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Journal of Ethnopharmacology 131 (2010) 146–153

Contents lists available at ScienceDirect

Journal of Ethnopharmacology

journa l homepage: www.e lsev ier .com/ locate / je thpharm

ffect of Mouriri pusa tannins and flavonoids on prevention and treatmentgainst experimental gastric ulcer

.C.P. Vasconcelosa, M.A. Andreob, W. Vilegasb, C.A. Hiruma-Limaa, C.H. Pellizzona,∗

Botucatu Institute of Biosciences, UNESP – São Paulo State University, Botucatu, SP, BrazilAraraquara Institute of Chemistry, UNESP – São Paulo State University, Araraquara, SP, Brazil

r t i c l e i n f o

rticle history:eceived 14 April 2010eceived in revised form 7 June 2010ccepted 11 June 2010vailable online 18 June 2010

eywords:anninslavonoidselastomataceae

a b s t r a c t

Aim of the study: Mouriri pusa, popularly known as “manapucá” or “jaboticaba do mato”, is a plant fromBrazilian cerrado that has been found to be commonly used in the treatment of gastrointestinal disturbs inits native region. The present work was carried out to investigate the effect of tannins (TF) and flavonoids(FF) fractions from Mouriri pusa leaves methanolic extract on the prevention and cicatrisation process ofgastric ulcers, and also evaluate possible toxic effects.Materials and methods: The following protocols were taken in rats: acute assay, in which ulcers wereinduced by oral ethanol after pre-treatment with the fractions; and 14 days treatment assay, in whichulcers were treated for 14 days after induction by local injection of acetic acid.Results: In the acute model, treatment with either, TF (25 mg/kg) or FF (50 mg/kg), was able to reduce

astric ulcerouriri pusa

lesion area, showing gastroprotective effect. In addition, FF proved itself anti-inflammatory by reducingCOX-2 levels. In acetic acid model, both fractions exhibited larger ulcers’ regenerative mucosa, indicatingcicatrisation enhancement. FF group also showed augmented cell proliferation, anti-inflammatory actionand enhanced angiogenesis as well as increased mucus secretion. Moreover, concerning the toxicityparameters analyzed, no alteration in the fractions groups was observed.Conclusions: Tannins and flavonoids from Mouriri pusa provide beneficial effects against gastric ulcers

with relative safety.

. Introduction

Medicinal plants are frequently used in gastrointestinal dis-urbs. Many different substances found in these plants haveastroprotective effects but few are shown to accelerate ulcer heal-ng (Zayachkivska et al., 2005). An ethnopharmacologic inventoryarried out in the Cerrado of the central region of Brazil showedgreat number of species used against stomachic pain and gas-

ric injuries in general (Silva et al., 2000), including the speciesouriri pusa Gardn. (Melastomataceae). Mouriri pusa is a small tree

5–7 m) from “Cerrado” region of Brazil popularly known as “jabu-icaba do mato”, “manapucá” or “moroso cigano”. The tea madesing its leaves is commonly used in folk medicine to the treatment

f gastritis and ulcers, illnesses that are largely distributed amongopulation mainly because of the increasing use of tobacco andon-steroidal anti-inflammatory drugs, complemented by alcoholnd stress (Taylor and Blaser, 1991; Grob, 2003).

∗ Corresponding author at: Departamento de Morfologia, Instituto de Biociên-ias de Botucatu, cp. 510, Rubião Junior, 18618-000 Botucatu, SP, Brazil. Tel.: +55438116264x116; fax: +55 1438116264x120.

E-mail address: claudia@ibb.unesp.br (C.H. Pellizzon).

378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2010.06.017

© 2010 Elsevier Ireland Ltd. All rights reserved.

Andreo et al. (2006) showed positive effect against acute gas-tric ulcer, probably due to an enhancement of the gastrointestinalmucosa defence mechanism against aggressive factors, since it wasobserved a participation of NO and SH groups to prevent or atten-uate the ulcer process. Moreover, Vasconcelos et al. (2008) showedpositive data in the treatment of 14 and 30 days with this extractto evaluate cicatrisation process.

