ratio (%) were subsequently determined (Takagi et al., 1969). 2.7.7. Determination of mucous in gastric content Mice (n = 67) were fasted for 24 h, under anesthesia, their abdomen incised and pylorus ligated. The vehicle (saline), indomethacin (30 mg/kg), carbenoxolone (250 mg/kg) and EFF (100 mg/kg) were administered intraduodenally after the pylorus ligature. The animals were killed by cervical dislocation 4 h after the drug treatments. The stomach content was immersed in 10 ml of 0.02% Alcian Blue in 0.16 M sucrose/0.05 M sodium acetate, pH 5.8, and incubated for 24 h at 20 C. The Alcian Blue binding extract was centrifuged at 3000 rpm for 10 min. The absorbency of supernatant was measured at 615 nm using a light spectrophotometer U/2000 (Hitachi, Japan). Free mucous in the gastric content was calculated from the amount of Alcian Blue binding [mg/wt tissue (g)] (Sun et al., 1991). 2.7.8. Determination of prostaglandin synthesis Thirty minutes after treatment with the vehicle, indomethacin (30 mg/kg, s.c.), vehicle (10 ml/kg) and EFF (100 mg/kg, p.o.), the rats were killed by cervical dislocation and the abdomen was opened. The Sham group, without treatment, experienced general conditions of the experimental group. Samples of the corpus (full thickness) were excised, weighed and suspended in 1 ml of 10 mM sodium phosphate buffer, pH 7.4. The tissue was minced nely with scissors and then incubated at 37 C for 20 min (Curtis et al., 1995). Prostaglandin in the buffer was measured using an enzyme immunoassay (RPN 222-Amersham). 2.7.9. Blood collection The heparinized blood samples of Sham rats and treated with vehicle, lansoprazole (30 mg/kg) and EFF (100 mg/kg) were obtained from the cardiac puncture (Krous, 1980). Immediately after puncture, the blood was removed and submitted to centrifugation (3000 rpm, at 6 C) for 10 min. After the centrifugation, the serum obtained was preserved at 20 C until use. 2.7.10. Somatostatin dosage After induction of gastric ulcer by ethanol, somatostatin hormone was measured in serum of Sham rats and treated with vehicle, lansoprazole and EFF (100 mg/kg) through radioimmunoassay kit to somatostatin (RB-306, EURO-Diagonostica) (Krous, 1980; Motegi et al., 1998). 2.7.11. Gastrin dosage After induction of gastric ulcer by ethanol, the gastrin hormone was measured in serum of animals Sham, and treated with vehicle, lansoprazole and EFF (100 mg/kg) through radioimmunoassay kit to gastrin (CIS Bio International GASK-PR) (Mayer et al., 1974; Motegi et al., 1998). 2.8. Statistical analysis Results were expressed as mean S.D. Statistical signicance was determined by one-way analysis of variance followed

by Dunnetts or Tukeys test, with the level of signicance at P < 0.05. 3. Results The effect of the two preparations from Alchornea castaneaefolia, HEL and HEB, on gastric ulcers induced by different damaging agents (HCl/ethanol and NSAIDs) was investigated rst in mice and the results are shown in Fig. 1. Pretreatment with HEL and HEB, both given orally at doses of 1000 mg/kg, and lansoprazole (positive control) administered at a dose of 30 mg/kg, induced a signicant protective effect in variable degrees. The antiulcer drugs, lansoprazole, HEL and HEB signicantly inhibited ulcer formation by 68, 88 and 86%, respectively. No signicant differences were observed between the groups treated with HEL and HEB (P > 0.05). The obtained results suggest that the crude extract of leaves (HEL) and bark (HEB) of Alchornea castaneaefolia present a signicant antiulcer effect in these ulcer-induced models. The effect of HEL and HEB on the gastric lesions induced by indomethacin/bethanechol is also shown in Fig. 1. Signicant inhibition of gastric ulcer was observed in pretreatment with HEL or HEB from Alchornea castaneaefolia. These extracts (1000 mg/kg) exhibited a protective effect by 62 and 60%, respectively. Animals treated with saline (negative control) showed extensive lesion area (38.3 2.1 mm2 ). Our results showed that both extracts HEL and HEB presented similar antiulcer properties in both models (HCl/ethanolinduced and NSAIDs-induced gastric lesions). Therefore, we chose to continue the pharmacological assays using only HEL. The oral toxicity dose of HEL was evaluated (results not shown) with a dose ve-fold higher than the highest tested antiulcer dose (1000 mg/kg, p.o.). At 5000 mg/kg, no signs and symptoms of acute toxicity were observed in all treated mice (n = 10). No signicant macroscopic differences were observed in heart, liver, kidney or lung. None of the treated mice died during the 14 days of observation after the administration of HEL. These preliminary results indicated the absence of acute toxic effects of HEL and they motivated us to continue with the assays.

