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Research Paper

Terminalia catappa L A medicinal plant from the Caribbean pharmacopeia with anti-Helicobacter pylori and antiulcer actionin experimental rodent models

Laiacutesa Pinheiro Silva a Ceacutelio Damacena de Angelis a Flavia Bonamin a Heacutelio Kushima aFrancisco Joseacute Mininel b Lourdes Campaner dos Santos b Flavia Karina Delella cSergio Luis Felisbino c Wagner Vilegas d Lucia Regina Machado da Rocha aMatheus Aparecido dos Santos Ramos f Tais Maria Bauab f Walber Toma en1 Clelia Akiko Hiruma-Lima an1

a

Univ Estadual Paulista-UNESP ndash

Departamento de Fisiologia Instituto de Biociecircncias CEP 18618-970 Botucatu SP Brazilb Univ Estadual Paulista-UNESP ndash Departamento de Quiacutemica Orgacircnica Instituto de Quiacutemica CEP 14800-900 Araraquara SP Brazilc Univ Estadual Paulista-UNESP ndash Departamento de Morfologia Instituto de Biociecircncias CEP 18618-970 Botucatu SP Brazild Univ Estadual Paulista-UNESP ndash Campus Experimental do Litoral Paulista CEP 11330-900 Satildeo Vicente SP Brazile Universidade Santa Ceciacutelia ndash Poacutes-Graduaccedilatildeo em Sustentabilidade de Ecossistemas Costeiros e Marinhos Rua Oswaldo Cruz 266

Boqueiratildeo CEP 11045907 Santos SP Brazilf Univ Estadual Paulista-UNESP ndash Departamento de Ciecircncias Bioloacutegicas Faculdade de Ciecircncias Farmacecircuticas CEP 14801-902 Araraquara SP Brazil

a r t i c l e i n f o

Article history

Received 22 August 2014Received in revised form3 November 2014Accepted 13 November 2014

Available online 24 November 2014Keywords

Terminalia catappa LCombretaceaeGastric healing actionHelicobacter pylori

MMP-9MMP-2

a b s t r a c t

Ethnopharmacological relevance Terminalia catappa L (Combretaceae) is a medicinal plant listed as apharmacopeia vegetable from Caribbean to treat gastritis The objective of this study was to evaluate thegastroprotective and healing effect of the aqueous fraction (FrAq) obtained from the leaves of Terminalia

catappa and to determine the antiulcer mechanism of action in experimental rodent models and itsactivity to Helicobacter pylori

Material and methods In rodents the FrAq was challenged by different necrotizing agents such asabsolute ethanol and ischemiandashreperfusion injury The antiulcer mechanism of action of FrAq wasassessed and the healing effects of the fraction after seven and 14 days of treatment was evaluated bymatrix metalloproteinase activity (MMP-2 and MMP-9) The toxicological effect of subacute treatmentwith FrAq during 14 days of treatment was also analyzed The anti-Helicobacter pylori activity wasdetermined by microdilution The phytochemical study of the fraction was analyzed by experiments withFIA-ESI-IT-MSn (Direct Flow Analysis-ionization Electrospray Ion Trap Tandem Mass Spectrometry) andhigh performance liquid chromatography (HPLC) coupled to a photodiode array (PDA)Results Oral treatment with FrAq (25 mgkg) signi1047297cantly decreased the number of ulcerative lesionsinduced by ethanol and ischemiareperfusion injury The action of FrAq was mediated by the activation of defensive mucosa-protective factors such as increases in mucus production the nitric oxide (NO)pathway and endogenous prostaglandins Oral treatment with FrAq for seven and 14 days signi1047297cantlyreduced the lesion area (80 and 37 respectively) compared to the negative control group Analyses of MMP-9 and MMP-2 activity from gastric mucosa con1047297rmed the accelerated gastric healing effect of FrAqThis extract also presented considerable activity against Helicobacter pylori The mass spectrum and

MSMS of the aqueous fraction indicates the existence of many different phenolic compounds includingpunicalagin punicalin and gallagic acid among othersConclusions We concluded that FrAq from Terminalia catappa leaves has excellent preventive andcurative effects on acute and chronic induced gastric ulcers and showed an important pro1047297le againstHelicobacter pylori

amp 2014 Elsevier Ireland Ltd All rights reserved

Contents lists available at ScienceDirect

journal homepage wwwelseviercomlocatejep

Journal of Ethnopharmacology

httpdxdoiorg101016jjep2014110250378-8741amp 2014 Elsevier Ireland Ltd All rights reserved

n Corresponding authors Tel thorn55 14 38800312 fax thorn55 1438153744E-mail addresses walbertomagmailcom (W Toma) hirumaibbunespbr (C Akiko Hiruma-Lima)1 Contributed equally to the supervision of this study

Journal of Ethnopharmacology 159 (2015) 285ndash295

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1 Introduction

Terminalia catappa is a species of the family Combretaceae andpopularly known in Brazil as ldquoamendoeirardquo ldquoamendoeira-da-praiardquoldquoamendoeira-da-Iacutendiardquo ldquocucardquo ldquoguarda-solrdquo ldquocastanheira da Iacutendiardquoldquocastanholardquo and ldquochapeacuteu-de-Solrdquo This plant is widely distributedin countries with tropical and subtropical climates especially incoastal regions due to the plants ability to easily adapt to salinity

and winds (Thomson and Evans 2006) In Asian countries theleaves of this species are commonly used for the treatment of dermatitis hepatitis diarrhea and pyresis (Chen et al 2000) Thisplant was also listed in Pharmacopeia vegetables of the Caribbeanwhere the leaves of this plant are used in a decoction for gastritisand urinary infection (Germoseacuten-Robineau 2014) The literaturealso shows that the polar extract from different parts (leaves fruitsand bark) of Terminalia catappa have shown the following biologicalactivities antimicrobial antifungal (Fyhrquist et al 2002) antiox-idant (Masuda et al 1999 Chen and Li 2005 Pandya et al 2013 )antimetastatic (Yeh et al 2012 2014) anti-in1047298ammatory (Fan et al2004 Lin et al 1999) hepatoprotective (Lin et al 1997 Chen et al2000 Tang et al 2006 Chen and Li 2005) mutagenic (Mininelet al 2014) aphrodisiac (Ratnasooriya and Dharmasiri 2000) andantidiabetic (Nagappa et al 2003) Nunes et al (2012) described thegastroprotective effect of the ethanolic extract obtained from barkof this species and Kumar et al (2014) previously reported theantisecretory effect of the ethanolic extract from leaves althoughthe mechanism responsible for this preventive effect remainsunknown thus the use of this specie against gastric bacteria isrelevant Infection caused to Helicobacter pylori is considered themost prevalent cause of gastric diseases such as ulcers dyspepsiaand stomach cancer (Camargo et al 2014) The drug therapy availa-ble for the treatment of diseases caused by this microorganism haslimitation such as the high rate of resistance to conventional drugsand inappropriate patient treatment as presented to numerous sideeffects (Megraud et al 2013) In this sense the use of naturalproducts as an alternative to control this bacterium has shown to besatisfactory what drives the improvement of investigations of medi-cinal plants as adjuvant or new antimicrobial drugs (Parreira et al2014 Takeuchi et al 2014)

The aims of this present work were to characterize the anti-Helicobacter pylori activity and antiulcer effect of the aqueousfraction obtained from leaves of Terminalia catappa and determinethe mechanism of action for this medicinal species

2 Material and methods

21 Preparation of the aqueous fraction

The leaves of Terminalia catappa were collected in January

(2010) by Msc Laiacutesa Pinheiro Silva in SantosSP Brazil The speci-men was identi1047297ed by Msc Paulo Salles Penteado Sampaio andthe voucher specimen was deposited to the Herbarium at theUniversidade Santa Ceciacutelia Santos SP Brazil under the registerM Tomaz 01 for future reference The leaves from Terminaliacatappa (37854 g) were dried for six days at 50 1C powdered(3 mm) and subjected to percolation with absolute ethanol (2 L)for 2 h with a 1047298ux of 20 mLminkg The hydroalcoholic extractsubmitted to the rotary evaporator (45 1C) resulted in 331 g of product (a yield of 875) This extract was partitioned into threefractions a hexane fraction ndash FrHex (709 g 2496) an ethylacetate fraction ndash FrEtOAc (1222 g 4302) and an aqueousfraction ndash FrAq (843 g 2616) As the commonly used formula-tion of this medicinal plant is a decoction we chose to utilize the

FrAq in the experimental pharmacological protocols

22 Chemicals and reagents

HPLCndashgrade methanol (MeOH) and acetonitrile were purchasedfrom JT Baker (Baker-Mallinckrodt Phillipsburg NJ USA) HPLCndash

grade water (18 MΩ cm) was obtained using a direct Milli-Q pur-i1047297cation system (Millipore Co Bedford MA USA) Sep-Pak RP18cartridges (500 mgmL) for solid-phase extraction (SPE) were pur-chased from Phenomenex Co (Torrance CA USA)

23 Apparatus

The mass spectrometry experiments were performed on LCQ Fleet equipment (Thermo Scienti1047297cs) equipped with the dispersal of the directly introduced sample via 1047298ow injection analysis (FIA) Thestudied matrix was analyzed by electrospray ionization (ESI) andmultiple stages of fragmentation (MS2 MS3 MSn) were performed atan ion trap (IT) interface The negative mode was selected for thegeneration and analysis of the mass spectra for the 1047297rst order (MS)and for the remaining multi-stage experiments under the followingconditions capillary voltage 25 V voltage spray 5 kV capillarytemperature 275 1C A carrier gas (N2) with a 1047298ow of 8 arbitraryunits (AU) was used and the collision gas was helium (He) The

track acquisition was 100ndash

2000 mz Xcalibur version 13 software(Thermo Finigans) was used to acquire and process the dataFor the FIA-ESI-IT-MSn assay 10 mg of the aqueous fraction was

dissolved in 1 mL of MeOHH2O (11 vv) after using an ultrasonicbath for 5 min The samples were then 1047297ltered through a 022 mmPTFE 1047297lter and aliquots of 20 mL were directly injected into theFIA-ESI-IT-MSn system

For the HPLCndashPDA a clean-up step was performed to remove anycontaminants the solution was puri1047297ed by solid phase extraction(SPE) using Phenomenex Strata C18 cartridges (500 mg of stationaryphase) that were previously activated with 5 mL of MeOH andequilibrated with 5 mL of MeOHH2O (11 vv) The dried aqueousfraction was diluted to 10 mgmL in HPLC solvent A 20 mL aliquotwas injected directly into the HPLCndashPDA with detection at 270 nm

The identi1047297

cation of the different compounds in the chromato-graphic pro1047297le of the aqueous fraction was done by comparing theirretention times (t r ) and the UV spectra with those isolates pre-viously described in the literature (Mininel et al 2014)

24 Animals

Male Wistar rats weighing 180ndash250 g were obtained from breed-ing at the Bioteacuterio Central at the Universidade Estadual Juacutelio deMesquita Filho (UNESP) Botucatu-SP Animals were fed with acerti1047297ed Nuvilab CR-diet with free access to tap water and werehoused on a 12 h lightdark cycle at 6071 humidity and atemperature of 2272 1C The UNESP Institutional Animal Care andUse Committee following the recommendations of the Canadian

Council on Animal Care (Olfert et al 1993) approved all of theemployed protocols under number 1805

25 Evaluation of the gastroprotection activity of the FrAq

251 Gastric ulcer induced by absolute ethanolThe rats were divided into 1047297ve treatment groups (nfrac147ndash14)

and fasted for 12 h prior to receiving an oral dose of the vehicle(saline 10 mLkg) carbenoxolone (100 mgkg) and FrAq (125 25and 100 mgkg) After 60 min all groups were orally treated with1 mL of absolute ethanol for the gastric ulcer induction One hourlater animals were killed and their stomachs excised The incisioninto each stomach was performed along the greater curvature andexamined for linear hemorrhagic lesions in the glandular region

(Morimoto et al 1991) Then the stomachs were photographed and

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the extent of the lesions was measured by ulcerative lesion area(ULA) in mm2 by the program AVSoft BioView Spectras

252 Gastric ulcer induced by ischemiandashreperfusionIschemiandashreperfusion damage was produced in rats by a

method proposed by Ueda et al (1989) Rats (nfrac147ndash9) were orallyadministered saline (10 mLkg) lansoprazole (30 mgkg) and FrAq(the lower effective dose of 25 mgkg) Thirty minutes later the

animals were anaesthetized by intramuscular injection of keta-mine (50 mgkg) and xylazine (10 mgkg) The left side of theabdomen was shaved and an incision was made Brie1047298y the celiacartery was dissected freed of excess fat and clamped for 30 min(ischemia phase) using a micro-bulldog clamp Re-oxygenationwas allowed by removing the clamp for 60 min (reperfusionphase) At the end of this period the animals were killed andthe stomachs were excised and opened along the great curvaturefor the detection of the ULA

26 Determination of mechanisms of action from FrAq

261 Gastric secretion in lesions induced by pylorus ligatureThe method of Shay et al (1945) was used with modi1047297cation Rats

(nfrac146ndash8) were fasted for 12 h and immediately after pylorus ligaturesaline (10 mLkg) cimetidine (100 mgkg) or FrAq (25 mgkg) wasadministered intraduodenally or orally The rats were killed 4 h latertheir abdomens were opened and the stomachs removed The gastriccontent was collected to determine the total amount of gastric-juiceacid (mL) Distilled water was added and the resultant solution wascentrifuged at 3000 g for 10 min The hydrogen ion concentrations(mEqmL4 h) were recorded in the gastric secretion by adjusting thesupernatant volume by titration to pH 70 with 001 N NaOH

262 Determination of the gastric mucus content

This assay was done as described by Rafatullah et al (1990)with modi1047297cation After a 12 h fast rats (nfrac146) received saline

(10 mLkg) carbenoxolone (100 mgkg) and FrAq from Terminaliacatappa (25 mgkg) orally The pylorus was ligated thirty minutesafter treatment The animals were killed 4 h after pylorus ligationand the glandular portion of the stomachs was removed andweighed Each segment was immediately immersed in 10 mL of 01 Alcian blue solution (016 M sucrose005 M sodium acetatepH 58) for 2 h followed by two successive rinses with 10 mL of 025 M sucrose (the 1047297rst for 15 min and the second for 45 min) toremove the excess dye Each stomach was then transferred to05 M magnesium chloride solution for 2 h Four milliliters of thedye solution was then vigorously shaken with an equal volume of ether the resulting emulsion was centrifuged at 2000 g and theabsorbance of the aqueous layer was measured at 580 nm Theamount of blue dye extracted per gram of wet glandular tissue

was then calculated from a standard curve of dye prepared in asucrosendashacetate solution

263 Determination of the role of nitric oxide (NO) prostaglandin

(PGE) and sulfhydryl compounds (SH) in gastric protectionMale rats (nfrac145) were divided into nine groups and pretrea-

ted with either saline L -NAME (N -nitro-L -arginine methyl ester70 mgkg) ndash an inhibitor of NO synthesis INDO (indomethacin30 mgkg) ndash an inhibitor of PGE or NEM (N -ethylmaleimide10 mgkg) ndash a blocker of SH compounds (Arrieta et al 2003)Thirty minutes after the pretreatment the animals were adminis-tered (po) saline (10 mLkg) carbenoxolone (100 mgkg) and FrAq(25 mgkg) After 60 min all groups received 1 mL absoluteethanol to induce gastric ulcers One hour after receiving ethanol

the rats were killed for the determination of gastric lesions

27 Effect of FrAq healing acetic acid-induced gastric lesions

The experiment was performed according to the method descri-bed by Okabe et al (1971) Six groups (nfrac145ndash6) of male Wistar ratswere fasted for 12 h before this experiment Under anesthesia alaparotomy was done in all animals through a midline epigastricincision A plastic 42 mm internal diameter tube was 1047297rmly appliedto the serosal surface of the stomach wall and 70 ml of an 80

solution of acetic acid was applied for 20 s on the serosal surface of the stomach and then completely removed The stomach was bathedwith saline (20 1C) to avoid adherence to the external surface of theulcerated region and then the abdomen was then closed and allthe animals were fed normally This process resulted in a chroniculceration of the mucosa and submucosa with an approximate ulcerarea of 138 mm2 FrAq (25 mgkg) from Terminalia catappa lanso-prazole (30 mgkg) or saline (10 mLkg) were administered for thedetermination of the healing effects by the subacute treatmentduring 7 and 14 days All treatments were done orally once a daybeginning one day after surgery One day after the last drugadministration the rats were killed and the stomachs were removedThe gastric lesions were evaluated by examining the inner gastricsurface with a dissecting magnifying glass

271 Extraction of total protein and zymographyTissue with gastric ulcer from each experimental group described

previously was used to extract total protein The extraction wascarried out following the ratio of 30 mg of tissue 01 mL of 50 mMTrisndashHCl solution pH 75 containing 025 Triton X-100 10 mMCaCl2 and protease inhibitor cocktail (P-8849 ndash Sigma-AldrichSt Louis MO USA) by crushing with the Polytron type homogenizerThe homogenate was centrifuged at 4000 g for 20 min at 4 1C Thesupernatant was collected and protein content was quanti1047297ed by theBradford (1976)

Samples of extracted proteins (28 μg) of the gastric ulcer fromdifferent experimental groups treated during 7 and 14 days weresubjected to electrophoresis under non-reducing conditions on 8

polyacrylamide gel copolymerized with 01 puri1047297ed gelatin(Sigma-Aldrich Co LLC St Louis MO USA) After electrophoresisthe gels were subjected to two washes of 15 min in a solution of 25 Triton X-100 to remove SDS and two washes of 5 min in50 mM pH 80 TrisndashHCl buffer Subsequently gels were incubatedin 50 mM TrisndashHCl buffer pH 80 containing 5 mM of CaCl2 for22 h at 37 1C Finally the gels were stained with CoomassieBrilliant Blue R-250 (Sigma-Aldrich Co LLC St Louis MO USA)The relative molecular weight of the bands was determinedaccording to the molecular weight standard (Precision Plus Pro-teintrade BIO-RAD) used in electrophoresis The bands obtainedthrough zymography were scanned and analyzed by densitometryThe bands representative of the gelatinase activity of MMP-2 and-9 were analyzed by obtaining the integrated optical density (IOD)

of the bands using Image J software Due to limited amount of tissue for this analysis the tissue from 5 different animals fromeach experimental group was pooled together for extraction Thezymography with pooled samples was repeated three time Valueswere plotted in a histogram showing the ratio of the IOD of thetreated groups to the control group IOD