Aiming to develop the understanding of mechanisms of action,in this study fractions of flavonoids and tannins from the methano-lic extract were used to evaluate their gastroprotective and healingeffects against gastric ulcers induced in rats. These fractionswere chosen because gastroprotective polyphenolic substances areshown to be involved in anti-inflammatory action in gastrointesti-nal tract (GIT), promoting tissue repair though expression of severalgrowth factors, presenting antioxidant activity, scavenging reac-tive oxygen species (ROS), showing anti-nucleolytic, cytochomeP450 and inhibitory activities as well as anti-necrotic and anti-carcinogenic activities (Zayachkivska et al., 2005).

The objective of this study was to evaluate the potential ofthe flavonoids and tannins fractions from the methanolic extractof Mouriri pusa leaves in the prevention (by the absolute ethanolmodel of ulcer induction) and in the treatment of experimentalgastric ulcers (by the acetic acid model of ulcer induction). We also

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sed the 14-day experimental model to evaluate possible subacuteoxic effect of the fractions.

. Materials and methods

.1. Vegetal extract and fractionating

Mouriri pusa was collected in Porto Nacional, Tocantins, Braziln July 2006 by Dr. Hiruma-Lima, being the botanical identificationarried by Professor Solange F. Lolis from the Tocantins Federal Uni-ersity. A voucher was deposited in the HTINS herbarium under theumber 4548.

Aerial parts of Mouriri pusa, properly dried and triturated in knifeill, were put in contact with methanol solvent in the ratio of 500 g

f vegetal raw material to 2 L of solvent. After 7 days of extraction,he content was vacuum-filtered and then the extract was con-entrated by evaporation in the rotavapor at 45 ◦C, resulting in theethanolic extract of Mouriri pusa. The methanol extract of Mouriri

usa (10 g) was then dissolved in 50 mL of methanol; 400 mL ethylcetate was added to the solution, leading to precipitation. Afterentrifugation, the precipitate was separated and dried to give theannins fraction (6.2 g). The supernatant was evaporated to drynessnd washed with hexane/dichloromethane 1:1 (v/v), which wasractionated in a column of Sephadex LH-20 eluted with methanol,esulting in 3 fractions. TLC analysis of these fractions showed thatavonoids had been concentrated in fraction 2, called flavonoids

raction (1.0 g).

.2. HPLC analysis

All samples were prepared by clean-up in C-18 cartridges200 mg, Phenomenex®) and the resulting solutions filteredhrough discs of 0.45 mm in pore size (Millipore®). The extract,ractions and flavonoids were analyzed by HPLC-DAD. The condi-ions used, after optimization, were the same as used by Santos etl. (2008). Briefly, the HPLC-DAD analyses were performed with aiquid chromatograph (Jasco) with a PU-2089 four-solvent pump,photodiode array detector (DAD, MD-2010, Jasco) and a circularichroism detector (CD - 2095, Jasco). The analytical columnas a Phenomenex Luna RP18 (250 mm × 4.6 mm i.d., 5 �m),

quipped with a Phenomenex security guard 4.0 mm × 2.0 mmTorrance, CA, USA). The mobile phase was a continuous gradientf water (eluent A) and acetonitrile (eluent B), both with 0.05%rifluoroacetic acid (TFA). The gradient program was as follows:7–34% B (3 min), 34–65% B (42 min) and 65–100% (5 min). Totalun time was 55 min. The flow rate of the mobile phase was.0 mL/min. Star LC Workstation software was used for the opera-ion of the detector and solvent delivery and for data processing.he following flavonoids previously isolated from the methanolxtract of Mouriri pusa were used in the analysis: myricetin--O-�-galactopyranoside, myricetin-3-O-�-glycopyranoside,utin, myricetin-3-O-�-rhamnopyranoside, quercetin-3--�-galactopyranoside, quercetin-3-O-�-xylopyranoside,uercetin-3-O-�-arabinopyranoside, quercetin-3-O-�-rabinofuranoside, kaempferol-3-O-�-galactopyranoside, anduercetin-3-O-�-rhamnopyranoside. Determination of theavonoids was performed by retention time and by spikingith isolated compounds under the same conditions.

.3. Animals

We used male Wistar rats (150–200 g) from Central Animalouse of UNESP-Botucatu, acclimatized to controlled temperature

23 ± 2 ◦C) and light/dark cycle of 12 h. The animals were fed onation and water ad libitum. All the experiments were in accor-ance with the experimental protocols previously approved by the

harmacology 131 (2010) 146–153 147

Commission of Ethics in Animal Experimentation from UNESP -Botucatu, protocol n◦ 002/04-CEEA.

