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Journal of Ethnopharmacology 130 (2010) 85–92

Contents lists available at ScienceDirect

Journal of Ethnopharmacology

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ntiulcerogenic activity of hydroalcoholic extract of Achillea millefolium L.:nvolvement of the antioxidant system

rancine Bittencourt Potricha , Alexandra Allemanda , Luísa Mota da Silvaa , Ana Cristina dos Santosa ,ristiane Hatsuko Baggioa, Cristina Setim Freitasa, Daniel Augusto Gasparin Bueno Mendesa,unice Andreb, Maria Fernanda de Paula Wernera, Maria Consuelo Andrade Marquesa,∗

Department of Pharmacology, Universidade Federal do Paraná, Curitiba, PR, BrazilDepartament of Biophysics and Pharmacology, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil

r t i c l e i n f o

rticle history:eceived 25 November 2009eceived in revised form 29 March 2010ccepted 17 April 2010vailable online 24 April 2010

eywords:chillea millefoliumastric ulcercetic acidthanolntioxidants enzymessteraceae

a b s t r a c t

Achillea millefolium L. is a member of the Asteraceae family that is commonly referred to as “yarrow” andhas been used in folk medicine against several disturbances including skin inflammations, spasmodicand gastrointestinal disorders, as well as hepato-biliary complaints.Aim of the study: In the present study, we evaluated the efficacy of a hydroalcoholic extract from theAchillea millefolium (HE) for gastroprotective properties and additional mechanism(s) involved in thisactivity.Material and methods: Rats were treated with HE and subsequently exposed to both acute gastriclesions induced by ethanol P.A. and chronic gastric ulcers induced by 80% acetic acid. Following treat-ment, glutathione (GSH) levels and superoxide dismutase (SOD) activity were measured. The activity ofmyeloperoxidase (MPO) and histological and immunohistochemical analysis were performed in animalswith acetic acid-induced gastric ulcers.Results: Oral administration of HE (30, 100 and 300 mg/kg) inhibited ethanol-induced gastric lesionsby 35, 56 and 81%, respectively. Oral treatment with HE (1 and 10 mg/kg) reduced the chronic gastric

ulcers induced by acetic acid by 43 and 65%, respectively, and promoted significant regeneration of thegastric mucosa after ulcer induction denoting increased cell proliferation, which was confirmed by PCNAimmunohistochemistry. HE treatment prevented the reduction of GSH levels and SOD activity after aceticacid-induced gastric lesions. In addition, HE (10 mg/kg) inhibited the MPO activity in acetic acid-inducedgastric ulcers.Conclusions: The results of the present study indicate that the antioxidant properties of HE may contribute

tivity

to the gastroprotective ac

. Introduction

Achillea millefolium L. is a member of the Asteraceae familyhat is commonly referred to as “yarrow”. Achillea millefolium haseen used in folk medicine against several disturbances includingkin inflammations, spasmodic and gastrointestinal disorders, andepato-biliary complaints (Benedek and Koop, 2007). Phytochem-

cal studies carried out with Achillea millefolium have identifiedeveral components, including essential oils, sesquiterpenes, andhenolic compounds such as flavonoids and phenolcarbonic acidsBenedek et al., 2007). Moreover, the presence of sesquiter-

∗ Corresponding author at: Department of Pharmacology, Sector of Biological Sci-nces, Universidade Federal do Paraná, CP 19031, 81531-990, Curitiba, PR, Brazil.el.: +55 41 3361 1721; fax: +55 41 3266 2042.

E-mail address: mconsu@ufpr.br (M.C.A. Marques).

378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.jep.2010.04.014

of this extract.© 2010 Elsevier Ireland Ltd. All rights reserved.

pene lactones, azulene, flavonoids such as apigenin, luteolin andrutin sustains the pharmacological activity of Achillea millefolium(Kocevar et al., 2008). Different preparations of Achillea millefoliumhave demonstrated anti-inflammatory, antitumor, antimicrobial,liver protective, and antioxidant properties (Goldberg et al., 1969;Tozyo et al., 1994; Gagdoli and Mishra, 1995; Tunon et al., 1995;Lin et al., 2002; Candan et al., 2003). In addition, our laboratorypreviously reported the potent gastric anti-secretory and gastro-protective activity of an aqueous extract of Achillea millefolium inmodels of acute (Baggio et al., 2002) and chronic gastric injury(Cavalcanti et al., 2006).