272 Toxicological evaluationSome toxicological parameters were also obtained from three

groups of animals subjected to the healing gastric ulcer model andtreated orally during 14 consecutive days once a day with FrAq(25 mgkg) from Terminalia catappa leaves lansoprazole (30 mgkg)and saline (10 mLkg) Body weight was recorded daily throughoutthe experimental period and the macroscopic analyses and weight of

vital organs (liver kidneys heart spleen and lungs) were compared

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between either the FrAq or lansoprazole and the vehicle-treatedgroups to evaluate subacute toxicity On the day after the last drugadministration the rats from each treatment group were killed andtheir blood was collected The blood samples were then centrifuged(3000 g for 10 min) and the serum obtained was frozen at 20 1Cuntil biochemical analysis Serum biochemical parameters includingglucose urea creatinine γ-glutamyl transpeptidase (γ-GT) aspartateaminotransferase (AST) and alanine aminotransferase (ALT) were

measured using an automated biochemical analyzer (SBA-200Companhia Equipadora de Laboratoacuterios Modernos Satildeo Paulo Brazil)

28 Minimum inhibitory concentration (MIC)

We used Helicobacter pylori ATCC 43504 using a microdilutiontechnique following by CLSI (2006) with modi1047297cations to determinethe minimal inhibitory concentration (MIC) values Helicobacter pyloriwas inoculated on Mueller-Hinton agar plates containing 5 sheepblood and incubated at 36 1C for 72 h in 10 CO2 atmosphereInoculates were prepared in the same medium at a density adjustedto a 20 McFarland turbidity standard The working suspension formicroorganism was diluted 110 and a 100 mL volume was added toeach well of microplates A 100 mL volume of Mueller-Hinton broth

supplemented with 10 fetal bovine serum was added each well of microplates The concentrations for each substance prepared in 2DMSO ranging from 05 to 1000 mgmL were obtained when a 100 mL volume of the fraction was transferred to the 1047297rst well of each rowand serial 2-fold dilutions were performed Amoxicillin was used asreference antimicrobial compound and the MICs were recorded afterincubation of the microplates at 36 1C for 72 h in a 10 CO2 atmo-sphere The MICs were recorded as the lowest concentration atwhich no growth was observed This record was facilitated byaddition of 20 mL of resazurin solution (100 mgmL) as revelator toeach of well and incubation until 2 h A pink color indicated bacterialgrowth and a blue color indicated a non-bacterial growth

29 Statistical analysis

The results were expressed as the mean7standard error of themean (SEM) of the parameters obtained Statistical comparisonswere done by one-way analysis of variance (ANOVA) followed byDunnetts or Tukeys post hoc test with the level of signi1047297cance setat n po005 nn po001 and nnn po0001

3 Results

In the present study we evaluated the protective effect of FrAqobtained from the leaves of Terminalia catappa against the gastricmucosa damage induced by absolute ethanol To establish a dosendashresponse pro1047297le for the antiulcer activity of FrAq we used varyingdoses of FrAq (125 25 and 100 mgkg body weight) to identify the

lowest dose that could elicit an optimal gastroprotective effect(Fig 1) The oral administration of ethanol rapidly penetrated thegastric mucosa and caused membrane damage erosion and hemor-rhagic ulcerations The pre-treatment of rats with FrAq signi1047297cantlyinhibited the formation of gastric lesions by 41 (25 mgkg) and 36(100 mgkg) compared to vehicle-treated control group ( po0001)A lower dose of FrAq (125 mgkg) evaluated in this study did notshow a gastroprotective effect against ethanol ( p4005) We alsoobserved no signi1047297cant differences in the gastroprotective effect of FrAq at doses of 25 or 100 mgkg ( p4005) Thus the subsequentexperiments with FrAq were carried out the lower dose of 25 mgkgadministered orally

This study evaluated the ability of FrAq to protect the gastricmucosa of rats from oxidative damage induced during an IR proce-

dure (Fig 2) We observed that oral treatment with FrAq (25 mgkg) as

well as lansoprazole (30 mgkg) signi1047297cantly decreased the extent of ulcerative lesions by 33 and 71 respectively when compared tostomachs from the vehicle-treated control group ( po001)

After con1047297rming the gastroprotective effect of FrAq againstlesions induced by ethanol and IR the next step was to evaluate

the antiulcer mechanisms of action for this fraction We studied theeffect of FrAq on gastric juice parameters to evaluate their possibleanti-secretory action by the pylorus ligation procedure (Table 1) Weobserved that the administration of FrAq by an intraduodenal ororal route was not able to change the Hthorn concentration of gastric

juice when compared to vehicle-treated animals ( p4005) Thisresult excluded the possibility of the antisecretory effect of FrAqHowever this assay also revealed that the oral treatment with FrAqwas able to signi1047297cantly increase gastric volume (42) This effectcould represent that FrAq was able to induce an increase in theamount of adherent gastric mucus Next we evaluated the effects of the oral administration of FrAq on this gastric mucus in pyloricligature rats (Fig 3) We observed that oral treatment with FrAqcaused an increase (43) in the amount of adherent gastric mucus

compared to the vehicle-treated group ( po005) con1047297rming our

0

100

200

300

400

Carbenoxolone100 mgkg

Vehicle 125 25 100

FrAq (mgkg)

U

L A ( m m

2 )

ns

Fig 1 Effect of pretreatment with the aqueous fraction (FrAq) obtained from theleaves of Terminalia catappa on ethanol-induced gastric ulcers in rats The animalsorally received saline solution (vehicle) carbenoxolone or FrAq The results areexpressed as the mean7SEM (nfrac147ndash14) and statistical signi1047297cance was deter-mined by one-way analysis of variance (ANOVA) followed by Dunnetts test(nn po001 nnn po0001 and ns ndash no signi1047297cant differences)

0

20

40

60

80

Vehicle Lansoprazole

30 mgkg

FrAq

25 mgkg

U L A ( m m sup2 )

Fig 2 Effect of pretreatment with the aqueous fraction (FrAq) obtained from theleaves of Terminalia catappa on ischemiandashreperfusion induced gastric ulcers in ratsThe animals orally received saline solution (vehicle) lansoprazole or FrAq Theresults are expressed as the mean7SEM (nfrac147ndash9) and statistical signi1047297cance wasdetermined by one-way analysis of variance (ANOVA) followed by Dunnetts testwith the level of signi1047297cance set at nn po001 and nnn po0001

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hypothesis As expected carbenoxolone (used as positive control)enhanced the mucus production and there was no statisticaldifference between the groups treated with carbenoxolone andFrAq ( p4005)

In addition to mucus as defense mechanism elicited by FrAqother factors involved with the strengthening of gastric mucosa werealso investigated such as NO (nitric oxide) sulfhydryl (SH) com-pounds and PGs which are factors that maintain mucosal integrityOur results (Fig 4a) shown that the pretreatment of animals withNEM (sulfhydryl inhibitor) did not change the gastroprotective effect

of FrAq ( p4005) compared to the vehicle-pretreated FrAq groupTherefore this result excluded the involvement of sulfhydryl groupsin the gastroprotective effect of FrAq However the group of animalspretreated with L -NAME (a NO-synthase inhibitor) and treated withFrAq display a signi1047297cant increase in gastric lesions (Fig 4b) A similarpicture emerged when the effect of PGs was studied Fig 4c showsthat administration of indomethacin markedly increased the size of the ulcerative lesion and completely abolished the previouslyobserved protective effect of FrAq Our results illustrated that besidethe increase in gastric mucus barrier induced by FrAq the gastro-protective effect of this fraction also involved the action of NO andendogenous PGs levels After determining the factors involved withFrAq action the possible healing effect of FrAq was evaluated withthe acetic acid method (the most similar to human gastric ulcer) Oral

treatment with FrAq during 7 consecutive days (Fig 5a) and also

14 days (Fig 5b) demonstrated that this fraction was able toaccelerate the healing of chronic gastric ulcers in rats At the doseof 25 mgkg FrAq 7 and 14 days of treatment signi1047297cantly decreasedthe area of lesion in 80 and 37 respectively when compared to thecontrol group treated with vehicle ( po001) In the group treatedwith lansoprazole at 30 mgkg the healing effect was also observedwith the decrease of gastric lesion at 83 (7 days) and 64 (14 days)To determine in better detail the healing process of FrAq we also

analyzed the stomach by zymograph Fig 6 presents the activities of MMP-2 and MMP-9 on the gastric mucosa after treatment withvehicle lansoprazole and FrAq for 7 or 14 days Our results show thatMMP-9 activity was present only after treatment during 7 days instomachs from the vehicle and lansoprazole-group We also observedthat this MMP-9 activity was absence for all treatments during 14days In contrast MMP-2 activity was present in all treated groupsincluding the sham group (without gastric lesion) The treatment of animals that exhibited major MMP-2 activity after 7 and 14 days wascompared to the animals treated with vehicle Lower activity of MMP-2 was observed after treatment of the animals with lansopra-zole and FrAq for 7 days (Fig 7a 40 and 26 respectively in rela-tion to the vehicle) and 14 days (Fig 7b 54 and 73 respectivelyin relation to the vehicle)

Other important data obtained from this test based on thetreatment of animals with FrAq for 7 and 14 consecutive days wasthe absence of death in these groups and also absence bodyweight changes (data not shown) and some organ weights andbiochemical parameters of serum (Table 2) These results there-fore ensure that based on the parameters used in this study theFrAq at doses of 25 mgkg does not cause signi1047297cant toxic effectsafter 7 or 14 consecutive days of treatment

This study also evaluated the activity of FrAq against Helico-bacter pylori and presented minimal inhibitory concentration(MIC) of 1250 mgmL considered a accentuated activity and thepositive control (amoxicillin) presented MIC of 150 mgmL

The analysis of mass spectra of FrAq (Fig 9) showed the samecompounds identi1047297ed in the hydroalcoholic extract studied byMininel et al (2014) as shown in Table 3 The spectra (Fig 8) showthe ions with mz 1083 (punicalagin) mz 601 attributed togallagic acid and at mz 301 attributed to ellagic acid illustratingthe similarity with the hydroalcoholic extract (Mininel et al2014) The fragmentation of the second order (MS2) ion at m z 1083 (punicalagin) leads to ions at m z 781 (punicalin) and the ionwith m z 601 (gallagic acid) as shown in Fig 9 The chromato-graphic pro1047297le of the water fraction is also shown in Fig 10 In thechromatogram two peaks are highlighted eluting at tr 172 minand tr 233 min indicating the presence of the major compoundpunicalagin (α and β anomers) Two peaks with tr 280 min and tr301 min (region 2) were observed corresponding to the com-pound punicalin (α and β anomers) derived from punicalagin

4 Discussion

The development of gastric ulcers is a complex and multi-factorial process including bacterial infections the increase of acidsecretion generation of reactive oxygen species (ROS) inhibitionof the endogenous PGs and the degradation of the extracellularmatrix (ECM) (Laine et al 2008 Mei et al 2013) Research duringthe last decade has offered new insights in the preventativetherapy and the healing of gastric ulcers and the synergisticef 1047297ciency of a multi-target approach based on individual mechan-isms of action could be the new perspective for treatment of thisdisease (Wagner 2011 de Carvalho et al 2014)

Terminalia catappa is a medicinal plant widely distributed intropical and subtropical regions and it has been previously reported

that this species exhibits a variety of biological effects However the

Table 1

Effects of the aqueous fraction (FrAq) from the leaves of Terminalia catappa (25 mgkg)administered through the intraduodenal (id) or oral (po) route on the biochemicalparameters of gastric juice obtained from pylorus-ligature rats (nfrac146ndash8)

Treatment Route Dose (mgkg) Gastric juice (mL) [Hthorn] (μEqmL4 h)

Vehicle id ndash 7027225 6997184Cimetidine 100 4297119nn 2777130nn

FrAq 25 8027218 7347065

Vehicle po ndash 3747101 5257151Cimetidine 100 2637067 1947087nn

FrAq 25 5307167 n 6337043

Data represents the means7SEM The asterisks denote the signi1047297cance levelswhen compared with the control group

n po005nn po001 by one-way ANOVA followed by Dunnetts test

0

1000

2000

3000

Vehicle Carbenoxolone

100 mgkg

FrAq

25 mgkg

A l c i a n b l u e

( m g g w e t t i s s u e )

ns

Fig 3 Effects of the aqueous fraction (FrAq) obtained from the leaves of Terminalia

catappa (25 mgkg) on the production of adherent gastric mucus (measured as the

amount of bound Alcian blue) in pylorus-ligated rats relative to the control values(nfrac146) The results are the mean7SEM Statistical signi1047297cance was determined byANOVA followed by Dunnetts t test (n po005 nnn po001 and ns ndash no signi1047297cantdifferences)

L Pinheiro Silva et al Journal of Ethnopharmacology 159 (2015) 285ndash 295 289

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underlying mechanisms of the gastroprotective and healing effectsof the aqueous fraction (FrAq) obtained from the leaves have yet tobe evaluated

In the present study FrAq showed a marked gastroprotectiveeffect evidenced by the dramatic inhibition of the gastric damageproduced by ethanol Ethanol induced erosion ulcerative lesionsand petechial bleeding in the mucosa of the stomach in humansand an ethanol-induced gastric ulcer model is commonly used tostudy both the pathogenesis and new therapeutics for the treat-ment of gastric ulcer disease (Oyagi et al 2010)

Our results have shown the effective gastroprotective effect of theaqueous fraction obtained from the leaves of Terminalia catappa

A previous study by Nunes et al (2012) evaluated the ethanolic

extract of bark from this species against gastric ulcers induced byethanol Aside from using a different part of plant (the bark insteadleaves) this study has demonstrated gastroprotection at a dose 10times higher than our study (250 mgkg vs 25 mgkg) Our studiesalso highlighted the use of the renewable part of this plant (leaves)that would enable the management of this plant for the futureproduction of a phytotherapeutic against gastric ulcer

Gastric ulcer induced by ethanol is commonly associated withreduced mucosal blood 1047298ow and ischemia that causes deleteriouseffects on the gastric mucosa mainly in the aging gastric mucosa(Tarnawski et al 2014) Ischemia weakens the gastric mucosal barrierand increases the acid back-diffusion predisposing the gastric mucosa

to damage (Rao and Vijayakumar 2007) The restoration of blood 1047298ow

Fig 4 The ulcerative lesion area (ULA-mm2) for gastric ulcers induced by ethanol in rats pretreated with L -NAME (panel a) with vehicle or together with carbenoxolone(100 mgkg) or the aqueous fraction (FrAq) obtained from leaves of Terminalia catappa (25 mgkg) pretreated with N -ethylmaleimide (panel b) with vehicle or together withcarbenoxolone (100 mgkg) and FrAq (25 mgkg) or pretreated with INDO ndash indomethacin (panel c) with vehicle or together with carbenoxolone (100 mgkg) or FrAq(25 mgkg) The results are reported as the mean7SEM Statistical signi1047297cance was determined by ANOVA followed by Dunnetts test n po005 nn po001 nnn po0001compared to the corresponding vehicle group o005 po001 compared to the corresponding NEMthornvehicle L -Namethornvehicle or INDOthornvehicle NS ndash no signi1047297cantdifferences

0

2

4

6

8

10

Vehicle Lansoprazole

30 mgkg

FrAq

25 mgkg

U L A ( m m sup2 )

0

5

10

15

20

Vehicle Lansoprazole

30 mgkg

FrAq

25 mgkg

U L A ( m m sup2 )

Fig 5 Effect of the oral administration of the aqueous fraction (FrAq) obtained from the leaves of Terminalia catappa (25 mgkg) on the healing of ulcers produced by theintroduction of acetic acid solution into the stomachs of rats The ulceration was scored on the 7th day (panel a) and 14th day (panel b) after surgery The results are reported

as the mean7

SEM Statistical signi1047297

cance was determined by ANOVA followed by Dunnetts test

nn

po

001

nnn

po

0001 compared to the corresponding vehicle group

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(reperfusion) after a period of ischemia initiates a cascade of changesincluding the release of local ROS in1047298ammatory responses tissuedamage by fragmenting cellular DNA and an increase in the adhesionof neutrophils to endothelial cells a phenomenon known as ische-miandashreperfusion (IR) (Smith et al 1996 De Foneska and Kaunitz2010)

The current study shows that oral treatment with FrAq (25 mgkg)signi1047297cantly decreased the extent of ulcerative lesions in the gastrictissue submitted to IR injury compared to stomachs from the vehicle-treated control group Based on the physiopathology of the IR experimental model our results are in concordance with Chen et al

(2012) and Wang et al (2013) in which they observed that punicalagin

and punicalin both polyphenols presents in Terminalia catappa wereable to attenuate oxidative stress and hypoxia-induced apoptosis

These results therefore con1047297rm the gastroprotective effect of this fraction and encourage the continuation of the studies of theeffects of FrAq treatment and the characterization of its mechan-isms of action

Studies already performed with the hydroalcoholic extract of Terminalia catappa and other species of this genus such as

Terminalia chebula and Terminalia fagifolia have demonstrated theantisecretory effect of these species (Kumar et al 2014 Mishraet al 2013 Nunes et al 2014) Unlike these studies our resultshave shown that the oral and intraduodenal treatment with FrAq(25 mgkg) does not promote the antiulcerogenic effect in anantisecretory manner However our result also illustrated thatoral treatment with FrAq was able to signi1047297cantly increase thegastric volume in relation to the control group treated with vehicle(530 mL and 374 mL respectively) Our hypothesis was that theFrAq elicits the increase of gastric juice because of the amount of adherent gastric mucus

The success of the pharmacological treatment for gastric ulcersrelies on the augmentation of the protective factors of the gastricmucosa such as the mucus barrier (Al-Jiboury and Kaunitz 2012Brzozowska et al 2013) The adherent gastric mucus is the 1047297rst lineof defense against acid and serves as a barrier against self-digestionthe functional integrity of the gastric mucosa depends on thisbarrier (Wallace 2008) Our study reveals a signi1047297cant increase inthe amount of adherent mucus in the animals treated with FrAqthus justifying the increase in gastric volume by treatment with thisfraction This study involving the FrAq is highly signi1047297cant in theongoing search for an antiulcerogenic therapy as the prolonged useof antisecretory drugs such as proton-pump inhibitors and H2

blockers can provoke serious side effects (Ozdil et al 2013 Eomet al 2011)

In addition to the increased production of the mucus barrierseveral studies have demonstrated a complex defense system invol-ving the gastric mucosa which include the regulation of gastricmucosal blood 1047298ow the formation of sulfhydryl compounds NO andendogenous PGs (Brzozowski et al 2000 Pawlik et al 2001)