2.4. Evaluation of the acute effect of fractions on ethanol-inducedulcers

This method was based on modifications by Morimoto et al.(1991). After 24 h of fasting, the experimental groups were treatedwith the respective fractions. An interval of 1 h for induction ofulcers with 1 mL of absolute ethanol was adopted. Groups of ani-mals received saline (negative control), carbenoxolone 100 mg/kg(positive control), and the fractions of flavonoids and tannins atdoses of 25, 50 and 100 mg/kg. All treatments were made by gavage,in final volume dose of 10 mL/kg.

The animals were killed 1 h after the administration of the inju-rious agent. The stomachs were removed and opened by the greatcurvature, pressed in glass plates, photo-documented in HP scan-jet 2400® scanner. The measure of the areas of the injuries weremade using AVsoft BioView 4® software. After that, the stomachswere routinely processed for histology to inclusion in paraplast.The material was thin-sliced and the cuts were placed in slides.To the execution of immunohistochemistry methods, we used theprotocols bellow with some modifications for each antibody [HSP70 (heat shock protein 70), COX-2 (ciclooxigenase-2) and SOD(superoxide dismutase)], depending on the recommendation of themanufacturer.

2.5. Immunohistochemical methods

The histological cuts were deparaffinized and re-hydrated asstandard of the histology laboratory; then they were submitted toantigenic recovery by the method of 0.1 M citrate buffer in hightemperature microwave oven for 10 min. After the antigenic recov-ery, the cuts were placed in the reaction blocking solution, whichwas made of 1% skimmed milk, 3% hydrogen peroxide and 1% goatnormal serum in PBS for 1 h. These cuts were washed in PBS andlater they were incubated for 2 h, in ambient temperature, withprimary antibody diluted in solution PBS plus 1% BSA.

2.6. Evaluation of the healing effect of the fractions on ulcersinduced by acetic acid in rats

The experiment was made as described by Takagi et al. (1969)and Okabe and Amagase (2005). Male Wistar rats (150–200 g) hadbeen fasted for 24 h before the experiment and had free access towater. A longitudinal incision below the xiphoid process was madein the anesthetized animals. The anterior wall of the stomach wasexposed and 0.02 mL of 30% acetic acid was injected with a microsy-ringe in the subserosal layer in the junction of the fundus with theantrum and later washed with saline. In the following day, the dailytreatments were initiated (once a day for 14 days) in four groups:tannins fraction 25 mg/kg, flavonoids fraction 50 mg/kg (concen-trations chosen for being the lesser effective doses on study ofprotection by the absolute ethanol model), cimetidine 100 mg/kgand saline (10 mL/kg).

During this period, we also measured the weight evolution ofthe animals. After treatment, they were killed and their stom-achs removed; the pH of the gastric juice was measured and thestomachs were opened throughout the great curvature. The injurywas located and its internal (only exposed submucosa) and exter-nal (including regenerative mucosa) areas were macroscopically

measured. Then, the samples were fixed in ALFAC and enclosed inparaplast for immunohistochemical and histological analyses, afterHE (Behmer et al., 1976) and PAS (Vacca, 1985) staining.

The morphometric analysis was made using Leica Q-Win imageanalyzer, which was made by measuring the regenerative mucosa

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egion, which borders the injury. For this, we made linear measuresrom the lumen to the muscularis mucosa.

The immunohistochemical methods were made as previouslyescribed, however, this time we used antibodies anti-PCNAcell division marker, to evaluate healing potential), anti COX-

(ciclooxigenase-2, to evaluate effect over inflammation) andnti-CXCR4 (endothelial marker, to evaluate the effect over angio-enesis). The slides were analyzed in Leica microscope. PCNAnalysis was made by PCNA-positive cells counting per mm2 inandom fields acquired in regenerative mucosa of the four groups.n COX-2 analysis, its expression was measured as �m2 per ran-om field of the regenerative mucosa. In vascular evident analysis,he number of newly formed vessels was measured per mm2 inandom fields of the submucosa.