Fig. 1. Effects of cimetidine, lansoprazole and the hydroalcoholic extract of leaves (HEL) and bark (HEB) of Alchornea castaneaefolia on indomethacin/bethanechol-induced and HCl/ethanol gastric lesions in mice. Results are mean S.D. ANOVA followed by the Dunnetts test: * P < 0.001.

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Fig. 2. Effects of cimetidine, lansoprazole and the doseresponse curve of hydroalcoholic extract of leaves (HEL) of Alchornea castaneaefolia on indomethacin/bethanechol-induced and HCl/ethanol gastric lesions in mice. Results are mean S.D determined by ANOVA followed by Dunnetts test: * P < 0.01.

Evaluation of different doses of HEL in the HCl/ethanol and NSAID models showed a dose-dependent curve with no signicant gastric protection at a dose of 250 mg/kg, in both models (Fig. 2). HEL (1000 and 500 mg/kg) effectively reduced the gastric ulcers produced by indomethacin/bethanechol and by HCl/ethanol by 62 and 30%, and 88 and 63%, respectively. No signicant differences between the doses of 500 and 1000 mg/kg were observed using HEL (P > 0.05). The aforementioned results indicate that further experiments should be conducted using HEL at the dose of 500 mg/kg, which presented signicant results at lower doses in the evaluated models. Our data also showed that pretreatment with HEL signicantly protected (55%) the gastric mucosa against hypothermicrestraint stress-induced ulcers in mice (Table 1) and pretreatment with HEL also exhibits a protective prole (34%) in ulcers

induced in mice by pylorus ligature (Table 1). However, administration of HEL by different routes (oral and intraduodenal) did not produce signicant modication in gastric juice content, as cimetidine did (Table 2). Thus, an antisecretory activity may not be involved. The healing effect of HEL was demonstrated for the rst time when the healing of chronic gastric ulcer induced by acetic acid in rats was accelerated. Postoperative treatment with cimetidine and HEL (500 mg/kg) for 14 consecutive days accelerated ulcer healing. On day 14 after surgery, the percentage of rats with cicatrized ulcers in both experimental groups was signicantly higher than in the control group (Table 3). In addition, HEL signicantly decreased the mean area of chronic ulcer (2.40 0.81 mm2 versus 6.86 1.46 mm2 in the control, P < 0.05). The average of body weight taken daily before the treatment indicated no toxicity sign from the animals treated with HEL, cimetidine and saline during 14 days (data not shown). These data, in association with those observed from acute toxicity were additional evidences for absence of toxicity of this plant. The possible mechanism of action was investigated using an enriched avonoidic fraction (EFF) obtained from HEL. The pretreatment with EFF (100 mg/kg) presented 52 and 79% of gastroprotection against HCl/ethanol and NSAIDs-induced gastric lesions, respectively (Fig. 3). We also observed the effect of EFF on free mucus production by the gastric mucosa (Table 4). Pretreatment with EFF did not induce signicant increase in mucoprotective effect in animals submitted to pylorus ligature.

Table 1 Effects of cimetidine and a single dose of hydroalcoholic extract of leaves (HEL) of Alchornea castaneaefolia on stress-induced (cold/restraint) and pylorus ligature gastric lesions in mice Models Stress Treatments (p.o.) Control Cimetidine HEL Pylorus ligature Control Cimetidine HEL Dose (mg/kg) 100 500 100 500 N 10 9 9 8 9 8 Ulcer index 17.40 2.45 7.56 2.59* 7.89 2.57* 42.10 7.81 29.80 6.92* 28.0 10.50* Inhibition (%) 56.6 54.7 29.2 33.5