NO is considered to be one of the most important defensiveendogenous agents in the gastric mucosa and plays a physiologicalrole in the homeostasis of the gastrointestinal tract (Brzozowskiet al 2006) Together with the endogenous prostaglandins NO alsopreserves the gastric mucosa integrity and the inhibition of NOrelease can result in disturbances in the gastrointestinal motilityblood 1047298ow and acid secretion (Brzozowski et al 2006 Khalifaet al 2002 Wallace and Ma 2001) In addition to the increase ingastric mucus our results show that the restoration of the endo-genous NO represents an additional mechanism responsible for thegastroprotective effects of FrAq as a NO-synthase inhibitor elicits asigni1047297cant increase in gastric lesion in stomachs previously treatedwith the fraction The gastroprotective effect of the FrAq through

endogenous NO agrees with the protective effect of this fractionagainst IR in rats by inhibiting the excessive formation of ROS

Our study is also in concordance with those performed byPandya et al (2013) in which they evaluated the status of theethanolic extract of Terminalia catappa (at dose of 200 mgkg) andillustrated an augmentation of the antioxidant defense systemagainst Ehrlich carcinoma Kinoshita et al (2007) also describedthe antioxidant and hepatoprotective actions from the aqueousextract of the leaves of Terminalia catappa As well as NO theincrease in gastric SH content is important for the limiting theproduction of oxygen-derived free radicals (Genestra 2007)Our study showed that the pre-treatment of animals with NEM(a sulfhydryl inhibitor) did not change the gastroprotective effectof the FrAq ( p4005) Therefore our results exclude the involve-

ment of SH content in the gastroprotective effect of FrAq

Fig 6 Representative zymography results of the gastric ulcers from the differentgroups illustrating MMP-2 and MMP-9 activity Treatment for 7 days ndash vehicle (line 1)lansoprazole (line 2) aqueous fraction (FrAq) obtained from leaves of Terminalia

catappa at dose of 25 mgkg (line 3) and sham (line 4) Treatment for 14 days ndash vehicle(line 5) lansoprazole (line 6) aqueous fraction (FrAq) obtained from leaves of Terminalia catappa at dose of 25 mgkg (line 7) and sham (line 8) Gelatinolytic bandsof 92 72 64 and 57 kDa were observed which correspond to active-MMP-9pro-enzyme intermediate and active-MMP-2 respectively

Fig 7 Effect of treatment with the vehicle lansoprazole (30 mgkg) or the aqueousfraction (FrAq) obtained from Terminalia catappa (FrAq) on the gelatinolytic activityof MMP-9 (black column) and MMP-2 (white column) in the gastric mucosa after 7

(panel a) and 14 days (panel b) of treatment Values show the ratio of the IOD fromthe treatment groups to the control group (vehicle) IOD value

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Among the humoral factors in the gastric mucosa endogenousPGs play an important role in the protective effect by stimulatingthe secretion of mucus maintaining the local blood 1047298ow andincreasing the resistance of epithelial cells to potential damage bycytotoxins (Takeuchi 2010) Our results illustrate that the admin-istration of a non-selective COX inhibitor (indomethacin) comple-tely abolished the gastroprotective action of the FrAq This resulthighlights the relevance of PGs in the antiulcer action of thisfraction and agreement with the previously results of fraction inincrease in the amount of adherent mucus According to Takeuchi(2010) one of the mechanisms underlying the action of PGE2 isexactly the increase in mucus secretion that augments the pro-tective factors on the gastric mucosa

Based on the results regarding the gastroprotective effect of theFrAq against gastric injury induced by different ulcerogenic agentswe con1047297rmed that the action of this fraction was mediated by the

activation of defensive mucosa-protective factors such as increasedmucus production NO pathways and endogenous PGs However itis desirable for any new antiulcer drug that the preventive effect isalso accompanied by ulcer healing effects

Antiulcer drugs such as H2-receptor antagonists fail in promot-ing the healing of gastric ulcers when the regenerated mucosapresents with poor histological maturity and ulcer relapse is notprevented by cimetidine (Arakawa et al 2012)

This study also determined the effect of the FrAq on the healingof gastric ulcers induced by acetic acid in rats treated for 7 or 14consecutive days The acetic acid induced a deep necrotic lesioninvolving the entire mucosal depth and penetrating through themuscularis mucosae Ulcer healing is a dynamic process of 1047297llingmucosal defects with proliferating and migrating epithelial cells

and connective tissue which results in the reconstruction of the

mucosal architecture (Okabe and Amagase 2005) Chronic treat-ment with FrAq (25 mgkg) demonstrated a dramatic reduction inthe ulcerative lesion area by treatment for 7 days (80) which wasmore effective than 14 days of treatment (37) This resultdemonstrated the FrAq (25 mgkg) accelerates the healing within7 days of treatment and the healing effect remains after treatmentof 14 days when compared to control group or lansoprazole group

The injection of acetic acid into gastric mucosa induces thedevelopment of deep gastric ulceration and gastric mucosal damagedirectly associated with ECM degradation in which the zinc-dependent matrix metalloproteinases (MMPs) play a crucial role

In several animal studies of gastric ulcer attention has focusedon the role of MMPs mainly MMP-2 and MMP-9 (Li et al 2013)Wound formation and the following healing are dynamic processesof ECM remodeling that are mainly in1047298uenced by MMP-2 andMMP-9 (Gyenge et al 2013)

According to Yeh et al (2012) MMP-9 is the protease mostsigni1047297cantly involved in the degradation of the basement membraneand is associated with pathological states that include in1047298ammationand cancer Li et al (2013) described enhanced expression of MMP-9in the margin of the ulcer in patients with this disease Their studysuggested that the presence of MMP-9 at the margin of the ulcer maybe indicative of in1047298ammation and poor wound healing Ulcerogenicagents such as indomethacin exhibited 12-fold higher pro-MMP-9activity and ethanol exhibited 22-fold higher pro-MMP-9 activity inrat gastric tissues relative to untreated tissues (Mei et al 2013) Ourresults showed MMP-9 activity only after treatments with the vehicleand lansoprazole (7 days) These results determined that the presenceof MMP-9 activity at the gastric mucosa in groups treated with the

vehicle was related with the absence of healing in ulcers of the gastricmucosa Our results also show that the seemingly healing macro-scopic ulcer observed by treating animals with lansoprazole (7 days)may not represent a good wound healing because the presence of MMP-9 activity in gastric damage further demonstrates the persis-tence of the in1047298ammatory process at the gastric mucosa Our resultsshown that treatment of the acetic acid induced chronic ulcers withFrAq (25 mgkg) for 7 and 14 days inhibited the MMP-9 activity Ourresults could be strengthened by the study from Yeh et al (2012) inwhich extract from Terminalia catappa leaves exerts an antimetasticeffect by attenuating MMP-9 mediated in1047298ammation in hepatocellu-lar carcinoma In addition to our results obtained regarding MMP-9our results also reported high MMP-2 activity in gastric ulcers of thecontrol group treated with vehicle (7 and 14 days) and lower MMP-2

activity was observed after the treatment of animals with

Table 2

Effects of the aqueous fraction (FrAq) obtained from leaves of Terminalia catappa (25 mgkg) and lansoprazole (30 mgkg) administered orally for 14 consecutive days onselected toxicological parameters (nfrac145ndash6)

Treatment (po)

Vehicle Lansoprazole FrAq

Organ weights (g)Heart 3857048 3887032 3677013

Lung 4497064 5347143 4207040Kidney 5147063 4917018 4917007Liver 10107123 9997028 9707017Spleen 2697028 2837024 2677027

Biochemical parameterGlucose 136907700 136407580 151007850Creatinine 0567002 0567003 0537004Urea 38107112 39407535 39607423γ-GT 0967017 2027055 1247025AST 157117134 1812071751 1906071450ALT 44337251 48007504 4317311

Results are expressed as the means7SEM obtained from different groups The units for the biochemical parameters of serum are UL (ALT ndash alanine aminotransferaseAST ndash aspartate aminotransferase γndashGT ndash gamma-glutamyl transpeptidase) and mgdL (urea and creatinine)

Table 3

Identi1047297cation of the substances obtained in the aqueous fraction (FrAq) from theleaves of Terminalia catappa by FIA-ESI-IT-MSn

[MndashH] MSn ions Identi1047297cation

1083 781 [M-152-152-H] Punicalagin (1)

601 [M-152-152-180-H]

781 601 [M-180-H] Punicalin (2)

601 409 [M-191-H] Gallagic acid (3)

301 257 [M-44-H] Ellagic acid (4)

229 [M-44-28-H]

Adapted from Mininel et al (2014)

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lansoprazole and FrAq Recent studies have highlighted the functionof MMP-2 in the healing process of rat gastric ulcers induced by aceticacid (Gyenge et al 2013) but the function of MMP-2 is not wellunderstood

The antiulcerogenic actions demonstrated by FrAq could beattributed to the phenolic compounds present in this fractionMininel et al (2014) analyzed mass spectra in a full-scan of theextract and this fraction and showing similarity to each otherhighlighted by the precursor ions m z 1083 (punicalagin) m z 781(pulicalin) m z 601 (gallagic acid) and m z 301 attributed to ellagicacid In these studies the chromatogram from HPLC-PDA of the

aqueous fraction of Terminalia catappa con1047297rmed the majority of the

compounds punicalagin (anomers α and β) and punicalin (anomers αand β) are similar to the hydroalcoholic extract studied by Mininelet al (2014) Ellagic acid (EA) is one of the naturally occurringpolyphenols found in the FrAq from Terminalia catappa leavesNumerous pharmacological studies have suggested that EA providesmucosal protective action in the stomach against ethanol or ische-miandashreperfusion injury (Iino et al 2001 Iino et al 2002) and severalgastroprotective mechanisms are attributed to this compound Forexample Chatterjee et al (2012) showed that EA enhanced prosta-glandins when compared with the ulcerated untreated group

In our 1047297nal experimental series we evaluated the subacute

toxicological parameters from groups that received vehicle

500 1000 1500 2000

mz

0

5

10

15

20

25

30

35

40

45

50

55

60

6570

75

80

85

90

95

100108339

7814554115

10131395449

11329163927

13498731207

139719156207

171229

R e l a t i v e A b u n d a n c e

Fig 8 First-order mass spectrum of the aqueous fraction (FrAq) obtained from Terminalia catappa (FrAq) at the negative mode Range of ions with m z 100ndash2000 Da

T_130129165016 1-1000 RT060-096 AV70 NL271E2

F ITMS - c ESI Full ms2 108300cid3000 [29500-120000]

300 400 500 600 700 800 900 1000 1100 1200

mz

0

5

10

15

20

25

30

35

40

45

50

55

6065

70

75

80

85

90

95

100

R e l a t i v e A b u n d a n c

e

Fig 9 Mass spectrum of the second order (MS2) of the precursor ion m z 1083 from the aqueous fraction (FrAq) obtained in the negative mode of leaves from Terminalia

catappa Range of ions with mz 300-1200 Da

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lansoprazole or the FrAq over 14 days Important toxicity para-meters including changes in body weight mortality and weight of the vital organs were determined as shown in Table 2 Treatmentwith FrAq for 14 days with 25 mgkg did not signi1047297cantly alter thebody weight of animals compared to the vehicle control over the14-days treatment period (data not shown) and the averageweights of the major organs of the FrAq-treated subjects werecomparable to those of the control group Table 2 also shows thatFrAq did not affect several biochemical parameters Treatment didnot signi1047297cantly alter the body weight the weight of vital organsand biochemical parameters of serum However Mininel et al(2014) evaluated the mutagenicity of the hydroalcoholic extractfrom the leaves of Terminalia catappa with the Ames test andillustrated that this extract exhibited mutagenic activity in vitro athigh concentrations leading them to recommend caution whenusing this compound for medicinal purposes

This study also evaluated the activity of aqueous fraction (FrAq)against Helicobacter pylori and determination of minimal inhibitoryconcentration (MIC) of 1250 mgmL considered an important anti-microbial activity The literature do not show a consensus in relationto MIC values obtained for natural products Aligannis et al (2001)consider MICs with values equal to or less than 500 mgmL as potentinhibitors MICs between 600 and 1500 mgmL and MIC moderateinhibitors as above 1600 mgmL as weak inhibitors Webster et al(2008) established as another satisfactory MIC value equal to or lessthan 1000 mgmL In this sense the result reported in this investiga-tion demonstrated the inhibitory potential of Terminalia catappaagainst this important bacteria responsible to gastric ulcer

5 Conclusions

We determined that the FrAq from Terminalia catappa leaveshas an important anti-Helicobacter pylori activity excellent pre-ventive and curative effects on acute and chronically inducedgastric ulcers The mechanisms involved in the gastroprotectionare related to the NO pathway an increase in the mucus and theendogenous prostaglandins and this fraction was able to healulcers through the inhibition of MMP-9 and MMP-2 activities

Acknowledgments

This study was supported by the Biota-FAPESP project (Fundaccedilatildeo

de Amparo agrave Pesquisa do Estado de Satildeo Paulo) CNPq (Conselho

Nacional de Desenvolvimento Cientiacute1047297co e Tecnoloacutegico) and CAPES(Coordenaccedilatildeo de Aperfeiccediloamento Pessoal de Niacutevel Superior)

References

Aligannis N Kalpotzakis E Mitaku S Chinou IB 2001 Composition andantimicrobial activity of the essential oils of two Origanum species Journal of

Agricultural and Food Chemistry 49 4168ndash

4170Al-Jiboury H Kaunitz JD 2012 Gastroduodenal mucosal defense CurrentOpinion of Gastroenterology 28 594ndash601

Arakawa T Watanabe T Tanigawa T Tominaga K Fujiwara Y Morimoto K2012 Quality of ulcer healing in gastrointestinal tract its pathophysiology andclinical relevance World Journal of Gastroenterology 18 4811ndash4822

Arrieta J Benitez J Flores E Castillo C Navarrete A 2003 Puri1047297cation of gastroprotective triterpenoids from stem bark of Amphipterygium adstringensroles of prostaglandins sulfhydryls nitric oxide and capsaicin neurons PlantaMedica 69 905ndash909

Bradford MM 1976 A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye bindingAnalytical Biochemistry 72 248ndash254

Brzozowska I Strzalka M Drozdowicz D Konturek SJ Brzozowski T 2013Mechanisms of esophageal protection gastroprotection and ulcer healing bymelatonin Implications for the therapeutic use of melatonin in gastroesopha-geal re1047298ux disease (GERD) and peptic ulcer disease Current PharmaceuticalDesign httpdxdoi org1021741381612819666131119110258 (Epub ahead of print)

Brzozowski T Konturek PC Konturek SJ Pajdo R Schuppan D Drozdowicz DPtak A Pawlik M Nakamura T Hahn EG 2000 Involvement of cycloox-ygenase (COX)-2 products in acceleration of ulcer healing by gastrin andhepatocyte growth factor Journal of Physiology and Pharmacology 51 751ndash773

Brzozowski T Konturek PC Sliwowski Z Pajdo R Drozdowicz D Kwiecien SBurnat G Konturek SJ Pawlik WW 2006 Prostaglandincyclooxygenasepathway in ghrelin-induced gastroprotection against ischemia-reperfusioninjury Journal of Pharmacology and Experimental Therapeutics 319 477ndash487

Camargo MC Garciacutea A Riquelme A Otero W Camargo CA Garciacutea THCandia R Bruce MG Rabkin CS 2014 The problem of Helicobacter pyloriresistance to antibiotics a systematic review in Latin America Clinical andSystematic Reviews 109 485ndash495

Chatterjee A Chatterjee S Das S Saha A Chattopadhyay S Bandyopadhyay SK2012 Ellagic acid facilitates indomethacin-induced gastric ulcer healing via COX-2 up-regulation Acta Biochimica et Biophysica Sinica 44 565ndash576

Chen PS Li JH 2005 Chemopreventive effect of punicalagin a novel tannincomponent isolated from Terminalia catappa on H-ras-transformed NIH3T3cells Toxicology Letter 163 44ndash53

Chen PS Li JH Liu TY Lin TC 2000 Folk medicine Terminalia catappa and its

major tannin component punicalagin are effective against bleomycin-inducedgenotoxicity in Chinese hamster ovary cells Cancer Letter 152 115ndash122

Chen B Tuuli MG Longtine MS Shin JS Lawrence R Inder T Michael ND2012 Pomegranate juice and punicalagin attenuate oxidative stress andapoptosis in human placenta and in human placental trophoblasts American

Journal of Physiology and Endocrinology Metabolism 302 E1142ndashE1152CLSI Manual Clinical and Laboratory Standards Institute 2006 Methods for

dilution antimicrobial susceptibility tests for bacteria that grow aerobicallyapproved standards 6ordf ed Performance standards for antimicrobial suscept-ibility testing Sixteenth informational supplement M100-S16 (tab 2J) Clinicaland Laboratory Standards Institute Wayne PA

De Foneska A Kaunitz JD 2010 Gastroduodenal mucosal defense CurrentOpinion Gastroenterology 26 604ndash610

de Carvalho KI Bonamin F Dos Santos RC Peacuterico LL Beserra FP de Sousa DPFilho JM da Rocha LR Hiruma-Lima CA 2014 Geraniol-a 1047298avoring agentwith multifunctional effects in protecting the gastric and duodenal mucosaNaunyn Schmiedebergs Archieves of Pharmacology 387 355ndash365

Eom CS Park SM Myung SK Yun JM Ahn JS 2011 Use of acid-suppressive

drugs and risk of fracture a meta-analysis of observational studies AnnalsFamily Medicine 9 257ndash267Fan YM Xu LZ Gao J Wang Y Tang XH Zhao XN Zhang ZX 2004

Phytochemical and antiin1047298ammatory studies on Terminalia catappa Fitoterapia75 253ndash260

Fyhrquist P Mwasumbi L Haeggstroumlm CA Vuorela H Hiltunen R Vuorela P2002 Ethnobotanical and antimicrobial investigation on some species of Terminalia and Combretum (Combretaceae) growing in Tanzania Journal of Ethnopharmacology 79 169ndash177

Genestra M 2007 Oxyl radicals redox-sensitive signalling cascades and antiox-idants Cell Signalling 19 1807ndash1819

Germoseacuten-Robineau L 2014 Farmacopea Vegetal Carbentildea CICY editorial YucataacutenMexico p 360

Gyenge M Amagase K Kunimi S Matsuoka R Takeuchi K 2013 Roles of pro-angiogenic and anti-angiogenic factors as well as matrix metalloproteinases inhealing of NSAID-induced small intestinal ulcers in rats Life Science 93441ndash447