.7. Subacute toxicity assay

By assessing healing effect of the fractions from methanolicxtract of Mouriri pusa on gastric ulcers induced by acetic acidn rats with daily treatments, we are simulating not only the use

anner of many drugs for gastric ulcers in humans, but also theirossible toxic and cumulative effects for their use by the methodsescribed by Souza-Brito (1994). Considering that we had alreadyade the acute toxicity assay (unique dose), subacute (14 days of

dministration) and prolonged (30 days of administration) withhe methanolic extract, in which no toxic effect was observedVasconcelos et al., 2008), the fractions of tannins and flavonoidsrom the Mouriri pusa methanolic extract were tested for their pos-ible toxicity in the cicatrisation model, above described.

During the period of 14 days, possible effects of Mouririusa fractions (evolution of body weight, alterations of hair anducosae) were daily observed in the treated rats. After the death

f the animal, besides organs weighing (heart, liver, kidneys, lungsnd spleen), their blood serum was collected for biochemicalssays, in which we quantified AST – aspartate aminotransferase,LT – alanine aminotransferase and �-GT – gamma glutamyltrans-

erase to assess liver damage, urea and creatinine and to assessidneys damage, using the automated biochemical analyzer SBA-00, CELM, Brazil.

.8. Statistical analysis

Results are mean ± standard error of means (S.E.M.). The resultsere submitted to one-way analysis of variance (ANOVA) followed

y Dunnett post test, under level of significance of p < 0.05. Theercentage relation between organs weight and body weight wasreviously transformed into arcsin.

. Results and discussion

.1. HPLC analysis

The evaluation of retention times and UV spectra allowed theetermination of the main classes of compounds present in theamples analyzed. Fig. 1 shows the chromatograms obtained. Com-ounds present in the methanol extract (chromatogram 1) inhe flavonoid fraction (chromatogram 2) and in the tannin frac-ion (chromatogram 3) were characterized by the analysis of theV spectra of each peak, and by spiking with authentic sam-

les of the flavonoids previously isolated (Andreo et al., 2006;antos et al., 2008). Condensed tannins could be detected due tolarge band at 254 nm at retention time between 30 and 45 min.ydrolyzable tannins eluted at the beginning of the chromato-raphic run and were characterized by their UV spectra, with bandst 280 nm.

harmacology 131 (2010) 146–153

3.2. Ethanol-induced ulcers

The ethanol-induced ulcer model is useful to measure thecytoprotection activity of a substance or element, as previouslydescribed by Robert et al. (1979) when the authors verified mucosadamage preventive activity (Brzozowski, 2003).

The ulcerogenic activity of ethanol is due to its capacity of dis-solving the constituents of stomach mucus with a concomitantlowering in the potential of action in the transmucosa, rising Na+

and H+ flux in the lumen and stimulating histamine, pepsin andH+ ions secretion (Szabo, 1987). To determine dose–effect curve,we tested 25, 50 and 100 mg/kg doses of Flavonoids Fraction (FF)and Tannins Fraction (TF) from Mouriri pusa. Fig. 2 shows thestomachs submitted to the different treatments by the ethanol-induced ulcer model. By the determination of the damaged area,we observed that FF presented a typical dose–effect curve, so thatthe least dose considered active was 50 mg/kg (Fig. 3). Flavonoidshave well described antioxidant activity, which may explain thegastroprotective effect from FF in doses of 50 mg/kg or more(Zayachkivska et al., 2005). However, TF groups resulted in aninverted curve, so that the best gastroprotective effect was obtainedin the least dose tested (25 mg/kg) (Fig. 3), what is an interestingdata – four times lesser than the dose used for positive control(Carbenoxolone). Tannins have the property of precipitating micro-proteins in the site of the ulcer, building a protective pellicle thatprevents toxic substances from being absorbed and promotes resis-tance to harmful agents (John and Onabanjo, 1990; Nwafor et al.,1996).

The doses of the fractions that presented best results concern-ing cytoprotective effect were immunohistochemically analyzedfor determination of HSP70 (heat shock protein 70) and COX-2(ciclooxigenase-2).