Results are mean S.D. Stress: ANOVA for ulcer index F(2,26) = 2.75 (P < 0.05) in stress-induced and F(2,22) = 4.68 (P < 0.05) in pylorus ligature. * Dunnetts test: P < 0.01. Table 2 Effects of cimetidine and a single dose of hydroalcoholic extract leaves (HEL) of Alchornea castaneaefolia administered orally (p.o.) or intraduodenally (i.d.) on gastric juice parameters in pylorus-ligature-induced gastric lesions in mice Treatments Control Cimetidine HEL Control Cimetidine HEL Route p.o. Dose (mg/kg) 100 500 100 500 N 8 9 8 8 8 7 pH (unit) 4.43 1.15 6.63 1.42* 3.44 1.33 4.84 0.69 7.19 0.19* 4.36 0.73 Volume 0.492 0.113 0.436 0.164 0.729 0.331 0.538 0.070 0.663 0.064 0.596 0.075 [H+ ] (mEq/(ml 4 h)) 7.02 4.96 3.54 2.14* 8.62 4.12 8.12 1.32 4.12 1.24* 7.87 1.32

i.d.

Results are mean S.D. ANOVA (p.o.) F(2,22) = 8.06 (P < 0.05) for pH; 2.79 for gastric volume (P > 0.05); 2.67 (P < 0.05) for [H+ ]. ANOVA (i.d.) F(2,20) = 3.52 (P < 0.05) for pH; 0.55 for gastric volume (P > 0.05); 2.79 (P < 0.05) for a [H+ ]. * Dunnetts test: P < 0.01.

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Fig. 3. Effects of cimetidine, lansoprazole and an enriched avonoidic fraction (EFF) obtained from HEL of Alchornea castaneaefolia on indomethacin/bethanechol-induced and HCl/ethanol gastric lesions in mice. Results are mean S.D. ANOVA followed by Dunnetts test: * P < 0.001, ** P < 0.0001.

Pretreatment of rats with indomethacin markedly reduced the gastric mucosal prostaglandin contents from 73.8 to 30.0 pg/mg (Table 5). On the other hand, pretreatment of rats with EFF (100 mg/kg) induced drastic increase of PGE2 levels as compared to rats treated only with saline (P < 0.01). After pretreatment of rats with EFF, it was possible to observe a marked decrease in the serum gastrin level when compared to the negative control (Table 6), thus indicating the action of EFF in more than one antiulcer mechanism. In our work, the serum level of somatostatin hormone in rats treated with EFF increased almost three times when compared to the negative control (Table 6, P < 0.001), thus reaching the same serum level of those rats treated with lansoprazole (P < 0.001). It is possible that the high levels of somatostatin induced by EFF may inhibit gastric acid secretion induced by gastrin. Preparative chromatographic separation of an aliquot of EFF led to the isolation of quercetin-3-O- -d-galactopyranoside 1 (15 mg), quercetin-3-O- -l-arabinopyranoside 2 (20 mg),

Fig. 4. HPLC-PDA-UV chromatographic prole of the EFF from Alchornea castaneaefolia. (A) Gallic acid derivatives (gallic acid 6 and gallic acid methyl ester 7) and (B) avonoids derivatives (quercetin-3-O- -d-galactopyranoside 1, quercetin-3-O- -l-arabinopyranoside 2, myricetin-3-O- -l-arabinopyranoside 3, quercetin 4 and amentoavone 5). RP-18, 250 mm 4.6 mm d.i., 5 m, elution: gradient acetonitrile/water 2535% in 30 min, ow rate 1.0 ml/min, detection 254 nm.

myricetin-3-O- -l-arabinopyranoside 3 (25 mg), quercetin 4 (8 mg), amentoavone 5 (4 mg), gallic acid 6 (20 mg), gallic acid methyl ester 7 (15 mg), besides glycolipids and free sugars. After the HPLC-UV-PDA chromatographic analysis of EFF (Fig. 4), the UV spectra of the peaks compared to those of standards available in our laboratory were used to characterize two regions: region A with gallic acid derivatives (225 and 275 nm) and region B, with avonoids (254 and 350 nm).

Table 3 Effects of cimetidine and hydroalcoholic extract (HEL) of leaves of Alchornea castaneaefolia on healing of ulcers produced by acetic acid in rats Treatment (p.o.) Control Cimetidine HEL N 7 8 5 Dose (mg/kg) 100 500 Lesion area (mm2 ) 6.86 1.46 1.63 1.11** 2.40 0.81* pH 3.67 1.21 2.89 0.78 2.60 1.52 Curative ratio (%) 76.2 65

ANOVA: F(2,17) = 20.5 (P < 0.05). * Dunnetts test: P < 0.05. ** Dunnetts test: P < 0.001.