Iino T Nakahara K Miki W Kiso Y Ogawa Y Kato S Takeuchi K 2001 Lessdamaging effect of whisky in rat stomachs in comparison with pure ethanol

Digestion 64 (214ndash221) 2001

1 - α e β punicalagin

2 - α e βpunicalin

00 50 100 150 200 250 300 350 400

Retention Time [min]

0

1000000

2000000

I n t e n

s i t y [ micro V ]

T catappa FrAq - CH5

Fig 10 Chromatogram from HPLCndashPDA of the aqueous fraction of the leaves of Terminalia catappa Hydro Column 1047298ow 1 mLmin gradient method 5ndash60 MeOH40 min HPLCndashPDA (Jascos) 270 nm

L Pinheiro Silva et al Journal of Ethnopharmacology 159 (2015) 285ndash 295294

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the extent of the lesions was measured by ulcerative lesion area(ULA) in mm2 by the program AVSoft BioView Spectras

252 Gastric ulcer induced by ischemiandashreperfusionIschemiandashreperfusion damage was produced in rats by a

method proposed by Ueda et al (1989) Rats (nfrac147ndash9) were orallyadministered saline (10 mLkg) lansoprazole (30 mgkg) and FrAq(the lower effective dose of 25 mgkg) Thirty minutes later the

animals were anaesthetized by intramuscular injection of keta-mine (50 mgkg) and xylazine (10 mgkg) The left side of theabdomen was shaved and an incision was made Brie1047298y the celiacartery was dissected freed of excess fat and clamped for 30 min(ischemia phase) using a micro-bulldog clamp Re-oxygenationwas allowed by removing the clamp for 60 min (reperfusionphase) At the end of this period the animals were killed andthe stomachs were excised and opened along the great curvaturefor the detection of the ULA

26 Determination of mechanisms of action from FrAq

261 Gastric secretion in lesions induced by pylorus ligatureThe method of Shay et al (1945) was used with modi1047297cation Rats

(nfrac146ndash8) were fasted for 12 h and immediately after pylorus ligaturesaline (10 mLkg) cimetidine (100 mgkg) or FrAq (25 mgkg) wasadministered intraduodenally or orally The rats were killed 4 h latertheir abdomens were opened and the stomachs removed The gastriccontent was collected to determine the total amount of gastric-juiceacid (mL) Distilled water was added and the resultant solution wascentrifuged at 3000 g for 10 min The hydrogen ion concentrations(mEqmL4 h) were recorded in the gastric secretion by adjusting thesupernatant volume by titration to pH 70 with 001 N NaOH

262 Determination of the gastric mucus content

This assay was done as described by Rafatullah et al (1990)with modi1047297cation After a 12 h fast rats (nfrac146) received saline

(10 mLkg) carbenoxolone (100 mgkg) and FrAq from Terminaliacatappa (25 mgkg) orally The pylorus was ligated thirty minutesafter treatment The animals were killed 4 h after pylorus ligationand the glandular portion of the stomachs was removed andweighed Each segment was immediately immersed in 10 mL of 01 Alcian blue solution (016 M sucrose005 M sodium acetatepH 58) for 2 h followed by two successive rinses with 10 mL of 025 M sucrose (the 1047297rst for 15 min and the second for 45 min) toremove the excess dye Each stomach was then transferred to05 M magnesium chloride solution for 2 h Four milliliters of thedye solution was then vigorously shaken with an equal volume of ether the resulting emulsion was centrifuged at 2000 g and theabsorbance of the aqueous layer was measured at 580 nm Theamount of blue dye extracted per gram of wet glandular tissue

was then calculated from a standard curve of dye prepared in asucrosendashacetate solution

263 Determination of the role of nitric oxide (NO) prostaglandin

(PGE) and sulfhydryl compounds (SH) in gastric protectionMale rats (nfrac145) were divided into nine groups and pretrea-

ted with either saline L -NAME (N -nitro-L -arginine methyl ester70 mgkg) ndash an inhibitor of NO synthesis INDO (indomethacin30 mgkg) ndash an inhibitor of PGE or NEM (N -ethylmaleimide10 mgkg) ndash a blocker of SH compounds (Arrieta et al 2003)Thirty minutes after the pretreatment the animals were adminis-tered (po) saline (10 mLkg) carbenoxolone (100 mgkg) and FrAq(25 mgkg) After 60 min all groups received 1 mL absoluteethanol to induce gastric ulcers One hour after receiving ethanol

the rats were killed for the determination of gastric lesions

27 Effect of FrAq healing acetic acid-induced gastric lesions

The experiment was performed according to the method descri-bed by Okabe et al (1971) Six groups (nfrac145ndash6) of male Wistar ratswere fasted for 12 h before this experiment Under anesthesia alaparotomy was done in all animals through a midline epigastricincision A plastic 42 mm internal diameter tube was 1047297rmly appliedto the serosal surface of the stomach wall and 70 ml of an 80

solution of acetic acid was applied for 20 s on the serosal surface of the stomach and then completely removed The stomach was bathedwith saline (20 1C) to avoid adherence to the external surface of theulcerated region and then the abdomen was then closed and allthe animals were fed normally This process resulted in a chroniculceration of the mucosa and submucosa with an approximate ulcerarea of 138 mm2 FrAq (25 mgkg) from Terminalia catappa lanso-prazole (30 mgkg) or saline (10 mLkg) were administered for thedetermination of the healing effects by the subacute treatmentduring 7 and 14 days All treatments were done orally once a daybeginning one day after surgery One day after the last drugadministration the rats were killed and the stomachs were removedThe gastric lesions were evaluated by examining the inner gastricsurface with a dissecting magnifying glass

271 Extraction of total protein and zymographyTissue with gastric ulcer from each experimental group described

previously was used to extract total protein The extraction wascarried out following the ratio of 30 mg of tissue 01 mL of 50 mMTrisndashHCl solution pH 75 containing 025 Triton X-100 10 mMCaCl2 and protease inhibitor cocktail (P-8849 ndash Sigma-AldrichSt Louis MO USA) by crushing with the Polytron type homogenizerThe homogenate was centrifuged at 4000 g for 20 min at 4 1C Thesupernatant was collected and protein content was quanti1047297ed by theBradford (1976)

Samples of extracted proteins (28 μg) of the gastric ulcer fromdifferent experimental groups treated during 7 and 14 days weresubjected to electrophoresis under non-reducing conditions on 8

polyacrylamide gel copolymerized with 01 puri1047297ed gelatin(Sigma-Aldrich Co LLC St Louis MO USA) After electrophoresisthe gels were subjected to two washes of 15 min in a solution of 25 Triton X-100 to remove SDS and two washes of 5 min in50 mM pH 80 TrisndashHCl buffer Subsequently gels were incubatedin 50 mM TrisndashHCl buffer pH 80 containing 5 mM of CaCl2 for22 h at 37 1C Finally the gels were stained with CoomassieBrilliant Blue R-250 (Sigma-Aldrich Co LLC St Louis MO USA)The relative molecular weight of the bands was determinedaccording to the molecular weight standard (Precision Plus Pro-teintrade BIO-RAD) used in electrophoresis The bands obtainedthrough zymography were scanned and analyzed by densitometryThe bands representative of the gelatinase activity of MMP-2 and-9 were analyzed by obtaining the integrated optical density (IOD)

of the bands using Image J software Due to limited amount of tissue for this analysis the tissue from 5 different animals fromeach experimental group was pooled together for extraction Thezymography with pooled samples was repeated three time Valueswere plotted in a histogram showing the ratio of the IOD of thetreated groups to the control group IOD

272 Toxicological evaluationSome toxicological parameters were also obtained from three

groups of animals subjected to the healing gastric ulcer model andtreated orally during 14 consecutive days once a day with FrAq(25 mgkg) from Terminalia catappa leaves lansoprazole (30 mgkg)and saline (10 mLkg) Body weight was recorded daily throughoutthe experimental period and the macroscopic analyses and weight of

vital organs (liver kidneys heart spleen and lungs) were compared

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between either the FrAq or lansoprazole and the vehicle-treatedgroups to evaluate subacute toxicity On the day after the last drugadministration the rats from each treatment group were killed andtheir blood was collected The blood samples were then centrifuged(3000 g for 10 min) and the serum obtained was frozen at 20 1Cuntil biochemical analysis Serum biochemical parameters includingglucose urea creatinine γ-glutamyl transpeptidase (γ-GT) aspartateaminotransferase (AST) and alanine aminotransferase (ALT) were

measured using an automated biochemical analyzer (SBA-200Companhia Equipadora de Laboratoacuterios Modernos Satildeo Paulo Brazil)

28 Minimum inhibitory concentration (MIC)

We used Helicobacter pylori ATCC 43504 using a microdilutiontechnique following by CLSI (2006) with modi1047297cations to determinethe minimal inhibitory concentration (MIC) values Helicobacter pyloriwas inoculated on Mueller-Hinton agar plates containing 5 sheepblood and incubated at 36 1C for 72 h in 10 CO2 atmosphereInoculates were prepared in the same medium at a density adjustedto a 20 McFarland turbidity standard The working suspension formicroorganism was diluted 110 and a 100 mL volume was added toeach well of microplates A 100 mL volume of Mueller-Hinton broth

supplemented with 10 fetal bovine serum was added each well of microplates The concentrations for each substance prepared in 2DMSO ranging from 05 to 1000 mgmL were obtained when a 100 mL volume of the fraction was transferred to the 1047297rst well of each rowand serial 2-fold dilutions were performed Amoxicillin was used asreference antimicrobial compound and the MICs were recorded afterincubation of the microplates at 36 1C for 72 h in a 10 CO2 atmo-sphere The MICs were recorded as the lowest concentration atwhich no growth was observed This record was facilitated byaddition of 20 mL of resazurin solution (100 mgmL) as revelator toeach of well and incubation until 2 h A pink color indicated bacterialgrowth and a blue color indicated a non-bacterial growth

29 Statistical analysis

The results were expressed as the mean7standard error of themean (SEM) of the parameters obtained Statistical comparisonswere done by one-way analysis of variance (ANOVA) followed byDunnetts or Tukeys post hoc test with the level of signi1047297cance setat n po005 nn po001 and nnn po0001

3 Results

In the present study we evaluated the protective effect of FrAqobtained from the leaves of Terminalia catappa against the gastricmucosa damage induced by absolute ethanol To establish a dosendashresponse pro1047297le for the antiulcer activity of FrAq we used varyingdoses of FrAq (125 25 and 100 mgkg body weight) to identify the

lowest dose that could elicit an optimal gastroprotective effect(Fig 1) The oral administration of ethanol rapidly penetrated thegastric mucosa and caused membrane damage erosion and hemor-rhagic ulcerations The pre-treatment of rats with FrAq signi1047297cantlyinhibited the formation of gastric lesions by 41 (25 mgkg) and 36(100 mgkg) compared to vehicle-treated control group ( po0001)A lower dose of FrAq (125 mgkg) evaluated in this study did notshow a gastroprotective effect against ethanol ( p4005) We alsoobserved no signi1047297cant differences in the gastroprotective effect of FrAq at doses of 25 or 100 mgkg ( p4005) Thus the subsequentexperiments with FrAq were carried out the lower dose of 25 mgkgadministered orally

This study evaluated the ability of FrAq to protect the gastricmucosa of rats from oxidative damage induced during an IR proce-

dure (Fig 2) We observed that oral treatment with FrAq (25 mgkg) as

well as lansoprazole (30 mgkg) signi1047297cantly decreased the extent of ulcerative lesions by 33 and 71 respectively when compared tostomachs from the vehicle-treated control group ( po001)

After con1047297rming the gastroprotective effect of FrAq againstlesions induced by ethanol and IR the next step was to evaluate

the antiulcer mechanisms of action for this fraction We studied theeffect of FrAq on gastric juice parameters to evaluate their possibleanti-secretory action by the pylorus ligation procedure (Table 1) Weobserved that the administration of FrAq by an intraduodenal ororal route was not able to change the Hthorn concentration of gastric

juice when compared to vehicle-treated animals ( p4005) Thisresult excluded the possibility of the antisecretory effect of FrAqHowever this assay also revealed that the oral treatment with FrAqwas able to signi1047297cantly increase gastric volume (42) This effectcould represent that FrAq was able to induce an increase in theamount of adherent gastric mucus Next we evaluated the effects of the oral administration of FrAq on this gastric mucus in pyloricligature rats (Fig 3) We observed that oral treatment with FrAqcaused an increase (43) in the amount of adherent gastric mucus

compared to the vehicle-treated group ( po005) con1047297rming our

0

100

200

300

400

Carbenoxolone100 mgkg

Vehicle 125 25 100

FrAq (mgkg)

U

L A ( m m

2 )

ns

Fig 1 Effect of pretreatment with the aqueous fraction (FrAq) obtained from theleaves of Terminalia catappa on ethanol-induced gastric ulcers in rats The animalsorally received saline solution (vehicle) carbenoxolone or FrAq The results areexpressed as the mean7SEM (nfrac147ndash14) and statistical signi1047297cance was deter-mined by one-way analysis of variance (ANOVA) followed by Dunnetts test(nn po001 nnn po0001 and ns ndash no signi1047297cant differences)

0

20

40

60

80

Vehicle Lansoprazole

30 mgkg

FrAq

25 mgkg

U L A ( m m sup2 )

Fig 2 Effect of pretreatment with the aqueous fraction (FrAq) obtained from theleaves of Terminalia catappa on ischemiandashreperfusion induced gastric ulcers in ratsThe animals orally received saline solution (vehicle) lansoprazole or FrAq Theresults are expressed as the mean7SEM (nfrac147ndash9) and statistical signi1047297cance wasdetermined by one-way analysis of variance (ANOVA) followed by Dunnetts testwith the level of signi1047297cance set at nn po001 and nnn po0001

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hypothesis As expected carbenoxolone (used as positive control)enhanced the mucus production and there was no statisticaldifference between the groups treated with carbenoxolone andFrAq ( p4005)

In addition to mucus as defense mechanism elicited by FrAqother factors involved with the strengthening of gastric mucosa werealso investigated such as NO (nitric oxide) sulfhydryl (SH) com-pounds and PGs which are factors that maintain mucosal integrityOur results (Fig 4a) shown that the pretreatment of animals withNEM (sulfhydryl inhibitor) did not change the gastroprotective effect

of FrAq ( p4005) compared to the vehicle-pretreated FrAq groupTherefore this result excluded the involvement of sulfhydryl groupsin the gastroprotective effect of FrAq However the group of animalspretreated with L -NAME (a NO-synthase inhibitor) and treated withFrAq display a signi1047297cant increase in gastric lesions (Fig 4b) A similarpicture emerged when the effect of PGs was studied Fig 4c showsthat administration of indomethacin markedly increased the size of the ulcerative lesion and completely abolished the previouslyobserved protective effect of FrAq Our results illustrated that besidethe increase in gastric mucus barrier induced by FrAq the gastro-protective effect of this fraction also involved the action of NO andendogenous PGs levels After determining the factors involved withFrAq action the possible healing effect of FrAq was evaluated withthe acetic acid method (the most similar to human gastric ulcer) Oral

treatment with FrAq during 7 consecutive days (Fig 5a) and also

14 days (Fig 5b) demonstrated that this fraction was able toaccelerate the healing of chronic gastric ulcers in rats At the doseof 25 mgkg FrAq 7 and 14 days of treatment signi1047297cantly decreasedthe area of lesion in 80 and 37 respectively when compared to thecontrol group treated with vehicle ( po001) In the group treatedwith lansoprazole at 30 mgkg the healing effect was also observedwith the decrease of gastric lesion at 83 (7 days) and 64 (14 days)To determine in better detail the healing process of FrAq we also

analyzed the stomach by zymograph Fig 6 presents the activities of MMP-2 and MMP-9 on the gastric mucosa after treatment withvehicle lansoprazole and FrAq for 7 or 14 days Our results show thatMMP-9 activity was present only after treatment during 7 days instomachs from the vehicle and lansoprazole-group We also observedthat this MMP-9 activity was absence for all treatments during 14days In contrast MMP-2 activity was present in all treated groupsincluding the sham group (without gastric lesion) The treatment of animals that exhibited major MMP-2 activity after 7 and 14 days wascompared to the animals treated with vehicle Lower activity of MMP-2 was observed after treatment of the animals with lansopra-zole and FrAq for 7 days (Fig 7a 40 and 26 respectively in rela-tion to the vehicle) and 14 days (Fig 7b 54 and 73 respectivelyin relation to the vehicle)

Other important data obtained from this test based on thetreatment of animals with FrAq for 7 and 14 consecutive days wasthe absence of death in these groups and also absence bodyweight changes (data not shown) and some organ weights andbiochemical parameters of serum (Table 2) These results there-fore ensure that based on the parameters used in this study theFrAq at doses of 25 mgkg does not cause signi1047297cant toxic effectsafter 7 or 14 consecutive days of treatment

This study also evaluated the activity of FrAq against Helico-bacter pylori and presented minimal inhibitory concentration(MIC) of 1250 mgmL considered a accentuated activity and thepositive control (amoxicillin) presented MIC of 150 mgmL

The analysis of mass spectra of FrAq (Fig 9) showed the samecompounds identi1047297ed in the hydroalcoholic extract studied byMininel et al (2014) as shown in Table 3 The spectra (Fig 8) showthe ions with mz 1083 (punicalagin) mz 601 attributed togallagic acid and at mz 301 attributed to ellagic acid illustratingthe similarity with the hydroalcoholic extract (Mininel et al2014) The fragmentation of the second order (MS2) ion at m z 1083 (punicalagin) leads to ions at m z 781 (punicalin) and the ionwith m z 601 (gallagic acid) as shown in Fig 9 The chromato-graphic pro1047297le of the water fraction is also shown in Fig 10 In thechromatogram two peaks are highlighted eluting at tr 172 minand tr 233 min indicating the presence of the major compoundpunicalagin (α and β anomers) Two peaks with tr 280 min and tr301 min (region 2) were observed corresponding to the com-pound punicalin (α and β anomers) derived from punicalagin

4 Discussion

The development of gastric ulcers is a complex and multi-factorial process including bacterial infections the increase of acidsecretion generation of reactive oxygen species (ROS) inhibitionof the endogenous PGs and the degradation of the extracellularmatrix (ECM) (Laine et al 2008 Mei et al 2013) Research duringthe last decade has offered new insights in the preventativetherapy and the healing of gastric ulcers and the synergisticef 1047297ciency of a multi-target approach based on individual mechan-isms of action could be the new perspective for treatment of thisdisease (Wagner 2011 de Carvalho et al 2014)