HSP70 is a 70 kDa protein from the HSP family present on mam-malian cells. It is the most conserved and abundantly producedprotein in response to different forms of stress (Shichijo et al., 2003),such as heat, toxic agents, infection and proliferation (Oberringer etal., 1995). These proteins are responsible to protect cellular home-ostatic processes from environmental and physiologic injuries bypreserving the structure of normal proteins and repairing or remov-ing damaged proteins (Tytell and Hooper, 2001), which makes thestudy of this protein an interesting element for possible mecha-nisms of action elucidation. Our results do not show significantalteration in HSP70 expression between saline and FF or TF, indicat-ing that it does not take part in the mechanisms of gastric mucosaprotection by both fractions (Fig. 4).

COX-2 is an inducible enzyme expressed in injuries by somecells for inflammatory processes or facing mutagenic agents (Halteret al., 2001; De Maria and Weir, 2003; Motilva et al., 2005).It is an important enzyme for the gastric mucosa because it,in cicatrisation process, induces angiogenesis, cell proliferationand cytoprotection, related to the production of prostaglandinsthat stimulates mucus and bicarbonate production (Brzozowskiet al., 2000). Kushima et al. (2009) studying Flavonoid fractionfrom Davilla elliptica observed that the number of COX-2 markedcells lowers dose-dependently, which confirms flavonoids anti-inflammatory effect by inhibition of COX-2 (Robak and Gryglewski,1996). In the study of the Tannins fraction from Davilla ellipticaby Kushima et al. (2009), COX-2 expression tended to rise alsodose dependently, although it was not related to the mechanismof action of this plant. In the FF group of this experiment, it is notnoticed any presence of COX-2 positive cells (Fig. 5), indicating

that this fraction may function as anti-inflammatory, corroborat-ing with Hashizume et al. (1978). In the TF group, it was noticeableCOX-2 positive cells in the gastric glands apex, what may indicatethat this enzyme is activated by TF for local epithelium protection(Fig. 5).

P.C.P. Vasconcelos et al. / Journal of Ethnopharmacology 131 (2010) 146–153 149

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ig. 1. Chromatograms obtained by HPLC-DAD. Chromatogram 1 – Methanol Extracobile phase – 27–34% B (3 min), 34–65% B (42 min) and 65–100% (5 min), water (

ime was 55 min. Flow rate – 1.0 mL/min.

.3. Acetic acid-induced ulcers

After determination of the best dose that exerts cytoprotectionn ethanol method, cicatrisation assay was performed by aceticcid method, which according to Okabe and Amagase (2005), is

matogram 2 – Flavonoids Fraction; Chromatogram 3 – Tannins Fraction; Conditions:A) and acetonitrile (eluent B), both with 0.05% trifluoroacetic acid (TFA); total run

the most similar to human gastric ulcer. Histologically, ulcers arestructured in two distinct regions: ulcer border, where the mucosahas dilated glands, and the granulation tissue, where submucosais exposed. Cicatrisation process generically involves cell migra-tion, proliferation, reepithelization, angiogenesis and deposition

150 P.C.P. Vasconcelos et al. / Journal of Ethnopharmacology 131 (2010) 146–153

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ig. 2. Representative stomachs of experimental gastric lesion induction by ethanohe arrows show areas of mucosal.

f extracellular matrix (Tarnawski, 2005). These events are reg-lated by many factors, such as cytokines, prostaglandins, nitricxide, growth factors and sensorial neurons (Tsukimi and Okabe,001). Moreover, this process is divided in two stages: the firsttage presents quick cicatrisation and depends on contraction ofhe ulcer basis; and second stage presents slow cicatrisation andepends additionally on mucosa regeneration. The epithelial recon-titution is performed by proliferation of undifferentiated epithelialrecursors, which migrate from the ulcer border to the granulationissue thus covering the base of the ulcer. The balance betweenpoptosis and proliferation is of outmost importance for the main-enance of basal membrane (Reed, 2000; Sánchez-Fidalgo et al.,004).

After the 14-day treatment, the macroscopic analysis of thexternal area of the ulcers showed no statistical difference amonghe four groups (Table 1). However, the Tannins group pre-ented smaller ulcer internal area when compared to Saline group,hat may indicate better cicatrisation in this experimental period

ig. 3. Gastric ulcers areas (mm2) after pre-treatments followed by ulcer induction by eth= 7. FF: Flavonoids Fraction; TF: Tannins Fraction; 25, 50 and 100: doses (mg/kg); saline

r pre-treatment with Flavonoids (FF) and Tannins fractions (TF) from Mouriri pusa.

comparing to control, even using a dose four times lower thanCimetidine.