Gastric ulcer is one of the major gastrointestinal disorders,

which occur due to an imbalance between the offensive (gastricacid secretion) and defensive (gastric mucosal integrity) factors(Laine et al., 2008). There is evidence for the participation of reac-tive oxygen species (ROS) in the etiology and pathophysiology ofhuman gastric ulcer development (Repetto and Llesuy, 2002). Many

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tudies have shown that ROS are involved in several models of gas-ric injury, such as ethanol, gastric lesions caused by stress, aceticcid, and the use of non-steroidal anti-inflammatory drugs (Das andanerjee, 1993; Odabasoglu et al., 2006; Ineu et al., 2008; Ishiharat al., 2008). These results indicate the involvement of antioxidantargeting enzymes in gastric injury models and the discovery ofrugs that also possess free-radical scavenging activity. In this con-ext, we have investigated the antioxidant mechanisms involved inhe gastroprotective effects of the hydroalcoholic extract of Achilleaillefolium (HE).

. Material and methods

.1. Botanical material

Achillea millefolium L. plants were collected in July 2007n the Botanical Garden of Universidade Paranaense (UNIPAR),ampus Umuarama (Brazil) at 430 m altitude above sea levelS23◦47′55–W53◦18′48). Dr. Mariza Barion Romagnolo identi-ed botanical material (Department of Botany, UNIPAR, Campusmuarama, Brazil), and a voucher specimen was deposited at theampus Umuarama Herbarium under number 1896.

.2. Preparation of the hydroalcoholic extract (HE)

The hydroalcoholic extract (HE) of aerial portions of the plantas prepared by maceration with 90% ethanol at room temper-

ture (1:1, v/v), initially for 48 h and thereafter until exhaustion.he hydroalcoholic extract obtained was filtered and concentratednder vacuum (at 50 ◦C) and stored at −20 ◦C. The concentratedxtract (yield of 17.4%) was diluted in distilled water immediatelyefore use.

.3. Animals

Experiments were conducted using female Wistar rats180–220 g), housed under standard laboratory conditions (12 hight/dark cycle, temperature 22 ± 2 ◦C) with free access to foodnd water. The rats were deprived of food for 16 h prior to thetart of experiments. The study was conducted in accordance withhe Ethical and Practical Principles of the Use of Laboratory Ani-

al guidelines and the experimental protocol was approved byhe Institutional Ethics Committee of the Universidade Federal doaraná (CEEA/UFPR; approval number 161).

.4. Induction of acute gastric lesion

Rats were orally treated with vehicle (water, 0.1 ml/100 g ofody weight), omeprazole (40 mg/kg), ascorbic acid (500 mg/kg) orE (30, 100 and 300 mg/kg) 1 h before intragastric administrationf ethanol P.A. (0.5 ml). The animals were sacrificed 1 h after ethanoldministration (Robert et al., 1979). The stomachs were removednd the area of ulceration (mm2) was measured by planimetrysing the program Image Tool 3.0.

.5. Induction of chronic gastric ulcer

Chronic gastric ulcers were induced with acetic acid asescribed previously, with modifications (Okabe et al., 1971;kabe and Amagase, 2005). Briefly, rats were anaesthetized withylazine/ketamine (7.5 mg/kg and 60 mg/kg, i.p., respectively). The

bdomen was opened, the stomach was exposed and the 80% glacialcetic acid (0.5 ml) was instilled into a cylinder (6 mm of diame-er) and applied to the serosal surface of the stomach for 1 min.he acetic acid was removed by aspiration and the area of con-act was washed with sterile saline. 48 h after the induction of

armacology 130 (2010) 85–92

ulcers, rats were treated orally with omeprazole (40 mg/kg), ascor-bic acid (250 mg/kg), HE (0.1, 1 and 10 mg/kg) or vehicle control(water, 0.1 ml/100 g of body weight), twice a day for 7 days. Oneday after the final treatment, the animals were sacrificed, the stom-achs removed, and the extent of the gastric lesion was measured asthe total injured area (mm3) = length (mm) × width (mm) × depth(mm).