Terminalia catappa is a medicinal plant widely distributed intropical and subtropical regions and it has been previously reported

that this species exhibits a variety of biological effects However the

Table 1

Effects of the aqueous fraction (FrAq) from the leaves of Terminalia catappa (25 mgkg)administered through the intraduodenal (id) or oral (po) route on the biochemicalparameters of gastric juice obtained from pylorus-ligature rats (nfrac146ndash8)

Treatment Route Dose (mgkg) Gastric juice (mL) [Hthorn] (μEqmL4 h)

Vehicle id ndash 7027225 6997184Cimetidine 100 4297119nn 2777130nn

FrAq 25 8027218 7347065

Vehicle po ndash 3747101 5257151Cimetidine 100 2637067 1947087nn

FrAq 25 5307167 n 6337043

Data represents the means7SEM The asterisks denote the signi1047297cance levelswhen compared with the control group

n po005nn po001 by one-way ANOVA followed by Dunnetts test

0

1000

2000

3000

Vehicle Carbenoxolone

100 mgkg

FrAq

25 mgkg

A l c i a n b l u e

( m g g w e t t i s s u e )

ns

Fig 3 Effects of the aqueous fraction (FrAq) obtained from the leaves of Terminalia

catappa (25 mgkg) on the production of adherent gastric mucus (measured as the

amount of bound Alcian blue) in pylorus-ligated rats relative to the control values(nfrac146) The results are the mean7SEM Statistical signi1047297cance was determined byANOVA followed by Dunnetts t test (n po005 nnn po001 and ns ndash no signi1047297cantdifferences)

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underlying mechanisms of the gastroprotective and healing effectsof the aqueous fraction (FrAq) obtained from the leaves have yet tobe evaluated

In the present study FrAq showed a marked gastroprotectiveeffect evidenced by the dramatic inhibition of the gastric damageproduced by ethanol Ethanol induced erosion ulcerative lesionsand petechial bleeding in the mucosa of the stomach in humansand an ethanol-induced gastric ulcer model is commonly used tostudy both the pathogenesis and new therapeutics for the treat-ment of gastric ulcer disease (Oyagi et al 2010)

Our results have shown the effective gastroprotective effect of theaqueous fraction obtained from the leaves of Terminalia catappa

A previous study by Nunes et al (2012) evaluated the ethanolic

extract of bark from this species against gastric ulcers induced byethanol Aside from using a different part of plant (the bark insteadleaves) this study has demonstrated gastroprotection at a dose 10times higher than our study (250 mgkg vs 25 mgkg) Our studiesalso highlighted the use of the renewable part of this plant (leaves)that would enable the management of this plant for the futureproduction of a phytotherapeutic against gastric ulcer

Gastric ulcer induced by ethanol is commonly associated withreduced mucosal blood 1047298ow and ischemia that causes deleteriouseffects on the gastric mucosa mainly in the aging gastric mucosa(Tarnawski et al 2014) Ischemia weakens the gastric mucosal barrierand increases the acid back-diffusion predisposing the gastric mucosa

to damage (Rao and Vijayakumar 2007) The restoration of blood 1047298ow

Fig 4 The ulcerative lesion area (ULA-mm2) for gastric ulcers induced by ethanol in rats pretreated with L -NAME (panel a) with vehicle or together with carbenoxolone(100 mgkg) or the aqueous fraction (FrAq) obtained from leaves of Terminalia catappa (25 mgkg) pretreated with N -ethylmaleimide (panel b) with vehicle or together withcarbenoxolone (100 mgkg) and FrAq (25 mgkg) or pretreated with INDO ndash indomethacin (panel c) with vehicle or together with carbenoxolone (100 mgkg) or FrAq(25 mgkg) The results are reported as the mean7SEM Statistical signi1047297cance was determined by ANOVA followed by Dunnetts test n po005 nn po001 nnn po0001compared to the corresponding vehicle group o005 po001 compared to the corresponding NEMthornvehicle L -Namethornvehicle or INDOthornvehicle NS ndash no signi1047297cantdifferences

0

2

4

6

8

10

Vehicle Lansoprazole

30 mgkg

FrAq

25 mgkg

U L A ( m m sup2 )

0

5

10

15

20

Vehicle Lansoprazole

30 mgkg

FrAq

25 mgkg

U L A ( m m sup2 )

Fig 5 Effect of the oral administration of the aqueous fraction (FrAq) obtained from the leaves of Terminalia catappa (25 mgkg) on the healing of ulcers produced by theintroduction of acetic acid solution into the stomachs of rats The ulceration was scored on the 7th day (panel a) and 14th day (panel b) after surgery The results are reported

as the mean7

SEM Statistical signi1047297

cance was determined by ANOVA followed by Dunnetts test

nn

po

001

nnn

po

0001 compared to the corresponding vehicle group

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(reperfusion) after a period of ischemia initiates a cascade of changesincluding the release of local ROS in1047298ammatory responses tissuedamage by fragmenting cellular DNA and an increase in the adhesionof neutrophils to endothelial cells a phenomenon known as ische-miandashreperfusion (IR) (Smith et al 1996 De Foneska and Kaunitz2010)

The current study shows that oral treatment with FrAq (25 mgkg)signi1047297cantly decreased the extent of ulcerative lesions in the gastrictissue submitted to IR injury compared to stomachs from the vehicle-treated control group Based on the physiopathology of the IR experimental model our results are in concordance with Chen et al

(2012) and Wang et al (2013) in which they observed that punicalagin

and punicalin both polyphenols presents in Terminalia catappa wereable to attenuate oxidative stress and hypoxia-induced apoptosis

These results therefore con1047297rm the gastroprotective effect of this fraction and encourage the continuation of the studies of theeffects of FrAq treatment and the characterization of its mechan-isms of action

Studies already performed with the hydroalcoholic extract of Terminalia catappa and other species of this genus such as

Terminalia chebula and Terminalia fagifolia have demonstrated theantisecretory effect of these species (Kumar et al 2014 Mishraet al 2013 Nunes et al 2014) Unlike these studies our resultshave shown that the oral and intraduodenal treatment with FrAq(25 mgkg) does not promote the antiulcerogenic effect in anantisecretory manner However our result also illustrated thatoral treatment with FrAq was able to signi1047297cantly increase thegastric volume in relation to the control group treated with vehicle(530 mL and 374 mL respectively) Our hypothesis was that theFrAq elicits the increase of gastric juice because of the amount of adherent gastric mucus

The success of the pharmacological treatment for gastric ulcersrelies on the augmentation of the protective factors of the gastricmucosa such as the mucus barrier (Al-Jiboury and Kaunitz 2012Brzozowska et al 2013) The adherent gastric mucus is the 1047297rst lineof defense against acid and serves as a barrier against self-digestionthe functional integrity of the gastric mucosa depends on thisbarrier (Wallace 2008) Our study reveals a signi1047297cant increase inthe amount of adherent mucus in the animals treated with FrAqthus justifying the increase in gastric volume by treatment with thisfraction This study involving the FrAq is highly signi1047297cant in theongoing search for an antiulcerogenic therapy as the prolonged useof antisecretory drugs such as proton-pump inhibitors and H2

blockers can provoke serious side effects (Ozdil et al 2013 Eomet al 2011)

In addition to the increased production of the mucus barrierseveral studies have demonstrated a complex defense system invol-ving the gastric mucosa which include the regulation of gastricmucosal blood 1047298ow the formation of sulfhydryl compounds NO andendogenous PGs (Brzozowski et al 2000 Pawlik et al 2001)

NO is considered to be one of the most important defensiveendogenous agents in the gastric mucosa and plays a physiologicalrole in the homeostasis of the gastrointestinal tract (Brzozowskiet al 2006) Together with the endogenous prostaglandins NO alsopreserves the gastric mucosa integrity and the inhibition of NOrelease can result in disturbances in the gastrointestinal motilityblood 1047298ow and acid secretion (Brzozowski et al 2006 Khalifaet al 2002 Wallace and Ma 2001) In addition to the increase ingastric mucus our results show that the restoration of the endo-genous NO represents an additional mechanism responsible for thegastroprotective effects of FrAq as a NO-synthase inhibitor elicits asigni1047297cant increase in gastric lesion in stomachs previously treatedwith the fraction The gastroprotective effect of the FrAq through

endogenous NO agrees with the protective effect of this fractionagainst IR in rats by inhibiting the excessive formation of ROS

Our study is also in concordance with those performed byPandya et al (2013) in which they evaluated the status of theethanolic extract of Terminalia catappa (at dose of 200 mgkg) andillustrated an augmentation of the antioxidant defense systemagainst Ehrlich carcinoma Kinoshita et al (2007) also describedthe antioxidant and hepatoprotective actions from the aqueousextract of the leaves of Terminalia catappa As well as NO theincrease in gastric SH content is important for the limiting theproduction of oxygen-derived free radicals (Genestra 2007)Our study showed that the pre-treatment of animals with NEM(a sulfhydryl inhibitor) did not change the gastroprotective effectof the FrAq ( p4005) Therefore our results exclude the involve-

ment of SH content in the gastroprotective effect of FrAq

Fig 6 Representative zymography results of the gastric ulcers from the differentgroups illustrating MMP-2 and MMP-9 activity Treatment for 7 days ndash vehicle (line 1)lansoprazole (line 2) aqueous fraction (FrAq) obtained from leaves of Terminalia

catappa at dose of 25 mgkg (line 3) and sham (line 4) Treatment for 14 days ndash vehicle(line 5) lansoprazole (line 6) aqueous fraction (FrAq) obtained from leaves of Terminalia catappa at dose of 25 mgkg (line 7) and sham (line 8) Gelatinolytic bandsof 92 72 64 and 57 kDa were observed which correspond to active-MMP-9pro-enzyme intermediate and active-MMP-2 respectively

Fig 7 Effect of treatment with the vehicle lansoprazole (30 mgkg) or the aqueousfraction (FrAq) obtained from Terminalia catappa (FrAq) on the gelatinolytic activityof MMP-9 (black column) and MMP-2 (white column) in the gastric mucosa after 7

(panel a) and 14 days (panel b) of treatment Values show the ratio of the IOD fromthe treatment groups to the control group (vehicle) IOD value

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Among the humoral factors in the gastric mucosa endogenousPGs play an important role in the protective effect by stimulatingthe secretion of mucus maintaining the local blood 1047298ow andincreasing the resistance of epithelial cells to potential damage bycytotoxins (Takeuchi 2010) Our results illustrate that the admin-istration of a non-selective COX inhibitor (indomethacin) comple-tely abolished the gastroprotective action of the FrAq This resulthighlights the relevance of PGs in the antiulcer action of thisfraction and agreement with the previously results of fraction inincrease in the amount of adherent mucus According to Takeuchi(2010) one of the mechanisms underlying the action of PGE2 isexactly the increase in mucus secretion that augments the pro-tective factors on the gastric mucosa

Based on the results regarding the gastroprotective effect of theFrAq against gastric injury induced by different ulcerogenic agentswe con1047297rmed that the action of this fraction was mediated by the

activation of defensive mucosa-protective factors such as increasedmucus production NO pathways and endogenous PGs However itis desirable for any new antiulcer drug that the preventive effect isalso accompanied by ulcer healing effects

Antiulcer drugs such as H2-receptor antagonists fail in promot-ing the healing of gastric ulcers when the regenerated mucosapresents with poor histological maturity and ulcer relapse is notprevented by cimetidine (Arakawa et al 2012)

This study also determined the effect of the FrAq on the healingof gastric ulcers induced by acetic acid in rats treated for 7 or 14consecutive days The acetic acid induced a deep necrotic lesioninvolving the entire mucosal depth and penetrating through themuscularis mucosae Ulcer healing is a dynamic process of 1047297llingmucosal defects with proliferating and migrating epithelial cells

and connective tissue which results in the reconstruction of the

mucosal architecture (Okabe and Amagase 2005) Chronic treat-ment with FrAq (25 mgkg) demonstrated a dramatic reduction inthe ulcerative lesion area by treatment for 7 days (80) which wasmore effective than 14 days of treatment (37) This resultdemonstrated the FrAq (25 mgkg) accelerates the healing within7 days of treatment and the healing effect remains after treatmentof 14 days when compared to control group or lansoprazole group

The injection of acetic acid into gastric mucosa induces thedevelopment of deep gastric ulceration and gastric mucosal damagedirectly associated with ECM degradation in which the zinc-dependent matrix metalloproteinases (MMPs) play a crucial role

In several animal studies of gastric ulcer attention has focusedon the role of MMPs mainly MMP-2 and MMP-9 (Li et al 2013)Wound formation and the following healing are dynamic processesof ECM remodeling that are mainly in1047298uenced by MMP-2 andMMP-9 (Gyenge et al 2013)

According to Yeh et al (2012) MMP-9 is the protease mostsigni1047297cantly involved in the degradation of the basement membraneand is associated with pathological states that include in1047298ammationand cancer Li et al (2013) described enhanced expression of MMP-9in the margin of the ulcer in patients with this disease Their studysuggested that the presence of MMP-9 at the margin of the ulcer maybe indicative of in1047298ammation and poor wound healing Ulcerogenicagents such as indomethacin exhibited 12-fold higher pro-MMP-9activity and ethanol exhibited 22-fold higher pro-MMP-9 activity inrat gastric tissues relative to untreated tissues (Mei et al 2013) Ourresults showed MMP-9 activity only after treatments with the vehicleand lansoprazole (7 days) These results determined that the presenceof MMP-9 activity at the gastric mucosa in groups treated with the

vehicle was related with the absence of healing in ulcers of the gastricmucosa Our results also show that the seemingly healing macro-scopic ulcer observed by treating animals with lansoprazole (7 days)may not represent a good wound healing because the presence of MMP-9 activity in gastric damage further demonstrates the persis-tence of the in1047298ammatory process at the gastric mucosa Our resultsshown that treatment of the acetic acid induced chronic ulcers withFrAq (25 mgkg) for 7 and 14 days inhibited the MMP-9 activity Ourresults could be strengthened by the study from Yeh et al (2012) inwhich extract from Terminalia catappa leaves exerts an antimetasticeffect by attenuating MMP-9 mediated in1047298ammation in hepatocellu-lar carcinoma In addition to our results obtained regarding MMP-9our results also reported high MMP-2 activity in gastric ulcers of thecontrol group treated with vehicle (7 and 14 days) and lower MMP-2

activity was observed after the treatment of animals with

Table 2

Effects of the aqueous fraction (FrAq) obtained from leaves of Terminalia catappa (25 mgkg) and lansoprazole (30 mgkg) administered orally for 14 consecutive days onselected toxicological parameters (nfrac145ndash6)

Treatment (po)

Vehicle Lansoprazole FrAq

Organ weights (g)Heart 3857048 3887032 3677013

Lung 4497064 5347143 4207040Kidney 5147063 4917018 4917007Liver 10107123 9997028 9707017Spleen 2697028 2837024 2677027

Biochemical parameterGlucose 136907700 136407580 151007850Creatinine 0567002 0567003 0537004Urea 38107112 39407535 39607423γ-GT 0967017 2027055 1247025AST 157117134 1812071751 1906071450ALT 44337251 48007504 4317311

Results are expressed as the means7SEM obtained from different groups The units for the biochemical parameters of serum are UL (ALT ndash alanine aminotransferaseAST ndash aspartate aminotransferase γndashGT ndash gamma-glutamyl transpeptidase) and mgdL (urea and creatinine)

Table 3

Identi1047297cation of the substances obtained in the aqueous fraction (FrAq) from theleaves of Terminalia catappa by FIA-ESI-IT-MSn

[MndashH] MSn ions Identi1047297cation

1083 781 [M-152-152-H] Punicalagin (1)

601 [M-152-152-180-H]

781 601 [M-180-H] Punicalin (2)

601 409 [M-191-H] Gallagic acid (3)

301 257 [M-44-H] Ellagic acid (4)

229 [M-44-28-H]

Adapted from Mininel et al (2014)

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lansoprazole and FrAq Recent studies have highlighted the functionof MMP-2 in the healing process of rat gastric ulcers induced by aceticacid (Gyenge et al 2013) but the function of MMP-2 is not wellunderstood

The antiulcerogenic actions demonstrated by FrAq could beattributed to the phenolic compounds present in this fractionMininel et al (2014) analyzed mass spectra in a full-scan of theextract and this fraction and showing similarity to each otherhighlighted by the precursor ions m z 1083 (punicalagin) m z 781(pulicalin) m z 601 (gallagic acid) and m z 301 attributed to ellagicacid In these studies the chromatogram from HPLC-PDA of the

aqueous fraction of Terminalia catappa con1047297rmed the majority of the

compounds punicalagin (anomers α and β) and punicalin (anomers αand β) are similar to the hydroalcoholic extract studied by Mininelet al (2014) Ellagic acid (EA) is one of the naturally occurringpolyphenols found in the FrAq from Terminalia catappa leavesNumerous pharmacological studies have suggested that EA providesmucosal protective action in the stomach against ethanol or ische-miandashreperfusion injury (Iino et al 2001 Iino et al 2002) and severalgastroprotective mechanisms are attributed to this compound Forexample Chatterjee et al (2012) showed that EA enhanced prosta-glandins when compared with the ulcerated untreated group

In our 1047297nal experimental series we evaluated the subacute

toxicological parameters from groups that received vehicle

500 1000 1500 2000

mz

0

5

10

15

20

25

30

35

40

45

50

55

60

6570

75

80

85

90

95

100108339

7814554115

10131395449

11329163927

13498731207

139719156207

171229

R e l a t i v e A b u n d a n c e

Fig 8 First-order mass spectrum of the aqueous fraction (FrAq) obtained from Terminalia catappa (FrAq) at the negative mode Range of ions with m z 100ndash2000 Da

T_130129165016 1-1000 RT060-096 AV70 NL271E2

F ITMS - c ESI Full ms2 108300cid3000 [29500-120000]

300 400 500 600 700 800 900 1000 1100 1200

mz

0

5

10

15

20

25

30

35

40

45

50

55

6065

70

75

80

85

90

95

100

R e l a t i v e A b u n d a n c

e

Fig 9 Mass spectrum of the second order (MS2) of the precursor ion m z 1083 from the aqueous fraction (FrAq) obtained in the negative mode of leaves from Terminalia

catappa Range of ions with mz 300-1200 Da

L Pinheiro Silva et al Journal of Ethnopharmacology 159 (2015) 285ndash 295 293

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lansoprazole or the FrAq over 14 days Important toxicity para-meters including changes in body weight mortality and weight of the vital organs were determined as shown in Table 2 Treatmentwith FrAq for 14 days with 25 mgkg did not signi1047297cantly alter thebody weight of animals compared to the vehicle control over the14-days treatment period (data not shown) and the averageweights of the major organs of the FrAq-treated subjects werecomparable to those of the control group Table 2 also shows thatFrAq did not affect several biochemical parameters Treatment didnot signi1047297cantly alter the body weight the weight of vital organsand biochemical parameters of serum However Mininel et al(2014) evaluated the mutagenicity of the hydroalcoholic extractfrom the leaves of Terminalia catappa with the Ames test andillustrated that this extract exhibited mutagenic activity in vitro athigh concentrations leading them to recommend caution whenusing this compound for medicinal purposes