The morphometric analysis has revealed a significant augmentin the regenerative mucosa comparing to control in both, Tanninsand Flavonoids groups (Fig. 6), what did not occur to Cimetidinegroup. These data suggest grater regeneration of the ulcers pro-moted by the fractions at lower doses than those of commercializedantiulcer drugs.

PCNA (Proliferating Cell Nuclear Antigen) is a highly conserved36 kDa polypeptide identified as auxiliary protein of DNA poly-merase delta (Nanji and Tahan, 1996; Bravo et al., 1987; Prelichet al., 1987). It is mainly expressed during S phase of cell cycle,hence, in dividing cells (Celis and Celis, 1985). Finn et al. (1996)

showed that cimetidine significantly inhibited the proliferation ofthree of five cell lines, what my indicate dependence by theselines on H2 receptors activation. Flavonoids fraction presentedsignificant augment on PCNA positive cells comparing to Saline(Table 1), indicating greater cell proliferation in the regenerative

anol in acute model. Results are mean ± S.E.M. ANOVA, Dunnett. *p < 0.05. **p < 0.01.10 mL/kg; cabernoxolone 100 mg/kg.

P.C.P. Vasconcelos et al. / Journal of Ethnopharmacology 131 (2010) 146–153 151

Fig. 4. Immunohistochemical analysis of expression of HSP-70 in the stomachs of rats from different treatments after ulcer induction by ethanol. Arrows indicate expression.Doses saline 10 mL/kg; carbenoxolone 100 mg/kg; flavonoids 50 mg/kg; tannins 25 mg/kg.

Fig. 5. Immunohistochemical analysis of expression of COX-2 in the stomachs from rats from different treatments after ulcer induction by ethanol. Arrows indicate expression.Doses saline 10 mL/kg; carbenoxolone 100 mg/kg; flavonoids 50 mg/kg; tannins 25 mg/kg.

Table 1Gastric lesions of rats that underwent ulcer induction by acetic acid. pH, internal and external area (mm2) and immunohistochemical analysis of ciclooxigenase 2 (COX-2),proliferating cell nuclear antigen (PCNA) and blood vessels evaluations of the stomachs after 14 days of treatments.

Saline Cimetidine Flavonoids Tannins

pH 3.57 ± 0.30 3.30 ± 0.42 3.57 ± 0.48 3.67 ± 0.56Internal lesion area 54.38 ± 6.19 35.65 ± 8.00 48.84 ± 8.07 28.55 ± 6.40*

External lesion area 153.65 ± 40.45 84.86 ± 16.70 133.89 ± 20.70 80.74 ± 24.54COX-2 (�m2/field) 13.89 × 103 ± 1805.6 9.91 × 103 ± 2050.2 2.99 × 103 ± 519.0** 5.96 × 103 ± 627.83**

PCNA (cells/mm2) 327.46 ± 41.4 607.63 ± 126.9 870.30 ± 56.8** 535.84 ± 30.1Vessels (vessels/mm2) 47.63 ± 4.1 39.22 ± 3.6 78.45 ± 6.0** 63.04 ± 4.4

Results are mean ± S.E.M. ANOVA, Dunnett. n = 7.* p < 0.05.

** p < 0.01.

152 P.C.P. Vasconcelos et al. / Journal of Ethnopharmacology 131 (2010) 146–153

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ig. 6. Stomachs regenerative mucosa size from rats that underwent acetic acidlcer induction after the different treatments. Doses saline 10 mL/kg; cimeti-ine 100 mg/kg; flavonoids 50 mg/kg; tannins 25 mg/kg. Results are mean ± S.E.M.NOVA, Dunnett. **p < 0.01, n = 5.

ucosa, which is a good effect for ulcer cicatrisation. Tannins frac-ion did not present statistic difference comparing to control at thisarameter.

CXCR4 (CXC chemokine receptor 4) and its activator SDF-1stromal cell-derived factor-1) play an important role in blood ves-el formation for stomach and intestine supply, and participatesctively in the development and maturation of blood vessels dur-ng regeneration process of gastric ulcers in humans (Akimoto etl., 2005). Again, flavonoid fraction presented significantly positiveesults for this marker (Table 1), indicating that its role in the ulcersegeneration involves angiogenesis activation. Tannins fraction didot present significant alteration, despite promoting visible rise inhis marker.