For histological evaluation, gastric ulcers were fixed in Alfacsolution for 16 h. After fixation, the tissue samples were dehy-drated with alcohol and xylene. Immediately after the dehydration,each sample was embedded in paraffin wax, sectioned at 5 �mand stained with hematoxylin/eosin. The gastric sections wereobserved and photographed under a stereomicroscope at 25-foldmagnification.

2.6. Immunohistochemistry

Proliferating cell nuclear antigen (PCNA) was used to determineproliferating cells in acetic acid-induced ulcers. Paraffin-embeddedsections were deparaffinized in xylene and hydrated through stan-dard graded ethanol solutions. Sections were rinsed 2 times for5 min each in PBS (pH 7.4), incubated in H2O2 solution for 10 minto inactivate endogenous peroxides and then heated in citric acidsodium solution in microwave oven at 100 ◦C to retrieve anti-gen for 10 min. Blocking of nonspecific reaction was performedwith blocking solution (1% BSA and 0.3% Triton X-100 in PBS)for 30 min. The sections were then incubated overnight at 4 ◦Cwith goat anti-PCNA (at 1:100; Santa Cruz Biotechnology Inc., CA,USA). After that, slides were rinsed in PBS (pH 7.4) and the sec-tions were incubated in secondary antibody at room temperaturefor 2 h. After washing, the immunoreacted cells were then devel-oped utilizing avidin-conjugated horseradish peroxidase (HRP)with diaminiobenzidine (DAB) as substrate (BD Biosciences, SanDiego, CA, USA). Finally, the specimens were counterstained withhematoxylin. PCNA-containing cells were identified by the pres-ence of a dark reddish-brown chromogen. The nuclear-positivestaining cells were observed under microscope (400×).

2.7. Preparation of subcellular fractions of stomachs

Tissue samples of stomachs were homogenized with 200 mMpotassium phosphate buffer, pH 6.5. The homogenate was used tomeasure the GSH levels and then centrifuged at 9000 × g for 20 min.The supernatant was used for the determination of SOD activity andthe pellet was used to determine the activity of MPO.

2.8. Determination of glutathione content

GSH levels in gastric mucosa were determined by the methodof Sedlak and Lindsay (1968). Aliquots of tissue homogenate weremixed with 12.5% trichloroacetic acid, vortexed for 10 min and cen-trifuged for 15 min at 900 × g. The supernatant was reserved, andTRIS buffer (0.4 M, pH 8.9) and 5,5′-dithiobis(2-nitrobenzoic acid)(DTNB, 0.01 M) were added to it. Absorbance was measured usinga spectrophotometer at 415 nm with a microplate reader. The pro-cedures were performed at 4 ◦C, and the individual values wereinterpolated into a standard curve of GSH and expressed as �g ofGSH/g of tissue.

2.9. Determination of enzymatic activities of SOD

SOD activity was measured according to Marklund andMarklund (1974) and Gao et al. (1998). Measurements were basedon the capacity of SOD to inhibit pyrogallol autoxidation. Pyrogal-lol (1 mM) was added to buffer solution (200 mM Tris HCl–EDTA,pH 8.5) and supernatant aliquots, and then vortexed for 1 min. The

F.B. Potrich et al. / Journal of Ethnopharmacology 130 (2010) 85–92 87

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ig. 1. Gastroprotective effects of the HE in acute gastric lesions induced by ethaxpressed as mean ± S.E.M. (n = 6–8). The animals received: vehicle (Veh – water), ooses of HE (30, 100 and 300; 0.1, 1 and 10 mg/kg, respectively). Statistical compariest. Differences from Veh group (*P < 0.05, **P < 0.01 and ***P < 0.001).

eaction was incubated for 20 min at room temperature, stoppedith the addition of 1 N HCl and centrifuged for 4 min at 18,700 × g.

he absorbance of the resulting supernatant was measured at05 nm using a spectrophotometer. The amount of SOD that inhib-

ted the oxidation of pyrogallol by 50% (relative to the control) wasefined as a unit of SOD activity.

.10. Determination of MPO activity

The activity of MPO (a marker of neutrophil infiltration) waseasured according to Bradley et al. (1982) and modified by De

oung et al. (1989). The pellet was re-suspended in 1 ml of 80 mMotassium phosphate buffer (pH 5.4) containing 0.5% hexade-yltrimethylammonium bromide (HTAB), and it was centrifugatedt 11,000 × g for 20 min at 4 ◦C. MPO activity in the supernatant wasetermined at 620 nm in presence of H2O2 and TMB. MPO activity

n tissues was expressed as units of milli optic density (mO.D.)/g ofissue.