This study also evaluated the activity of aqueous fraction (FrAq)against Helicobacter pylori and determination of minimal inhibitoryconcentration (MIC) of 1250 mgmL considered an important anti-microbial activity The literature do not show a consensus in relationto MIC values obtained for natural products Aligannis et al (2001)consider MICs with values equal to or less than 500 mgmL as potentinhibitors MICs between 600 and 1500 mgmL and MIC moderateinhibitors as above 1600 mgmL as weak inhibitors Webster et al(2008) established as another satisfactory MIC value equal to or lessthan 1000 mgmL In this sense the result reported in this investiga-tion demonstrated the inhibitory potential of Terminalia catappaagainst this important bacteria responsible to gastric ulcer

5 Conclusions

We determined that the FrAq from Terminalia catappa leaveshas an important anti-Helicobacter pylori activity excellent pre-ventive and curative effects on acute and chronically inducedgastric ulcers The mechanisms involved in the gastroprotectionare related to the NO pathway an increase in the mucus and theendogenous prostaglandins and this fraction was able to healulcers through the inhibition of MMP-9 and MMP-2 activities

Acknowledgments

This study was supported by the Biota-FAPESP project (Fundaccedilatildeo

de Amparo agrave Pesquisa do Estado de Satildeo Paulo) CNPq (Conselho

Nacional de Desenvolvimento Cientiacute1047297co e Tecnoloacutegico) and CAPES(Coordenaccedilatildeo de Aperfeiccediloamento Pessoal de Niacutevel Superior)

References

Aligannis N Kalpotzakis E Mitaku S Chinou IB 2001 Composition andantimicrobial activity of the essential oils of two Origanum species Journal of

Agricultural and Food Chemistry 49 4168ndash

4170Al-Jiboury H Kaunitz JD 2012 Gastroduodenal mucosal defense CurrentOpinion of Gastroenterology 28 594ndash601

Arakawa T Watanabe T Tanigawa T Tominaga K Fujiwara Y Morimoto K2012 Quality of ulcer healing in gastrointestinal tract its pathophysiology andclinical relevance World Journal of Gastroenterology 18 4811ndash4822

Arrieta J Benitez J Flores E Castillo C Navarrete A 2003 Puri1047297cation of gastroprotective triterpenoids from stem bark of Amphipterygium adstringensroles of prostaglandins sulfhydryls nitric oxide and capsaicin neurons PlantaMedica 69 905ndash909

Bradford MM 1976 A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye bindingAnalytical Biochemistry 72 248ndash254

Brzozowska I Strzalka M Drozdowicz D Konturek SJ Brzozowski T 2013Mechanisms of esophageal protection gastroprotection and ulcer healing bymelatonin Implications for the therapeutic use of melatonin in gastroesopha-geal re1047298ux disease (GERD) and peptic ulcer disease Current PharmaceuticalDesign httpdxdoi org1021741381612819666131119110258 (Epub ahead of print)

Brzozowski T Konturek PC Konturek SJ Pajdo R Schuppan D Drozdowicz DPtak A Pawlik M Nakamura T Hahn EG 2000 Involvement of cycloox-ygenase (COX)-2 products in acceleration of ulcer healing by gastrin andhepatocyte growth factor Journal of Physiology and Pharmacology 51 751ndash773

Brzozowski T Konturek PC Sliwowski Z Pajdo R Drozdowicz D Kwiecien SBurnat G Konturek SJ Pawlik WW 2006 Prostaglandincyclooxygenasepathway in ghrelin-induced gastroprotection against ischemia-reperfusioninjury Journal of Pharmacology and Experimental Therapeutics 319 477ndash487

Camargo MC Garciacutea A Riquelme A Otero W Camargo CA Garciacutea THCandia R Bruce MG Rabkin CS 2014 The problem of Helicobacter pyloriresistance to antibiotics a systematic review in Latin America Clinical andSystematic Reviews 109 485ndash495

Chatterjee A Chatterjee S Das S Saha A Chattopadhyay S Bandyopadhyay SK2012 Ellagic acid facilitates indomethacin-induced gastric ulcer healing via COX-2 up-regulation Acta Biochimica et Biophysica Sinica 44 565ndash576

Chen PS Li JH 2005 Chemopreventive effect of punicalagin a novel tannincomponent isolated from Terminalia catappa on H-ras-transformed NIH3T3cells Toxicology Letter 163 44ndash53

Chen PS Li JH Liu TY Lin TC 2000 Folk medicine Terminalia catappa and its

major tannin component punicalagin are effective against bleomycin-inducedgenotoxicity in Chinese hamster ovary cells Cancer Letter 152 115ndash122

Chen B Tuuli MG Longtine MS Shin JS Lawrence R Inder T Michael ND2012 Pomegranate juice and punicalagin attenuate oxidative stress andapoptosis in human placenta and in human placental trophoblasts American

Journal of Physiology and Endocrinology Metabolism 302 E1142ndashE1152CLSI Manual Clinical and Laboratory Standards Institute 2006 Methods for

dilution antimicrobial susceptibility tests for bacteria that grow aerobicallyapproved standards 6ordf ed Performance standards for antimicrobial suscept-ibility testing Sixteenth informational supplement M100-S16 (tab 2J) Clinicaland Laboratory Standards Institute Wayne PA

De Foneska A Kaunitz JD 2010 Gastroduodenal mucosal defense CurrentOpinion Gastroenterology 26 604ndash610

de Carvalho KI Bonamin F Dos Santos RC Peacuterico LL Beserra FP de Sousa DPFilho JM da Rocha LR Hiruma-Lima CA 2014 Geraniol-a 1047298avoring agentwith multifunctional effects in protecting the gastric and duodenal mucosaNaunyn Schmiedebergs Archieves of Pharmacology 387 355ndash365

Eom CS Park SM Myung SK Yun JM Ahn JS 2011 Use of acid-suppressive

drugs and risk of fracture a meta-analysis of observational studies AnnalsFamily Medicine 9 257ndash267Fan YM Xu LZ Gao J Wang Y Tang XH Zhao XN Zhang ZX 2004

Phytochemical and antiin1047298ammatory studies on Terminalia catappa Fitoterapia75 253ndash260

Fyhrquist P Mwasumbi L Haeggstroumlm CA Vuorela H Hiltunen R Vuorela P2002 Ethnobotanical and antimicrobial investigation on some species of Terminalia and Combretum (Combretaceae) growing in Tanzania Journal of Ethnopharmacology 79 169ndash177

Genestra M 2007 Oxyl radicals redox-sensitive signalling cascades and antiox-idants Cell Signalling 19 1807ndash1819

Germoseacuten-Robineau L 2014 Farmacopea Vegetal Carbentildea CICY editorial YucataacutenMexico p 360

Gyenge M Amagase K Kunimi S Matsuoka R Takeuchi K 2013 Roles of pro-angiogenic and anti-angiogenic factors as well as matrix metalloproteinases inhealing of NSAID-induced small intestinal ulcers in rats Life Science 93441ndash447

Iino T Nakahara K Miki W Kiso Y Ogawa Y Kato S Takeuchi K 2001 Lessdamaging effect of whisky in rat stomachs in comparison with pure ethanol

Digestion 64 (214ndash221) 2001

1 - α e β punicalagin

2 - α e βpunicalin

00 50 100 150 200 250 300 350 400

Retention Time [min]

0

1000000

2000000

I n t e n

s i t y [ micro V ]

T catappa FrAq - CH5

Fig 10 Chromatogram from HPLCndashPDA of the aqueous fraction of the leaves of Terminalia catappa Hydro Column 1047298ow 1 mLmin gradient method 5ndash60 MeOH40 min HPLCndashPDA (Jascos) 270 nm

L Pinheiro Silva et al Journal of Ethnopharmacology 159 (2015) 285ndash 295294

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between either the FrAq or lansoprazole and the vehicle-treatedgroups to evaluate subacute toxicity On the day after the last drugadministration the rats from each treatment group were killed andtheir blood was collected The blood samples were then centrifuged(3000 g for 10 min) and the serum obtained was frozen at 20 1Cuntil biochemical analysis Serum biochemical parameters includingglucose urea creatinine γ-glutamyl transpeptidase (γ-GT) aspartateaminotransferase (AST) and alanine aminotransferase (ALT) were

measured using an automated biochemical analyzer (SBA-200Companhia Equipadora de Laboratoacuterios Modernos Satildeo Paulo Brazil)

28 Minimum inhibitory concentration (MIC)

We used Helicobacter pylori ATCC 43504 using a microdilutiontechnique following by CLSI (2006) with modi1047297cations to determinethe minimal inhibitory concentration (MIC) values Helicobacter pyloriwas inoculated on Mueller-Hinton agar plates containing 5 sheepblood and incubated at 36 1C for 72 h in 10 CO2 atmosphereInoculates were prepared in the same medium at a density adjustedto a 20 McFarland turbidity standard The working suspension formicroorganism was diluted 110 and a 100 mL volume was added toeach well of microplates A 100 mL volume of Mueller-Hinton broth

supplemented with 10 fetal bovine serum was added each well of microplates The concentrations for each substance prepared in 2DMSO ranging from 05 to 1000 mgmL were obtained when a 100 mL volume of the fraction was transferred to the 1047297rst well of each rowand serial 2-fold dilutions were performed Amoxicillin was used asreference antimicrobial compound and the MICs were recorded afterincubation of the microplates at 36 1C for 72 h in a 10 CO2 atmo-sphere The MICs were recorded as the lowest concentration atwhich no growth was observed This record was facilitated byaddition of 20 mL of resazurin solution (100 mgmL) as revelator toeach of well and incubation until 2 h A pink color indicated bacterialgrowth and a blue color indicated a non-bacterial growth

29 Statistical analysis

The results were expressed as the mean7standard error of themean (SEM) of the parameters obtained Statistical comparisonswere done by one-way analysis of variance (ANOVA) followed byDunnetts or Tukeys post hoc test with the level of signi1047297cance setat n po005 nn po001 and nnn po0001

3 Results

In the present study we evaluated the protective effect of FrAqobtained from the leaves of Terminalia catappa against the gastricmucosa damage induced by absolute ethanol To establish a dosendashresponse pro1047297le for the antiulcer activity of FrAq we used varyingdoses of FrAq (125 25 and 100 mgkg body weight) to identify the

lowest dose that could elicit an optimal gastroprotective effect(Fig 1) The oral administration of ethanol rapidly penetrated thegastric mucosa and caused membrane damage erosion and hemor-rhagic ulcerations The pre-treatment of rats with FrAq signi1047297cantlyinhibited the formation of gastric lesions by 41 (25 mgkg) and 36(100 mgkg) compared to vehicle-treated control group ( po0001)A lower dose of FrAq (125 mgkg) evaluated in this study did notshow a gastroprotective effect against ethanol ( p4005) We alsoobserved no signi1047297cant differences in the gastroprotective effect of FrAq at doses of 25 or 100 mgkg ( p4005) Thus the subsequentexperiments with FrAq were carried out the lower dose of 25 mgkgadministered orally

This study evaluated the ability of FrAq to protect the gastricmucosa of rats from oxidative damage induced during an IR proce-

dure (Fig 2) We observed that oral treatment with FrAq (25 mgkg) as

well as lansoprazole (30 mgkg) signi1047297cantly decreased the extent of ulcerative lesions by 33 and 71 respectively when compared tostomachs from the vehicle-treated control group ( po001)

After con1047297rming the gastroprotective effect of FrAq againstlesions induced by ethanol and IR the next step was to evaluate

the antiulcer mechanisms of action for this fraction We studied theeffect of FrAq on gastric juice parameters to evaluate their possibleanti-secretory action by the pylorus ligation procedure (Table 1) Weobserved that the administration of FrAq by an intraduodenal ororal route was not able to change the Hthorn concentration of gastric

juice when compared to vehicle-treated animals ( p4005) Thisresult excluded the possibility of the antisecretory effect of FrAqHowever this assay also revealed that the oral treatment with FrAqwas able to signi1047297cantly increase gastric volume (42) This effectcould represent that FrAq was able to induce an increase in theamount of adherent gastric mucus Next we evaluated the effects of the oral administration of FrAq on this gastric mucus in pyloricligature rats (Fig 3) We observed that oral treatment with FrAqcaused an increase (43) in the amount of adherent gastric mucus

compared to the vehicle-treated group ( po005) con1047297rming our

0

100

200

300

400

Carbenoxolone100 mgkg

Vehicle 125 25 100

FrAq (mgkg)

U

L A ( m m

2 )

ns

Fig 1 Effect of pretreatment with the aqueous fraction (FrAq) obtained from theleaves of Terminalia catappa on ethanol-induced gastric ulcers in rats The animalsorally received saline solution (vehicle) carbenoxolone or FrAq The results areexpressed as the mean7SEM (nfrac147ndash14) and statistical signi1047297cance was deter-mined by one-way analysis of variance (ANOVA) followed by Dunnetts test(nn po001 nnn po0001 and ns ndash no signi1047297cant differences)

0

20

40

60

80

Vehicle Lansoprazole

30 mgkg

FrAq

25 mgkg

U L A ( m m sup2 )

Fig 2 Effect of pretreatment with the aqueous fraction (FrAq) obtained from theleaves of Terminalia catappa on ischemiandashreperfusion induced gastric ulcers in ratsThe animals orally received saline solution (vehicle) lansoprazole or FrAq Theresults are expressed as the mean7SEM (nfrac147ndash9) and statistical signi1047297cance wasdetermined by one-way analysis of variance (ANOVA) followed by Dunnetts testwith the level of signi1047297cance set at nn po001 and nnn po0001

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hypothesis As expected carbenoxolone (used as positive control)enhanced the mucus production and there was no statisticaldifference between the groups treated with carbenoxolone andFrAq ( p4005)

In addition to mucus as defense mechanism elicited by FrAqother factors involved with the strengthening of gastric mucosa werealso investigated such as NO (nitric oxide) sulfhydryl (SH) com-pounds and PGs which are factors that maintain mucosal integrityOur results (Fig 4a) shown that the pretreatment of animals withNEM (sulfhydryl inhibitor) did not change the gastroprotective effect

of FrAq ( p4005) compared to the vehicle-pretreated FrAq groupTherefore this result excluded the involvement of sulfhydryl groupsin the gastroprotective effect of FrAq However the group of animalspretreated with L -NAME (a NO-synthase inhibitor) and treated withFrAq display a signi1047297cant increase in gastric lesions (Fig 4b) A similarpicture emerged when the effect of PGs was studied Fig 4c showsthat administration of indomethacin markedly increased the size of the ulcerative lesion and completely abolished the previouslyobserved protective effect of FrAq Our results illustrated that besidethe increase in gastric mucus barrier induced by FrAq the gastro-protective effect of this fraction also involved the action of NO andendogenous PGs levels After determining the factors involved withFrAq action the possible healing effect of FrAq was evaluated withthe acetic acid method (the most similar to human gastric ulcer) Oral

treatment with FrAq during 7 consecutive days (Fig 5a) and also

14 days (Fig 5b) demonstrated that this fraction was able toaccelerate the healing of chronic gastric ulcers in rats At the doseof 25 mgkg FrAq 7 and 14 days of treatment signi1047297cantly decreasedthe area of lesion in 80 and 37 respectively when compared to thecontrol group treated with vehicle ( po001) In the group treatedwith lansoprazole at 30 mgkg the healing effect was also observedwith the decrease of gastric lesion at 83 (7 days) and 64 (14 days)To determine in better detail the healing process of FrAq we also

analyzed the stomach by zymograph Fig 6 presents the activities of MMP-2 and MMP-9 on the gastric mucosa after treatment withvehicle lansoprazole and FrAq for 7 or 14 days Our results show thatMMP-9 activity was present only after treatment during 7 days instomachs from the vehicle and lansoprazole-group We also observedthat this MMP-9 activity was absence for all treatments during 14days In contrast MMP-2 activity was present in all treated groupsincluding the sham group (without gastric lesion) The treatment of animals that exhibited major MMP-2 activity after 7 and 14 days wascompared to the animals treated with vehicle Lower activity of MMP-2 was observed after treatment of the animals with lansopra-zole and FrAq for 7 days (Fig 7a 40 and 26 respectively in rela-tion to the vehicle) and 14 days (Fig 7b 54 and 73 respectivelyin relation to the vehicle)

Other important data obtained from this test based on thetreatment of animals with FrAq for 7 and 14 consecutive days wasthe absence of death in these groups and also absence bodyweight changes (data not shown) and some organ weights andbiochemical parameters of serum (Table 2) These results there-fore ensure that based on the parameters used in this study theFrAq at doses of 25 mgkg does not cause signi1047297cant toxic effectsafter 7 or 14 consecutive days of treatment

This study also evaluated the activity of FrAq against Helico-bacter pylori and presented minimal inhibitory concentration(MIC) of 1250 mgmL considered a accentuated activity and thepositive control (amoxicillin) presented MIC of 150 mgmL

The analysis of mass spectra of FrAq (Fig 9) showed the samecompounds identi1047297ed in the hydroalcoholic extract studied byMininel et al (2014) as shown in Table 3 The spectra (Fig 8) showthe ions with mz 1083 (punicalagin) mz 601 attributed togallagic acid and at mz 301 attributed to ellagic acid illustratingthe similarity with the hydroalcoholic extract (Mininel et al2014) The fragmentation of the second order (MS2) ion at m z 1083 (punicalagin) leads to ions at m z 781 (punicalin) and the ionwith m z 601 (gallagic acid) as shown in Fig 9 The chromato-graphic pro1047297le of the water fraction is also shown in Fig 10 In thechromatogram two peaks are highlighted eluting at tr 172 minand tr 233 min indicating the presence of the major compoundpunicalagin (α and β anomers) Two peaks with tr 280 min and tr301 min (region 2) were observed corresponding to the com-pound punicalin (α and β anomers) derived from punicalagin

4 Discussion

The development of gastric ulcers is a complex and multi-factorial process including bacterial infections the increase of acidsecretion generation of reactive oxygen species (ROS) inhibitionof the endogenous PGs and the degradation of the extracellularmatrix (ECM) (Laine et al 2008 Mei et al 2013) Research duringthe last decade has offered new insights in the preventativetherapy and the healing of gastric ulcers and the synergisticef 1047297ciency of a multi-target approach based on individual mechan-isms of action could be the new perspective for treatment of thisdisease (Wagner 2011 de Carvalho et al 2014)