Anti-COX-2 immunohistochemistry showed again thatavonoids fraction has anti-inflammatory potential, since lessctivation of this enzyme was observed comparing to control

Table 1). Tannins fraction has also presented significant reductionn the expression of this marker, but not as much as flavonoidsraction, possibly because the cicatrisation in this group was moredvanced comparing to control and COX-2 levels, which had

ig. 7. Histological slices of the stomachs from rats that underwent ulcer induction by acetoses saline 10 mL/kg; flavonoids (FF) 50 mg/kg; tannins (TF) 25 mg/kg.

able 2iochemical quantification of AST (U/L), ALT (U/L), Gamma-GT (U/L), creatinine (mg/dL) acetic acid.

Treatment AST ALT

Saline 0.47 ± 0.03 44.7 ± 1.52Cimetidine 0.47 ± 0.01 44.4 ± 3.64Flavonoids 0.53 ± 0.06 43.4 ± 6.35Tannins 0.46 ± 0.02 42.4 ± 2.36

esults are mean ± S.E.M. ANOVA p > 0.05, n = 5. AST (aspartate aminotransferase), ALTreatinine and urea: mg/dL.

Fig. 8. Changes in body weight of rats that underwent ulcer induction by aceticacid. Arrows indicate weighing after 24 h of fasting. Doses saline 10 mL/kg; cimeti-dine 100 mg/kg; flavonoids 50 mg/kg; tannins 25 mg/kg. Results expressed as mean.ANOVA, p > 0.05. n = 7.

already lowered. These results show that cicatrizing effects fromthese fractions do not include COX-2 activation.

PH measures among the four groups in cicatrisation model didnot show any significant alteration, what indicates absence of anti-secretory action from the fractions.

PAS histology staining (Fig. 7) has shown an augmented mucusproduction in the stomachs of rats treated with the fractions,mainly the flavonoids fraction, what is a factor of great importancein the ulcer regeneration, because the mucus layer protects newlyformed cells against injuries caused by acid pH and proteolyticpotential of gastric secretions (Tarnawski et al., 2001; Valcheva-Kuzmanova et al., 2005).

Toxicity analysis performed in the cicatrisation model did notshow any toxic effects from the fractions: no behavioural alterationwas observed during treatments; the body weight of the animalsprogressed similarly in all groups (Fig. 8); and the organs weightwere also similar in all groups. Biochemical analysis of AST (hepatic

marker), Gamma-GT (hepatic toxicity initial indicator) creatinineand urea (renal lesion markers) (Dugo et al., 2004) in sera of theanimals did not show alterations between saline and other groups(Table 2). Similar data were observed for other plants studied in

ic acid. PAS staining. Note the large secretion of mucus in fractions groups (arrows).

nd urea (mg/dL) in serum from animals treated for 14 days after ulcer induction by

GGT Creatinine Urea

2.57 ± 0.20 0.47 ± 0.03 37.14 ± 2.401.86 ± 0.26 0.47 ± 0.01 37.57 ± 1.832.00 ± 0.50 0.47 ± 0.03 36.00 ± 1.312.71 ± 0.94 0.46 ± 0.02 40.37 ± 1.92

(alanine aminotransferase) and gamma-GT (gamma glutamyltransferase): U/L.

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ur laboratory, such as Qualea grandiflora (Mart.) extract (Hiruma-ima et al., 2006) and Mangifera indica (Lima et al., 2006) extract,ndicating that in the period tested and under the doses used, theres no toxic effect from the fractions for the parameters assessed.

. Conclusions

Based on these data, it is possible to conclude that both fractionsested exhibit cytoprotective and cicatrizing effect on gastric ulcers,videnced by the reduction on ulcer formation in acute model andugment of regenerative mucosa in 14-day treatment. Flavonoidsraction may act as anti-inflammatory and induce angiogenesis andell proliferation. Tannins fraction may promote a mechanic barrierhat protects the stomach from ulcer formation and facilitates ulcerealing. Moreover, none of the fractions showed toxic effect in thearameters tested. Future studies must be carried on more specificechanisms and active principles.

eferences

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