.11. DPPH free-radical scavenging assay

2,2-Diphenyl-1-picrylhydrazyl (DPPH) is a stable free radicalhat has been widely used as a tool to estimate the free-radical scav-nging activity of antioxidants. The reduction capacity of the DPPHadical was determined by the decrease of absorbance induced byntioxidants, according to Blois (1958) and Chen et al. (2004), withfew modifications. The reaction system consisted of 750 �l of HEiluted to different concentrations (3, 10, 30 and 300 �g/ml) and50 �l of DPPH methanolic solution. After 5 min, the absorbanceas measured at 517 nm using a spectrophotometer. Ascorbic acid

50 �g/ml) was used as positive control and distilled water wasmployed as negative control.

.12. Drugs and reagents

The following substances were used: hexadecyltrimethy-ammonium bromide, 3,3′,5,5′-tetramethylbenzidine, hydrogeneroxide, glutathione, pyrogallol, 5,5′-dithiobis(2-nitrobenzoiccid) and 2,2-diphenyl-1-picrylhydrazyl (all from Sigma, St. Louis,SA), acetic acid, sodium acetate, N,N-dimethylformamide, ascor-ic acid, xylene, formaldehyde, trichloroacetic acid and absolutethanol (Vetec, Rio de Janeiro, Brazil) and methanol (Tédia Brazil,io de Janeiro, Brazil).

.13. Statistical analysis

Data were expressed as means ± standard error of mean with–10 animals per group. Differences between means were deter-

. (A) and in chronic gastric ulcers induced by 80% acetic acid (B). The results areazole (Omep – 40 mg/kg), ascorbic acid (AA – 500 and 250 mg/kg, respectively) andas performed using analysis of variance (ANOVA) followed by post hoc Bonferroni’s

mined by one-way analysis of variance (ANOVA) followed byBonferroni’s post hoc test. In all cases, differences were consideredto be significant when value <0.05.

3. Results

3.1. Effect of HE on gastric lesions induced by ethanol

Oral administration of HE (30, 100 and 300 mg/kg), 1 h before theinduction of gastric lesions with ethanol P.A., significantly reducedlesion area by 35, 56 and 81%, respectively, compared to the con-trol group (87.1 ± 6.3 mm2) (Fig. 1A). Omeprazole (40 mg/kg, p.o.)and ascorbic acid (500 mg/kg, p.o.) have been previously demon-strated to inhibit ethanol-induced gastric lesion formation and sothey were used as positive controls of lesion inhibition. Omepra-zole and ascorbic acid inhibited the gastric lesions in 72 and 35%,respectively (Fig. 1A).

3.2. Effect of HE on gastric ulcers induced by 80% acetic acid

The results show that oral administration of HE (1 and 10 mg/kg)reduced the gastric ulcer formation caused by 80% acetic acidexposure by 43 and 65%, respectively (Fig. 1B). The positive con-trols for lesion inhibition, omeprazole (40 mg/kg p.o.) reducedthe gastric ulcer size by 69% when compared to control group(146.7 ± 6.8 mm3) (Fig. 1B). However, ascorbic acid (250 mg/kg p.o.)treatment did not reduce the acetic acid-induced gastric lesion for-mation.

Histological analysis of the gastric ulcers showed extensive deepdamage induced by acetic acid (Fig. 2A and E). Slices from ulcerstreated with HE (1 and 10 mg/kg, p.o.) (Fig. 2C, D, G and H) andomeprazole (40 mg/kg, p.o.) demonstrated a regenerated mucosa(Fig. 2B and F).

These results were confirmed by PCNA immunohistochemicalanalysis, where the HE (10 mg/kg) treatment increased the num-ber of proliferating cells in 112 ± 18% when compared to controlgroup (Fig. 3). However, PCNA expression in the omeprazole group(40 mg/kg, p.o.) was not different from control group (Fig. 3).