Terminalia catappa is a medicinal plant widely distributed intropical and subtropical regions and it has been previously reported

that this species exhibits a variety of biological effects However the

Table 1

Effects of the aqueous fraction (FrAq) from the leaves of Terminalia catappa (25 mgkg)administered through the intraduodenal (id) or oral (po) route on the biochemicalparameters of gastric juice obtained from pylorus-ligature rats (nfrac146ndash8)

Treatment Route Dose (mgkg) Gastric juice (mL) [Hthorn] (μEqmL4 h)

Vehicle id ndash 7027225 6997184Cimetidine 100 4297119nn 2777130nn

FrAq 25 8027218 7347065

Vehicle po ndash 3747101 5257151Cimetidine 100 2637067 1947087nn

FrAq 25 5307167 n 6337043

Data represents the means7SEM The asterisks denote the signi1047297cance levelswhen compared with the control group

n po005nn po001 by one-way ANOVA followed by Dunnetts test

0

1000

2000

3000

Vehicle Carbenoxolone

100 mgkg

FrAq

25 mgkg

A l c i a n b l u e

( m g g w e t t i s s u e )

ns

Fig 3 Effects of the aqueous fraction (FrAq) obtained from the leaves of Terminalia

catappa (25 mgkg) on the production of adherent gastric mucus (measured as the

amount of bound Alcian blue) in pylorus-ligated rats relative to the control values(nfrac146) The results are the mean7SEM Statistical signi1047297cance was determined byANOVA followed by Dunnetts t test (n po005 nnn po001 and ns ndash no signi1047297cantdifferences)

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underlying mechanisms of the gastroprotective and healing effectsof the aqueous fraction (FrAq) obtained from the leaves have yet tobe evaluated

In the present study FrAq showed a marked gastroprotectiveeffect evidenced by the dramatic inhibition of the gastric damageproduced by ethanol Ethanol induced erosion ulcerative lesionsand petechial bleeding in the mucosa of the stomach in humansand an ethanol-induced gastric ulcer model is commonly used tostudy both the pathogenesis and new therapeutics for the treat-ment of gastric ulcer disease (Oyagi et al 2010)

Our results have shown the effective gastroprotective effect of theaqueous fraction obtained from the leaves of Terminalia catappa

A previous study by Nunes et al (2012) evaluated the ethanolic

extract of bark from this species against gastric ulcers induced byethanol Aside from using a different part of plant (the bark insteadleaves) this study has demonstrated gastroprotection at a dose 10times higher than our study (250 mgkg vs 25 mgkg) Our studiesalso highlighted the use of the renewable part of this plant (leaves)that would enable the management of this plant for the futureproduction of a phytotherapeutic against gastric ulcer

Gastric ulcer induced by ethanol is commonly associated withreduced mucosal blood 1047298ow and ischemia that causes deleteriouseffects on the gastric mucosa mainly in the aging gastric mucosa(Tarnawski et al 2014) Ischemia weakens the gastric mucosal barrierand increases the acid back-diffusion predisposing the gastric mucosa

to damage (Rao and Vijayakumar 2007) The restoration of blood 1047298ow

Fig 4 The ulcerative lesion area (ULA-mm2) for gastric ulcers induced by ethanol in rats pretreated with L -NAME (panel a) with vehicle or together with carbenoxolone(100 mgkg) or the aqueous fraction (FrAq) obtained from leaves of Terminalia catappa (25 mgkg) pretreated with N -ethylmaleimide (panel b) with vehicle or together withcarbenoxolone (100 mgkg) and FrAq (25 mgkg) or pretreated with INDO ndash indomethacin (panel c) with vehicle or together with carbenoxolone (100 mgkg) or FrAq(25 mgkg) The results are reported as the mean7SEM Statistical signi1047297cance was determined by ANOVA followed by Dunnetts test n po005 nn po001 nnn po0001compared to the corresponding vehicle group o005 po001 compared to the corresponding NEMthornvehicle L -Namethornvehicle or INDOthornvehicle NS ndash no signi1047297cantdifferences

0

2

4

6

8

10

Vehicle Lansoprazole

30 mgkg

FrAq

25 mgkg

U L A ( m m sup2 )

0

5

10

15

20

Vehicle Lansoprazole

30 mgkg

FrAq

25 mgkg

U L A ( m m sup2 )

Fig 5 Effect of the oral administration of the aqueous fraction (FrAq) obtained from the leaves of Terminalia catappa (25 mgkg) on the healing of ulcers produced by theintroduction of acetic acid solution into the stomachs of rats The ulceration was scored on the 7th day (panel a) and 14th day (panel b) after surgery The results are reported

as the mean7

SEM Statistical signi1047297

cance was determined by ANOVA followed by Dunnetts test

nn

po

001

nnn

po

0001 compared to the corresponding vehicle group

L Pinheiro Silva et al Journal of Ethnopharmacology 159 (2015) 285ndash 295290

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(reperfusion) after a period of ischemia initiates a cascade of changesincluding the release of local ROS in1047298ammatory responses tissuedamage by fragmenting cellular DNA and an increase in the adhesionof neutrophils to endothelial cells a phenomenon known as ische-miandashreperfusion (IR) (Smith et al 1996 De Foneska and Kaunitz2010)

The current study shows that oral treatment with FrAq (25 mgkg)signi1047297cantly decreased the extent of ulcerative lesions in the gastrictissue submitted to IR injury compared to stomachs from the vehicle-treated control group Based on the physiopathology of the IR experimental model our results are in concordance with Chen et al

(2012) and Wang et al (2013) in which they observed that punicalagin

and punicalin both polyphenols presents in Terminalia catappa wereable to attenuate oxidative stress and hypoxia-induced apoptosis

These results therefore con1047297rm the gastroprotective effect of this fraction and encourage the continuation of the studies of theeffects of FrAq treatment and the characterization of its mechan-isms of action

Studies already performed with the hydroalcoholic extract of Terminalia catappa and other species of this genus such as

Terminalia chebula and Terminalia fagifolia have demonstrated theantisecretory effect of these species (Kumar et al 2014 Mishraet al 2013 Nunes et al 2014) Unlike these studies our resultshave shown that the oral and intraduodenal treatment with FrAq(25 mgkg) does not promote the antiulcerogenic effect in anantisecretory manner However our result also illustrated thatoral treatment with FrAq was able to signi1047297cantly increase thegastric volume in relation to the control group treated with vehicle(530 mL and 374 mL respectively) Our hypothesis was that theFrAq elicits the increase of gastric juice because of the amount of adherent gastric mucus

The success of the pharmacological treatment for gastric ulcersrelies on the augmentation of the protective factors of the gastricmucosa such as the mucus barrier (Al-Jiboury and Kaunitz 2012Brzozowska et al 2013) The adherent gastric mucus is the 1047297rst lineof defense against acid and serves as a barrier against self-digestionthe functional integrity of the gastric mucosa depends on thisbarrier (Wallace 2008) Our study reveals a signi1047297cant increase inthe amount of adherent mucus in the animals treated with FrAqthus justifying the increase in gastric volume by treatment with thisfraction This study involving the FrAq is highly signi1047297cant in theongoing search for an antiulcerogenic therapy as the prolonged useof antisecretory drugs such as proton-pump inhibitors and H2

blockers can provoke serious side effects (Ozdil et al 2013 Eomet al 2011)

In addition to the increased production of the mucus barrierseveral studies have demonstrated a complex defense system invol-ving the gastric mucosa which include the regulation of gastricmucosal blood 1047298ow the formation of sulfhydryl compounds NO andendogenous PGs (Brzozowski et al 2000 Pawlik et al 2001)

NO is considered to be one of the most important defensiveendogenous agents in the gastric mucosa and plays a physiologicalrole in the homeostasis of the gastrointestinal tract (Brzozowskiet al 2006) Together with the endogenous prostaglandins NO alsopreserves the gastric mucosa integrity and the inhibition of NOrelease can result in disturbances in the gastrointestinal motilityblood 1047298ow and acid secretion (Brzozowski et al 2006 Khalifaet al 2002 Wallace and Ma 2001) In addition to the increase ingastric mucus our results show that the restoration of the endo-genous NO represents an additional mechanism responsible for thegastroprotective effects of FrAq as a NO-synthase inhibitor elicits asigni1047297cant increase in gastric lesion in stomachs previously treatedwith the fraction The gastroprotective effect of the FrAq through

endogenous NO agrees with the protective effect of this fractionagainst IR in rats by inhibiting the excessive formation of ROS

Our study is also in concordance with those performed byPandya et al (2013) in which they evaluated the status of theethanolic extract of Terminalia catappa (at dose of 200 mgkg) andillustrated an augmentation of the antioxidant defense systemagainst Ehrlich carcinoma Kinoshita et al (2007) also describedthe antioxidant and hepatoprotective actions from the aqueousextract of the leaves of Terminalia catappa As well as NO theincrease in gastric SH content is important for the limiting theproduction of oxygen-derived free radicals (Genestra 2007)Our study showed that the pre-treatment of animals with NEM(a sulfhydryl inhibitor) did not change the gastroprotective effectof the FrAq ( p4005) Therefore our results exclude the involve-

ment of SH content in the gastroprotective effect of FrAq

Fig 6 Representative zymography results of the gastric ulcers from the differentgroups illustrating MMP-2 and MMP-9 activity Treatment for 7 days ndash vehicle (line 1)lansoprazole (line 2) aqueous fraction (FrAq) obtained from leaves of Terminalia

catappa at dose of 25 mgkg (line 3) and sham (line 4) Treatment for 14 days ndash vehicle(line 5) lansoprazole (line 6) aqueous fraction (FrAq) obtained from leaves of Terminalia catappa at dose of 25 mgkg (line 7) and sham (line 8) Gelatinolytic bandsof 92 72 64 and 57 kDa were observed which correspond to active-MMP-9pro-enzyme intermediate and active-MMP-2 respectively

Fig 7 Effect of treatment with the vehicle lansoprazole (30 mgkg) or the aqueousfraction (FrAq) obtained from Terminalia catappa (FrAq) on the gelatinolytic activityof MMP-9 (black column) and MMP-2 (white column) in the gastric mucosa after 7

(panel a) and 14 days (panel b) of treatment Values show the ratio of the IOD fromthe treatment groups to the control group (vehicle) IOD value

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Among the humoral factors in the gastric mucosa endogenousPGs play an important role in the protective effect by stimulatingthe secretion of mucus maintaining the local blood 1047298ow andincreasing the resistance of epithelial cells to potential damage bycytotoxins (Takeuchi 2010) Our results illustrate that the admin-istration of a non-selective COX inhibitor (indomethacin) comple-tely abolished the gastroprotective action of the FrAq This resulthighlights the relevance of PGs in the antiulcer action of thisfraction and agreement with the previously results of fraction inincrease in the amount of adherent mucus According to Takeuchi(2010) one of the mechanisms underlying the action of PGE2 isexactly the increase in mucus secretion that augments the pro-tective factors on the gastric mucosa

Based on the results regarding the gastroprotective effect of theFrAq against gastric injury induced by different ulcerogenic agentswe con1047297rmed that the action of this fraction was mediated by the

activation of defensive mucosa-protective factors such as increasedmucus production NO pathways and endogenous PGs However itis desirable for any new antiulcer drug that the preventive effect isalso accompanied by ulcer healing effects

Antiulcer drugs such as H2-receptor antagonists fail in promot-ing the healing of gastric ulcers when the regenerated mucosapresents with poor histological maturity and ulcer relapse is notprevented by cimetidine (Arakawa et al 2012)

This study also determined the effect of the FrAq on the healingof gastric ulcers induced by acetic acid in rats treated for 7 or 14consecutive days The acetic acid induced a deep necrotic lesioninvolving the entire mucosal depth and penetrating through themuscularis mucosae Ulcer healing is a dynamic process of 1047297llingmucosal defects with proliferating and migrating epithelial cells

and connective tissue which results in the reconstruction of the

mucosal architecture (Okabe and Amagase 2005) Chronic treat-ment with FrAq (25 mgkg) demonstrated a dramatic reduction inthe ulcerative lesion area by treatment for 7 days (80) which wasmore effective than 14 days of treatment (37) This resultdemonstrated the FrAq (25 mgkg) accelerates the healing within7 days of treatment and the healing effect remains after treatmentof 14 days when compared to control group or lansoprazole group

The injection of acetic acid into gastric mucosa induces thedevelopment of deep gastric ulceration and gastric mucosal damagedirectly associated with ECM degradation in which the zinc-dependent matrix metalloproteinases (MMPs) play a crucial role

In several animal studies of gastric ulcer attention has focusedon the role of MMPs mainly MMP-2 and MMP-9 (Li et al 2013)Wound formation and the following healing are dynamic processesof ECM remodeling that are mainly in1047298uenced by MMP-2 andMMP-9 (Gyenge et al 2013)

According to Yeh et al (2012) MMP-9 is the protease mostsigni1047297cantly involved in the degradation of the basement membraneand is associated with pathological states that include in1047298ammationand cancer Li et al (2013) described enhanced expression of MMP-9in the margin of the ulcer in patients with this disease Their studysuggested that the presence of MMP-9 at the margin of the ulcer maybe indicative of in1047298ammation and poor wound healing Ulcerogenicagents such as indomethacin exhibited 12-fold higher pro-MMP-9activity and ethanol exhibited 22-fold higher pro-MMP-9 activity inrat gastric tissues relative to untreated tissues (Mei et al 2013) Ourresults showed MMP-9 activity only after treatments with the vehicleand lansoprazole (7 days) These results determined that the presenceof MMP-9 activity at the gastric mucosa in groups treated with the

vehicle was related with the absence of healing in ulcers of the gastricmucosa Our results also show that the seemingly healing macro-scopic ulcer observed by treating animals with lansoprazole (7 days)may not represent a good wound healing because the presence of MMP-9 activity in gastric damage further demonstrates the persis-tence of the in1047298ammatory process at the gastric mucosa Our resultsshown that treatment of the acetic acid induced chronic ulcers withFrAq (25 mgkg) for 7 and 14 days inhibited the MMP-9 activity Ourresults could be strengthened by the study from Yeh et al (2012) inwhich extract from Terminalia catappa leaves exerts an antimetasticeffect by attenuating MMP-9 mediated in1047298ammation in hepatocellu-lar carcinoma In addition to our results obtained regarding MMP-9our results also reported high MMP-2 activity in gastric ulcers of thecontrol group treated with vehicle (7 and 14 days) and lower MMP-2

activity was observed after the treatment of animals with

Table 2

Effects of the aqueous fraction (FrAq) obtained from leaves of Terminalia catappa (25 mgkg) and lansoprazole (30 mgkg) administered orally for 14 consecutive days onselected toxicological parameters (nfrac145ndash6)

Treatment (po)

Vehicle Lansoprazole FrAq

Organ weights (g)Heart 3857048 3887032 3677013

Lung 4497064 5347143 4207040Kidney 5147063 4917018 4917007Liver 10107123 9997028 9707017Spleen 2697028 2837024 2677027

Biochemical parameterGlucose 136907700 136407580 151007850Creatinine 0567002 0567003 0537004Urea 38107112 39407535 39607423γ-GT 0967017 2027055 1247025AST 157117134 1812071751 1906071450ALT 44337251 48007504 4317311

Results are expressed as the means7SEM obtained from different groups The units for the biochemical parameters of serum are UL (ALT ndash alanine aminotransferaseAST ndash aspartate aminotransferase γndashGT ndash gamma-glutamyl transpeptidase) and mgdL (urea and creatinine)

Table 3

Identi1047297cation of the substances obtained in the aqueous fraction (FrAq) from theleaves of Terminalia catappa by FIA-ESI-IT-MSn

[MndashH] MSn ions Identi1047297cation

1083 781 [M-152-152-H] Punicalagin (1)

601 [M-152-152-180-H]

781 601 [M-180-H] Punicalin (2)

601 409 [M-191-H] Gallagic acid (3)

301 257 [M-44-H] Ellagic acid (4)

229 [M-44-28-H]

Adapted from Mininel et al (2014)

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lansoprazole and FrAq Recent studies have highlighted the functionof MMP-2 in the healing process of rat gastric ulcers induced by aceticacid (Gyenge et al 2013) but the function of MMP-2 is not wellunderstood

The antiulcerogenic actions demonstrated by FrAq could beattributed to the phenolic compounds present in this fractionMininel et al (2014) analyzed mass spectra in a full-scan of theextract and this fraction and showing similarity to each otherhighlighted by the precursor ions m z 1083 (punicalagin) m z 781(pulicalin) m z 601 (gallagic acid) and m z 301 attributed to ellagicacid In these studies the chromatogram from HPLC-PDA of the

aqueous fraction of Terminalia catappa con1047297rmed the majority of the

compounds punicalagin (anomers α and β) and punicalin (anomers αand β) are similar to the hydroalcoholic extract studied by Mininelet al (2014) Ellagic acid (EA) is one of the naturally occurringpolyphenols found in the FrAq from Terminalia catappa leavesNumerous pharmacological studies have suggested that EA providesmucosal protective action in the stomach against ethanol or ische-miandashreperfusion injury (Iino et al 2001 Iino et al 2002) and severalgastroprotective mechanisms are attributed to this compound Forexample Chatterjee et al (2012) showed that EA enhanced prosta-glandins when compared with the ulcerated untreated group

In our 1047297nal experimental series we evaluated the subacute

toxicological parameters from groups that received vehicle

500 1000 1500 2000

mz

0

5

10

15

20

25

30

35

40

45

50

55

60

6570

75

80

85

90

95

100108339

7814554115

10131395449

11329163927

13498731207

139719156207

171229

R e l a t i v e A b u n d a n c e

Fig 8 First-order mass spectrum of the aqueous fraction (FrAq) obtained from Terminalia catappa (FrAq) at the negative mode Range of ions with m z 100ndash2000 Da

T_130129165016 1-1000 RT060-096 AV70 NL271E2

F ITMS - c ESI Full ms2 108300cid3000 [29500-120000]