Table 4 Effects of carbenoxolone and enriched avonoidic fraction (EFF) obtained from the HEL of Alchornea castaneaefolia administered by the intraduodenal route on Alcian Blue binding to free gastric mucous from pylorus ligature mice Treatments Control Indomethacin Carbenoxolone EFF Dose (mg/kg) 30 250 100 N 7 6 6 6 Volume (ml) 0.40 0.08 0.55 0.08 0.83 0.07* 0.18 0.08* Alcian Blue bound [mg/wt tissue (g)] 4.04 0.44 2.14 0.47* 6.77 0.56* 4.86 0.47

ANOVA F(3,21) = 7.98 (P < 0.05) for Alcian Blue bound; 2.47 (P < 0.05) for volume. * Dunnetts test: P < 0.05.

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Table 5 Effects of indomethacin and enriched avonoidic fraction (EFF) obtained from the HEL of Alchornea castaneaefolia on prostaglandin synthesis by the gastric mucosa of rats Treatments Sham Vehicle (p.o.) Indomethacin (s.c.) EFF (p.o.) Dose 10 ml/kg 30 mg/kg 100 mg/kg N 4 4 4 3 Prostaglandin synthesis (pg/mg) 71.80 15.82 73.81 7.32 30.0 8.52# 110.70 2.08* Increase (%) 2.78 58.2 54.2

Results are mean S.D from ANOVA: F(5,11) = 18.2 (P < 0.05) for prostaglandin synthesis. * Tukeys test: P < 0.01. # Tukeys test: P < 0.001.

4. Discussion Although there are many products in the market for the treatment of gastric ulcers, including antacids, proton pump inhibitors, anticholinergics and histamine H2 -antagonists, most of these drugs produce several adverse reactions, such as gynecomastia, hematopoietic changes, acute interstitial nephritis (Ra and Tobe, 2004), thrombocytopenia (Zlabek and Anderson, 2002), anaphylaxis reactions (Gonzalez et al., 2002), nephrotoxicity and hepatotoxicity (Fisher and Le Couteur, 2001). Besides, in third world countries like Brazil, peptic ulcer treatment is very expensive. Thus, there is a need for more effective, less toxic and less expensive antiulcer agents. Medicinal plants are amongst the most attractive sources of new drugs, and have been shown to give promising results in treatment of gastric ulcers (Borrelli and Izzo, 2000). In Brazil, a large number of herbal extracts is used in folk medicine to treat various types of digestive disorders (Gracioso et al., 2002; Hiruma-Lima et al., 2002; Toma et al., 2002; Almeida et al., 2003). Alchornea castaneaefolia is a medicinal plant with extensive use as remedy in folk medicine, including gastric disorders. However, there are no data about its pharmacological effect on the gastrointestinal system or possible toxic properties. The effect of the two preparations from Alchornea castaneaefolia, HEL and HEB, on gastric ulcers induced by different damaging agents (HCl/ethanol and NSAIDs) was rst investigated in mice. Oral administration of HCl/ethanol solution to the control group clearly produced the expected characteristic

zonal necrotizing mucosal lesions. HCl/ethanol solution induced both long ulcers and petechial lesions within a relatively short time. Pretreatment with HEL and HEB (1000 mg/kg) induced a signicant protective effect in varying levels and no signicant differences were observed between the groups treated with HEL and HEB. The obtained results suggest that the crude extract of leaves (HEL) and bark (HEB) of Alchornea castaneaefolia induce a signicant antiulcer effect in these ulcer-induced models. Chronic administration of NSAIDs is often associated with the development of adverse gastrointestinal effects, such as gastric erosions, gastric or duodenal ulceration, and severe complications, such as gastrointestinal hemorrhage or perforation that often limited their widespread clinical use (Villegas et al., 2004). In our experimental model, the association between indomethacin and bethanechol increased the ulceration index by producing bleeding and damage in the glandular part of the stomach. Indomethacin is known to induce gastric ulcer by inhibition of prostaglandins, which are cytoprotective to gastric mucosa (Peskar and Maricic, 1998). Signicant inhibition of the gastric ulcer was also observed in the pretreatment with both preparations from Alchornea castaneaefolia. Although both extracts HEL and HEB showed a similar type of response to the ulcer model employed for the preliminary testing, it is decided to continue the pharmacological assay and phytochemical analyses on HEL in the present study. As part of this pharmacological study, the acute toxicity in mice of the HEL obtained from leaves of Alchornea castaneae-