3.3. Effects of HE on antioxidant system

Administration of ethanol P.A. decreased GSH levels and SODactivity by 51% and 37%, respectively, when compared with

untreated controls (Table 1). The treatment of animals withHE (300 mg/kg, p.o.) prevented the decrease in GSH levels andSOD activity. Omeprazole (40 mg/kg, p.o.) prevented decreasedSOD activity but failed to affect GSH levels, while ascorbic acid(500 mg/kg, p.o.) increased the levels of glandular GSH and SOD

88 F.B. Potrich et al. / Journal of Ethnopharmacology 130 (2010) 85–92

Fig. 2. The effect of treatment with HE on the regeneration of gastric mucosa 10 days after injury induced by 80% acetic acid. The images representing macroscopic photographof the vehicle group water p.o. (A), omeprazole 40 mg/kg p.o. (B), HE 1 mg/kg p.o. (C) or HE 10 mg/kg p.o. (D); and the histological sections (25×) of the vehicle group waterp The aB

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.o. (E), omeprazole 40 mg/kg p.o. (F), HE 1 mg/kg p.o. (G) or HE 10 mg/kg p.o. (H).ars = 1 mm (E–H).

ctivity by 45% and 43%, respectively, when compared to ethanol-nduced lesion controls (Table 1).

In gastric ulcers induced by acetic acid, GSH levels decreasedy 54% and SOD activity decreased by 33% (Table 2). Oral admin-

rrows represent the edge of the ulcer in the recovery process or failing to recover.

istration of HE (10 mg/kg, p.o.) restored the GSH levels and SODactivity to baseline levels. Omeprazole (40 mg/kg, p.o.) and ascorbicacid (250 mg/kg, p.o.) also restored the GSH levels and SOD activity(Table 2).

F.B. Potrich et al. / Journal of Ethnopharmacology 130 (2010) 85–92 89

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ig. 3. The effect of treatment with HE on the immunohistochemical staining for PCepict PCNA immunoreactivity in the vehicle group water p.o. (A), omeprazole 40 mg

.4. Effects of HE on MPO activity

The chronic gastric ulcer induced by acetic acid increasedPO activity by 82% when compared to untreated controls

1.3 ± 0.4 mO.D./g of tissue). HE (10 mg/kg, p.o.), omeprazole40 mg/kg, p.o.) and ascorbic acid (250 mg/kg, p.o.) inhibited MPOctivity by 68, 42 and 60%, respectively (Fig. 4).

.5. Effect of HE on free-radical DPPH

In the DPPH test, HE (3, 10, 30 and 300 �g/ml) scavenged thePPH radicals with IC50 value of 30.9 (19.3–49.5) �g/ml and an inhi-

gastric mucosa 10 days after injury induced by 80% acetic acid. Photomicrographs. (B), HE 1 mg/kg p.o. (C) or HE 10 mg/kg p.o. (D). Magnification, 400×, bars = 250 �m.

bition of 72%. Ascorbic acid was used as a positive control, and itwas able to reduce DPPH levels by 75% (Fig. 5).

4. Discussion

In the present study, we demonstrated that oral administra-tion of the hydroalcoholic extract from Achillea millefolium (HE)

effectively protects the animals against acute gastric lesions causedby ethanol and chronic gastric ulcer induced by acetic acid. Theseresults are in accordance with previous studies obtained with aque-ous extract of A. millefolium that presented potent anti-secretoryand gastroprotective activity in several models of acute and chronic

90 F.B. Potrich et al. / Journal of Ethnopharmacology 130 (2010) 85–92

Table 1Effects of HE on the quantity of GSH and SOD in rats with gastric ulcer induced byethanol P.A.

Treatment GSH (�g GSH/g tissue) SOD (U SOD/g tissue)

Veh/Veh 1572 ± 38 2.7 ± 0.1Veh/ethanol 764 ± 14### 1.7 ± 0.2##

Omeprazole (40 mg/kg) 1036 ± 133### 3.6 ± 0.1###,***

Ascorbic acid (500 mg/kg) 1394 ± 69*** 3.0 ± 0.1***

HE (30 mg/kg) 434 ± 61### 2.6 ± 0.1*

HE (100 mg/kg) 763 ± 71### 2.6 ± 0.2*

HE (300 mg/kg) 1187 ± 85#,* 2.5 ± 0.1*

The difference between groups was determined by ANOVA followed by Bonferroni’stest.

* P < 0.05 as compared with Veh/ethanol.*** P < 0.001, as compared with Veh/ethanol.

# P < 0.05 as compared with Veh/Veh.## P < 0.01 as compared with Veh/Veh.

### P < 0.001 as compared with Veh/Veh.

Table 2Effects of HE on the quantity of GSH and SOD in rats with gastric ulcer induced by80% acetic acid.

Treatment GSH (�g GSH/g tissue) SOD (U SOD/g tissue)

Veh/Veh 1595 ± 99 3.0 ± 0.1Veh/Acetic acid 728 ± 33### 2.0 ± 0.1#

Omeprazole (40 mg/kg) 1229 ± 109* 3.9 ± 0.1##,***

Ascorbic acid (250 mg/kg) 1329 ± 120** 3.9 ± 0.3***

HE (1 mg/kg) 1224 ± 95* 2.9 ± 0.2*

HE (10 mg/kg) 1597 ± 177*** 2.9 ± 0.2*

The difference between groups was determined by ANOVA followed by Bonferroni’stest.

* P < 0.05 as compared with Veh/acetic acid.** P < 0.01 as compared with Veh/acetic acid.

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Fig. 5. The effect of HE (3, 10, 30 and 300 �g/ml) or ascorbic acid (AA, 50 �g/ml)on the ability to scavenge the free-radical DPPH. The results are expressed as

*** P < 0.001 as compared with Veh/acetic acid.# P < 0.05 as compared with the Veh/Veh.

## P < 0.01 as compared with the Veh/Veh.## P < 0.001 as compared with the Veh/Veh.

njury (Baggio et al., 2002; Cavalcanti et al., 2006). Results obtainedrom this study reinforce the traditional use of Achillea millefolium

n the treatment of gastrointestinal disorders, an effect seeminglyelated to the antioxidant protection and radical scavenging activ-ty, which antagonizes oxidative stress-induced gastric damage.

HE protected rats from gastric lesions induced by the intragas-ric application of irritant agent, such as ethanol. It is known that

ig. 4. Effect of HE on MPO activity. The graph represents the activity of MPO in gas-ric damage induced by 80% acetic acid in the experimental groups: vehicle/vehicleVeh/Veh – water/water), vehicle (Veh – acetic acid/water), omeprazole (Omep –0 mg/kg p.o.), ascorbic acid (AA – 250 mg/kg p.o.) and HE (10 mg/kg p.o.). The resultsre expressed as mean ± S.E.M. (n = 8). Statistical comparison was performed usingnalysis of variance (ANOVA) followed by post hoc Bonferroni’s test. Difference fromeh/Veh group (###P < 0.001); difference from Veh group (***P < 0.001).

mean ± S.E.M. Statistical comparisons were performed using analysis of variance(ANOVA) followed by the Bonferroni’s post hoc test. Difference from Veh group(***P < 0.001).

ethanol rapidly penetrates the gastric mucosa, promoting mem-brane damage, erosion and ulcer formation through destruction ofthe mucus barrier (Hirschowitz, 1983), increases in vascular perme-ability (Szabo et al., 1985) and decreases in non-proteic sulphydrilicgroups (NP-SH) of the gastric mucosa (Repetto and Llesuy, 2002;Siegmund et al., 2003). In addition, it is proposed that ROS areinvolved in the development of gastric ulcers induced by ethanol,stress, H. pylori, acetic acid and non-steroidal anti-inflammatorydrugs (Shaw et al., 1990; Bandyopadhyay et al., 2001; Kwiecien etal., 2002, 2003; Ishihara et al., 2008).

Glutathione is the majority NP-SH of gastric mucosa and it,therefore, constitutes one of the most important cytoprotectivemechanisms against lesion formation (Cnubben et al., 2001), whileSOD-mediated catalysis of superoxide radical anion (O2