Table 6 Effects of enriched avonoidic fraction (EFF) of Alchornea castaneaefolia on serum somatostatin and gastrin in rats Treatment (p.o.) Sham Vehicle Lansoprazole EFF Treatment (p.o.) Sham Vehicle Lansoprazole EFF Dose 10 ml/kg 30 mg/kg 100 mg/kg Dose 10 ml/kg 30 mg/kg 100 mg/kg N 3 4 4 4 N 3 4 3 4 Gastrin ( U/ml) 333.61 15.10 349.20 22.20 46.61 7.01** 85.30 59.60** Somatostatin ( U/ml) 20.81 1.53 20.0 7.49 87.70 22.11** 82.50 8.21** Decrease (%) 4.67 86.0 74.4 Increase (%) 3 321.6 296.6

Results are mean S.D from ANOVA: F(5,11) = 0.27 (P < 0.05) for gastrin. ANOVA: F(5,11) = 28.6 (P < 0.05) for somatostatin. ** Tukeys test: P < 0.001.

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folia was rst investigated. A single oral administration of HEL (5 g/kg) did not produce any visible acute toxicity in the treated animals. These preliminary results indicated the absence of acute toxic effects of HEL and motivated us to continue with the assays. HEL at different doses showed a dose-dependent curve in preventive models (HCl/ethanol and NSAID-induced gastric lesions) and also indicated the involvement of this extract in the enhancement of cytoprotection effect and defensive factors of gastric mucosa, respectively. Hypothermic-restraint stress ulcers have been used experimentally for the evaluation of antiulcer activity in animals by the enhancement of acid secretion and reduction in mucous production mediated by histamine release. Free radicals may play a major role in stress-involved gastrointestinal injury (Bagchi et al., 1999). Stress is found to inactivate mucosal prostaglandin syntheses by accumulating H2 O2 , an inhibitor of the prostaglandin synthesis, which also favors the generation of reactive oxygen species (Bandyopadhyay et al., 1999). Our data showed that pretreatment with HEL signicantly protected the gastric mucosa against hypothermic-restraint stress-induced ulcers and by pylorus ligature. In this last case, the ligature of the pyloric sphincter led to an accumulation of gastric juice in the stomach. It is known that compounds that exert their activity through a mechanism affecting gastric acid content are effective in both models, i.e., an antisecretory activity may be considered. However, administration of HEL by different routes (oral and intraduodenal) did not produce signicant modication in gastric juice content, as cimetidine did. Thus, an antisecretory activity may not be involved. Acetic acid induced gastric ulcers by a perforative nature, spread over a relatively large area that does not heal with time. The application of glacial acetic acid to the gastric serous membrane caused ulcer with wall-encircled deep craters (Okabe et al., 1971). The results of the present study demonstrate for the rst time that HEL treatment accelerates the healing of chronic gastric ulcer in rats. The healing effect of the gastric mucosa did not change the pH of gastric juice but this result was foreseen based on preceeding results indicating that antisecretory activity might not be involved with HEL. Studies on cimetidine and omeprazole that are able to accelerate the healing of chronic gastric ulcers in rats, observed that the inhibition of acid secretion is poorly related to ulcer healing (Ito et al., 1994). The possible mechanism of action was investigated using an enriched avonoidic fraction (EFF) obtained from HEL. The pretreatment with EFF induced gastroprotection against HCl/ethanol and NSAIDs gastric lesion. Promising results can be observed from the pretreatment with EFF, since it exerted protective effect higher than those obtained with cimetidine (100 mg/kg) or lansoprazole (30 mg/kg), standard antiulcer drugs in the market. Gastric mucus is an important protective factor for the gastric mucosa and consists of a viscous, elastic, adherent and transparent gel formed by water and glycoproteins, which covers the entire gastrointestinal mucosa. The protective properties of the mucus barrier depend not only on the gel structure but also on the