•−) intoless noxious hydrogen peroxide (H2O2) represents the first line ofantioxidant defense. In our experiments, the GSH levels and SODactivity were significantly reduced after ethanol administration,and this reduction was prevented by pretreatment with ascorbicacid (P < 0.001) and HE (P < 0.05). Moreover, we hypothesized thatHE could be acting as a scavenger of free radicals because the chem-icals employed to induce ulceration invoke damage by reactiveoxygen species. Interestingly, the HE (300 �g/ml) presented a com-parable profile of action with ascorbic acid in reducing (P < 0.001)the levels of free-radical DPPH, suggesting a direct scavenger activ-ity. In fact, it is well known that the reduction of the DPPH moleculeby Achillea extracts is related to the presence of flavonoids andtannins (Giorgi et al., 2009; Tuberoso et al., 2009). Furthermore,the treatment of animals twice a day for seven days with HE andomeprazole significantly reduced the size of acetic acid-inducedgastric ulcers. In this model, the antioxidant assays showed thesame profile as the antioxidant system of ethanol-induced gas-tric lesions. In addition, in agreement with our data, Ishihara etal. (2008) showed that gastric ulcers induced by acetic acid havereduced SOD activity. Oral treatment with HE (1 and 10 mg/kg),omeprazole and ascorbic acid restored the acetic acid-dependentdecrease in GSH levels (P < 0.05, 0.001, 0.05 and 0.001, respectively)and SOD activity (P < 0.05, 0.05, 0.001, 0.001, respectively).

Unfortunately, the phytochemical components responsible forthe antioxidant effect of hydroalcoholic extract of A. millefolium ingastric ulcers are currently unknown; however, preliminary studiesdemonstrated the presence of flavonoids such as apigenin, lute-

olin and rutin, which show powerful antioxidant properties, in thisplant (Kocevar et al., 2008; Giorgi et al., 2009; Tuberoso et al., 2009).Therefore, the contribution of these chemicals to the antioxidativecapability of HE remains to be evaluated.

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It has been shown that the application of acetic acid on theurface of the serosa produces a gastric ulcer in rats that is very sim-lar to the human gastric ulcers in terms of location, severity andhronicity and which undergoes a similar healing process (Okabend Pfeiffer, 1972). The ulcer results from tissue necrosis trig-ered by mucosal ischemia, free-radical formation, and cessation ofutrient delivery, all of which are caused by vascular and microvas-ular injury such as thrombi, constriction, or other occlusionsTarnawski, 2005). Ulcer healing is a complex process that involvesell migration, proliferation, re-epithelization, angiogenesis, andatrix deposition, all of which ultimately lead to scar formation

Tarnawski, 2005). In addition, to promote the restoration of thentioxidant system, HE treatment also resulted in re-epithelizationf the gastric lesions induced by acetic acid. Interestingly, histolog-cal analysis of gastric ulcers revealed the presence of extensiveeep damage in the gastric mucosa after administration of aceticcid. The treatment with HE significantly regenerates the gastricucosa, which is similar to observations for animals treated with

meprazole. Moreover, cell proliferation plays an important rolen wound healing (Tarnawski, 2005) and it was shown that PCNA,tissue marker of cell proliferation, was increased during healingf gastric mucosal injury (Sun et al., 2003). We observed that HEdministration increased the number of PCNA-positive cells, sug-esting that HE treatment could promote gastric cell regenerationnd proliferation.

Recent studies have shown that the excessive recruitmentnd metabolic activation of neutrophils generates free radicalsn several models of gastric damage resulting in inflammation-ependent tissue damage (Fialkow et al., 2007). The MPO enzyme

s present in neutrophils and acts in the presence of superoxidenion and chloride anion (Cl−) to form the hypochloric acid (whichs toxic to bacteria); but is also harmful to host tissues (Halliwellnd Gutteridge, 2006). In this study, we observed that the treat-ent with HE decreased (P < 0.001) the activity of MPO, suggestingreduction of neutrophil infiltration into ulcerated tissue. This lackf activity may contribute to the reduction of neutrophil-dependentOS formation and therefore block the gastric inflammatory pro-ess induced by acetic acid.

Collectively, the results of the present study demonstrate thathe gastroprotective effects in rat stomach induced ulcers pro-

oted by a hydroalcoholic extract of A. millefolium (HE) could bettributed to antioxidant properties of this plant. However, furthertudies are required to investigate the active compound(s) and pre-ise mechanisms involved in the effects produced by A. millefolium.

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enedek, B., Koop, B., 2007. Achillea millefolium L. s.l. revisited: recent findings con-firm the traditional use. Wiener Medizinische Wochenschrift 157, 312–314.

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