amount or thickness of the layer covering the mucosal surface (Penisi and Piezzi, 1999). Pretreatment with EFF did not induce signicant increase in mucoprotective effect in animals submitted to pylorus ligature. But, different from the results obtained after administration of HEL, pretreatment of Shay animals with EFF induced an increase in the gastric pH and decrease in the gastric volume when compared to control group. It is known that the protective mechanism exhibited for mucus and bicarbonate secretion depends, to a large extent, on prostaglandin E2 secretion, and this in turn depends on the activity of the cyclooxygenase (COX) enzyme system (Peskar and Maricic, 1998). Our results prove that pretreatment of rats with EFF induced drastic increase of PGE2 levels compared to rats treated only with saline. Therefore, these results clearly indicate the involvement of PGE2 in the gastroprotective mechanism of EFF obtained from Alchornea castaneaefolia. There is an increased change of the mucosal blood ow around the ulcer in experimental animals (Skarstein, 1979). Skarstein (1979) also suggested that there is an increase in the concentration of prostaglandin in ulcerative regions, when compared to other parts of the gastric mucosa, since prostaglandin causes vasodilatation. The large blood supply seems to reect the active re-epithelization, which requires an abundant supply of glucose and oxygen (Sato et al., 1995). So, the increase in PGE2 production obtained in the treatment with EFF clearly indicated that stimulation of cytoprotective factors contributed to accelerate the healing of gastric ulcer observed in rats treated with HEL. Gastrin and somatostatin are gastrointestinal hormones closely related to the function of the gastrointestinal system. In rats with ulcer, the gastrin level in the plasma, gastric juice and the antral mucosa tissue increase, while the somatostatin level declines (Sun et al., 2002). The gastrin secreted by antral G cells is the principal stimulant of the gastric acid secretion. Duodenal ulcer patients were shown to have a larger capacity to secrete acid under maximal stimulation by gastrin (McColl et al., 2000). The present results showed that pretreatment of rats with EFF induced a marked decrease in the serum gastrin level when compared to the negative control, thus indicating the action of EFF in more than one antiulcer mechanism. Somatostatin, a regulatory peptide, acts on multiple targets throughout the body, including the gastrointestinal tract. This peptide has been reported to exert potent inhibitory effects on gastric acid, pepsin and gastrin secretion (Sun et al., 2002). Several studies have shown a possible inuence of a local decrease in gastric somatostatin in the physiopathologic characteristic of peptic ulcer disease. For the digestive system, as for virtually all other organs, somatostatin action has been generally characterized as inhibitory. For example, somatostatin strongly inhibits secretion of a number of hormones such as gastrin, motilin, cholecystokinin, insuline, glucacon, and reduces gastric acid secretion (Zavros et al., 1998). In our work, the serum level of somatostatin hormone in rats treated with EFF increased almost three times when

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compared to the negative control, reaching the same serum level of those rats treated with lansoprazole. We can speculate that the high levels of somatostatin induced by EFF may be responsible to inhibit gastric acid secretion induced by gastrin. Phytochemical analysis of EFF led to the isolation of the avonoids and gallic acid derivatives. Literature reports that avonoids, especially those with a catechol moiety, like quercetin and myricetin, can be related to the antiulcer activity, playing a major role in some mechanisms of action (LaCasa et al., 2000; Havsteen, 2002). Not only avonoids, but also other phenolic substances with an ortho-dihydroxy group (like gallic acid derivatives) are known for their antioxidant activities, which can be related to some antiulcer mechanisms (Repetto and Llesuy, 2002). 5. Conclusions In conclusion, all these results taken together suggest that the effectiveness of Alchornea castaneaefolia is based on its ability to strengthen defensive factors like prostaglandin synthesis, in addition to other gastroprotective actions, like a stimulant effect on somatostatin synthesis and an inhibitory effect on gastrin secretion. Therefore, results obtained with the administration of HEL of Alchornea castaneaefolia as well as its EFF in vivo models not only support the ethnopharmacological use of this species but also shows its potential as a new antiulcerogenic drug. Acknowledgements We are grateful to Prof. Dr. Luciana de Pietro Magri for the assistance with the EIA and RIA assays, Prof. Solange F. Lolis-UNITINS for the species identication, to FAPESP (Biota-Fapesp Program, Prof. 02/05503-6, Proc. 00/02772-0) for the nancial support and for fellowships to F.D.P.A., and to CNPQ for grants to A.R.M.S.B. and W.V. References
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