300 400 500 600 700 800 900 1000 1100 1200

mz

0

5

10

15

20

25

30

35

40

45

50

55

6065

70

75

80

85

90

95

100

R e l a t i v e A b u n d a n c

e

Fig 9 Mass spectrum of the second order (MS2) of the precursor ion m z 1083 from the aqueous fraction (FrAq) obtained in the negative mode of leaves from Terminalia

catappa Range of ions with mz 300-1200 Da

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lansoprazole or the FrAq over 14 days Important toxicity para-meters including changes in body weight mortality and weight of the vital organs were determined as shown in Table 2 Treatmentwith FrAq for 14 days with 25 mgkg did not signi1047297cantly alter thebody weight of animals compared to the vehicle control over the14-days treatment period (data not shown) and the averageweights of the major organs of the FrAq-treated subjects werecomparable to those of the control group Table 2 also shows thatFrAq did not affect several biochemical parameters Treatment didnot signi1047297cantly alter the body weight the weight of vital organsand biochemical parameters of serum However Mininel et al(2014) evaluated the mutagenicity of the hydroalcoholic extractfrom the leaves of Terminalia catappa with the Ames test andillustrated that this extract exhibited mutagenic activity in vitro athigh concentrations leading them to recommend caution whenusing this compound for medicinal purposes

This study also evaluated the activity of aqueous fraction (FrAq)against Helicobacter pylori and determination of minimal inhibitoryconcentration (MIC) of 1250 mgmL considered an important anti-microbial activity The literature do not show a consensus in relationto MIC values obtained for natural products Aligannis et al (2001)consider MICs with values equal to or less than 500 mgmL as potentinhibitors MICs between 600 and 1500 mgmL and MIC moderateinhibitors as above 1600 mgmL as weak inhibitors Webster et al(2008) established as another satisfactory MIC value equal to or lessthan 1000 mgmL In this sense the result reported in this investiga-tion demonstrated the inhibitory potential of Terminalia catappaagainst this important bacteria responsible to gastric ulcer

5 Conclusions

We determined that the FrAq from Terminalia catappa leaveshas an important anti-Helicobacter pylori activity excellent pre-ventive and curative effects on acute and chronically inducedgastric ulcers The mechanisms involved in the gastroprotectionare related to the NO pathway an increase in the mucus and theendogenous prostaglandins and this fraction was able to healulcers through the inhibition of MMP-9 and MMP-2 activities

Acknowledgments

This study was supported by the Biota-FAPESP project (Fundaccedilatildeo

de Amparo agrave Pesquisa do Estado de Satildeo Paulo) CNPq (Conselho

Nacional de Desenvolvimento Cientiacute1047297co e Tecnoloacutegico) and CAPES(Coordenaccedilatildeo de Aperfeiccediloamento Pessoal de Niacutevel Superior)

References

Aligannis N Kalpotzakis E Mitaku S Chinou IB 2001 Composition andantimicrobial activity of the essential oils of two Origanum species Journal of

Agricultural and Food Chemistry 49 4168ndash

4170Al-Jiboury H Kaunitz JD 2012 Gastroduodenal mucosal defense CurrentOpinion of Gastroenterology 28 594ndash601

Arakawa T Watanabe T Tanigawa T Tominaga K Fujiwara Y Morimoto K2012 Quality of ulcer healing in gastrointestinal tract its pathophysiology andclinical relevance World Journal of Gastroenterology 18 4811ndash4822

Arrieta J Benitez J Flores E Castillo C Navarrete A 2003 Puri1047297cation of gastroprotective triterpenoids from stem bark of Amphipterygium adstringensroles of prostaglandins sulfhydryls nitric oxide and capsaicin neurons PlantaMedica 69 905ndash909

Bradford MM 1976 A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye bindingAnalytical Biochemistry 72 248ndash254

Brzozowska I Strzalka M Drozdowicz D Konturek SJ Brzozowski T 2013Mechanisms of esophageal protection gastroprotection and ulcer healing bymelatonin Implications for the therapeutic use of melatonin in gastroesopha-geal re1047298ux disease (GERD) and peptic ulcer disease Current PharmaceuticalDesign httpdxdoi org1021741381612819666131119110258 (Epub ahead of print)

Brzozowski T Konturek PC Konturek SJ Pajdo R Schuppan D Drozdowicz DPtak A Pawlik M Nakamura T Hahn EG 2000 Involvement of cycloox-ygenase (COX)-2 products in acceleration of ulcer healing by gastrin andhepatocyte growth factor Journal of Physiology and Pharmacology 51 751ndash773

Brzozowski T Konturek PC Sliwowski Z Pajdo R Drozdowicz D Kwiecien SBurnat G Konturek SJ Pawlik WW 2006 Prostaglandincyclooxygenasepathway in ghrelin-induced gastroprotection against ischemia-reperfusioninjury Journal of Pharmacology and Experimental Therapeutics 319 477ndash487

Camargo MC Garciacutea A Riquelme A Otero W Camargo CA Garciacutea THCandia R Bruce MG Rabkin CS 2014 The problem of Helicobacter pyloriresistance to antibiotics a systematic review in Latin America Clinical andSystematic Reviews 109 485ndash495

Chatterjee A Chatterjee S Das S Saha A Chattopadhyay S Bandyopadhyay SK2012 Ellagic acid facilitates indomethacin-induced gastric ulcer healing via COX-2 up-regulation Acta Biochimica et Biophysica Sinica 44 565ndash576

Chen PS Li JH 2005 Chemopreventive effect of punicalagin a novel tannincomponent isolated from Terminalia catappa on H-ras-transformed NIH3T3cells Toxicology Letter 163 44ndash53

Chen PS Li JH Liu TY Lin TC 2000 Folk medicine Terminalia catappa and its

major tannin component punicalagin are effective against bleomycin-inducedgenotoxicity in Chinese hamster ovary cells Cancer Letter 152 115ndash122

Chen B Tuuli MG Longtine MS Shin JS Lawrence R Inder T Michael ND2012 Pomegranate juice and punicalagin attenuate oxidative stress andapoptosis in human placenta and in human placental trophoblasts American

Journal of Physiology and Endocrinology Metabolism 302 E1142ndashE1152CLSI Manual Clinical and Laboratory Standards Institute 2006 Methods for

dilution antimicrobial susceptibility tests for bacteria that grow aerobicallyapproved standards 6ordf ed Performance standards for antimicrobial suscept-ibility testing Sixteenth informational supplement M100-S16 (tab 2J) Clinicaland Laboratory Standards Institute Wayne PA

De Foneska A Kaunitz JD 2010 Gastroduodenal mucosal defense CurrentOpinion Gastroenterology 26 604ndash610

de Carvalho KI Bonamin F Dos Santos RC Peacuterico LL Beserra FP de Sousa DPFilho JM da Rocha LR Hiruma-Lima CA 2014 Geraniol-a 1047298avoring agentwith multifunctional effects in protecting the gastric and duodenal mucosaNaunyn Schmiedebergs Archieves of Pharmacology 387 355ndash365

Eom CS Park SM Myung SK Yun JM Ahn JS 2011 Use of acid-suppressive

drugs and risk of fracture a meta-analysis of observational studies AnnalsFamily Medicine 9 257ndash267Fan YM Xu LZ Gao J Wang Y Tang XH Zhao XN Zhang ZX 2004

Phytochemical and antiin1047298ammatory studies on Terminalia catappa Fitoterapia75 253ndash260

Fyhrquist P Mwasumbi L Haeggstroumlm CA Vuorela H Hiltunen R Vuorela P2002 Ethnobotanical and antimicrobial investigation on some species of Terminalia and Combretum (Combretaceae) growing in Tanzania Journal of Ethnopharmacology 79 169ndash177

Genestra M 2007 Oxyl radicals redox-sensitive signalling cascades and antiox-idants Cell Signalling 19 1807ndash1819

Germoseacuten-Robineau L 2014 Farmacopea Vegetal Carbentildea CICY editorial YucataacutenMexico p 360

Gyenge M Amagase K Kunimi S Matsuoka R Takeuchi K 2013 Roles of pro-angiogenic and anti-angiogenic factors as well as matrix metalloproteinases inhealing of NSAID-induced small intestinal ulcers in rats Life Science 93441ndash447

Iino T Nakahara K Miki W Kiso Y Ogawa Y Kato S Takeuchi K 2001 Lessdamaging effect of whisky in rat stomachs in comparison with pure ethanol

Digestion 64 (214ndash221) 2001

1 - α e β punicalagin

2 - α e βpunicalin

00 50 100 150 200 250 300 350 400

Retention Time [min]

0

1000000

2000000

I n t e n

s i t y [ micro V ]

T catappa FrAq - CH5

Fig 10 Chromatogram from HPLCndashPDA of the aqueous fraction of the leaves of Terminalia catappa Hydro Column 1047298ow 1 mLmin gradient method 5ndash60 MeOH40 min HPLCndashPDA (Jascos) 270 nm

L Pinheiro Silva et al Journal of Ethnopharmacology 159 (2015) 285ndash 295294

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hypothesis As expected carbenoxolone (used as positive control)enhanced the mucus production and there was no statisticaldifference between the groups treated with carbenoxolone andFrAq ( p4005)

In addition to mucus as defense mechanism elicited by FrAqother factors involved with the strengthening of gastric mucosa werealso investigated such as NO (nitric oxide) sulfhydryl (SH) com-pounds and PGs which are factors that maintain mucosal integrityOur results (Fig 4a) shown that the pretreatment of animals withNEM (sulfhydryl inhibitor) did not change the gastroprotective effect

of FrAq ( p4005) compared to the vehicle-pretreated FrAq groupTherefore this result excluded the involvement of sulfhydryl groupsin the gastroprotective effect of FrAq However the group of animalspretreated with L -NAME (a NO-synthase inhibitor) and treated withFrAq display a signi1047297cant increase in gastric lesions (Fig 4b) A similarpicture emerged when the effect of PGs was studied Fig 4c showsthat administration of indomethacin markedly increased the size of the ulcerative lesion and completely abolished the previouslyobserved protective effect of FrAq Our results illustrated that besidethe increase in gastric mucus barrier induced by FrAq the gastro-protective effect of this fraction also involved the action of NO andendogenous PGs levels After determining the factors involved withFrAq action the possible healing effect of FrAq was evaluated withthe acetic acid method (the most similar to human gastric ulcer) Oral

treatment with FrAq during 7 consecutive days (Fig 5a) and also

14 days (Fig 5b) demonstrated that this fraction was able toaccelerate the healing of chronic gastric ulcers in rats At the doseof 25 mgkg FrAq 7 and 14 days of treatment signi1047297cantly decreasedthe area of lesion in 80 and 37 respectively when compared to thecontrol group treated with vehicle ( po001) In the group treatedwith lansoprazole at 30 mgkg the healing effect was also observedwith the decrease of gastric lesion at 83 (7 days) and 64 (14 days)To determine in better detail the healing process of FrAq we also

analyzed the stomach by zymograph Fig 6 presents the activities of MMP-2 and MMP-9 on the gastric mucosa after treatment withvehicle lansoprazole and FrAq for 7 or 14 days Our results show thatMMP-9 activity was present only after treatment during 7 days instomachs from the vehicle and lansoprazole-group We also observedthat this MMP-9 activity was absence for all treatments during 14days In contrast MMP-2 activity was present in all treated groupsincluding the sham group (without gastric lesion) The treatment of animals that exhibited major MMP-2 activity after 7 and 14 days wascompared to the animals treated with vehicle Lower activity of MMP-2 was observed after treatment of the animals with lansopra-zole and FrAq for 7 days (Fig 7a 40 and 26 respectively in rela-tion to the vehicle) and 14 days (Fig 7b 54 and 73 respectivelyin relation to the vehicle)

Other important data obtained from this test based on thetreatment of animals with FrAq for 7 and 14 consecutive days wasthe absence of death in these groups and also absence bodyweight changes (data not shown) and some organ weights andbiochemical parameters of serum (Table 2) These results there-fore ensure that based on the parameters used in this study theFrAq at doses of 25 mgkg does not cause signi1047297cant toxic effectsafter 7 or 14 consecutive days of treatment

This study also evaluated the activity of FrAq against Helico-bacter pylori and presented minimal inhibitory concentration(MIC) of 1250 mgmL considered a accentuated activity and thepositive control (amoxicillin) presented MIC of 150 mgmL

The analysis of mass spectra of FrAq (Fig 9) showed the samecompounds identi1047297ed in the hydroalcoholic extract studied byMininel et al (2014) as shown in Table 3 The spectra (Fig 8) showthe ions with mz 1083 (punicalagin) mz 601 attributed togallagic acid and at mz 301 attributed to ellagic acid illustratingthe similarity with the hydroalcoholic extract (Mininel et al2014) The fragmentation of the second order (MS2) ion at m z 1083 (punicalagin) leads to ions at m z 781 (punicalin) and the ionwith m z 601 (gallagic acid) as shown in Fig 9 The chromato-graphic pro1047297le of the water fraction is also shown in Fig 10 In thechromatogram two peaks are highlighted eluting at tr 172 minand tr 233 min indicating the presence of the major compoundpunicalagin (α and β anomers) Two peaks with tr 280 min and tr301 min (region 2) were observed corresponding to the com-pound punicalin (α and β anomers) derived from punicalagin

4 Discussion

The development of gastric ulcers is a complex and multi-factorial process including bacterial infections the increase of acidsecretion generation of reactive oxygen species (ROS) inhibitionof the endogenous PGs and the degradation of the extracellularmatrix (ECM) (Laine et al 2008 Mei et al 2013) Research duringthe last decade has offered new insights in the preventativetherapy and the healing of gastric ulcers and the synergisticef 1047297ciency of a multi-target approach based on individual mechan-isms of action could be the new perspective for treatment of thisdisease (Wagner 2011 de Carvalho et al 2014)

Terminalia catappa is a medicinal plant widely distributed intropical and subtropical regions and it has been previously reported

that this species exhibits a variety of biological effects However the

Table 1

Effects of the aqueous fraction (FrAq) from the leaves of Terminalia catappa (25 mgkg)administered through the intraduodenal (id) or oral (po) route on the biochemicalparameters of gastric juice obtained from pylorus-ligature rats (nfrac146ndash8)

Treatment Route Dose (mgkg) Gastric juice (mL) [Hthorn] (μEqmL4 h)

Vehicle id ndash 7027225 6997184Cimetidine 100 4297119nn 2777130nn

FrAq 25 8027218 7347065

Vehicle po ndash 3747101 5257151Cimetidine 100 2637067 1947087nn

FrAq 25 5307167 n 6337043

Data represents the means7SEM The asterisks denote the signi1047297cance levelswhen compared with the control group

n po005nn po001 by one-way ANOVA followed by Dunnetts test

0

1000

2000

3000

Vehicle Carbenoxolone

100 mgkg

FrAq

25 mgkg

A l c i a n b l u e

( m g g w e t t i s s u e )

ns

Fig 3 Effects of the aqueous fraction (FrAq) obtained from the leaves of Terminalia

catappa (25 mgkg) on the production of adherent gastric mucus (measured as the

amount of bound Alcian blue) in pylorus-ligated rats relative to the control values(nfrac146) The results are the mean7SEM Statistical signi1047297cance was determined byANOVA followed by Dunnetts t test (n po005 nnn po001 and ns ndash no signi1047297cantdifferences)

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underlying mechanisms of the gastroprotective and healing effectsof the aqueous fraction (FrAq) obtained from the leaves have yet tobe evaluated

In the present study FrAq showed a marked gastroprotectiveeffect evidenced by the dramatic inhibition of the gastric damageproduced by ethanol Ethanol induced erosion ulcerative lesionsand petechial bleeding in the mucosa of the stomach in humansand an ethanol-induced gastric ulcer model is commonly used tostudy both the pathogenesis and new therapeutics for the treat-ment of gastric ulcer disease (Oyagi et al 2010)

Our results have shown the effective gastroprotective effect of theaqueous fraction obtained from the leaves of Terminalia catappa

A previous study by Nunes et al (2012) evaluated the ethanolic

extract of bark from this species against gastric ulcers induced byethanol Aside from using a different part of plant (the bark insteadleaves) this study has demonstrated gastroprotection at a dose 10times higher than our study (250 mgkg vs 25 mgkg) Our studiesalso highlighted the use of the renewable part of this plant (leaves)that would enable the management of this plant for the futureproduction of a phytotherapeutic against gastric ulcer

Gastric ulcer induced by ethanol is commonly associated withreduced mucosal blood 1047298ow and ischemia that causes deleteriouseffects on the gastric mucosa mainly in the aging gastric mucosa(Tarnawski et al 2014) Ischemia weakens the gastric mucosal barrierand increases the acid back-diffusion predisposing the gastric mucosa

to damage (Rao and Vijayakumar 2007) The restoration of blood 1047298ow

Fig 4 The ulcerative lesion area (ULA-mm2) for gastric ulcers induced by ethanol in rats pretreated with L -NAME (panel a) with vehicle or together with carbenoxolone(100 mgkg) or the aqueous fraction (FrAq) obtained from leaves of Terminalia catappa (25 mgkg) pretreated with N -ethylmaleimide (panel b) with vehicle or together withcarbenoxolone (100 mgkg) and FrAq (25 mgkg) or pretreated with INDO ndash indomethacin (panel c) with vehicle or together with carbenoxolone (100 mgkg) or FrAq(25 mgkg) The results are reported as the mean7SEM Statistical signi1047297cance was determined by ANOVA followed by Dunnetts test n po005 nn po001 nnn po0001compared to the corresponding vehicle group o005 po001 compared to the corresponding NEMthornvehicle L -Namethornvehicle or INDOthornvehicle NS ndash no signi1047297cantdifferences

0

2

4

6

8

10

Vehicle Lansoprazole

30 mgkg

FrAq

25 mgkg

U L A ( m m sup2 )

0

5

10

15

20

Vehicle Lansoprazole

30 mgkg

FrAq

25 mgkg

U L A ( m m sup2 )

Fig 5 Effect of the oral administration of the aqueous fraction (FrAq) obtained from the leaves of Terminalia catappa (25 mgkg) on the healing of ulcers produced by theintroduction of acetic acid solution into the stomachs of rats The ulceration was scored on the 7th day (panel a) and 14th day (panel b) after surgery The results are reported

as the mean7

SEM Statistical signi1047297

cance was determined by ANOVA followed by Dunnetts test

nn

po

001

nnn

po

0001 compared to the corresponding vehicle group

L Pinheiro Silva et al Journal of Ethnopharmacology 159 (2015) 285ndash 295290

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(reperfusion) after a period of ischemia initiates a cascade of changesincluding the release of local ROS in1047298ammatory responses tissuedamage by fragmenting cellular DNA and an increase in the adhesionof neutrophils to endothelial cells a phenomenon known as ische-miandashreperfusion (IR) (Smith et al 1996 De Foneska and Kaunitz2010)

The current study shows that oral treatment with FrAq (25 mgkg)signi1047297cantly decreased the extent of ulcerative lesions in the gastrictissue submitted to IR injury compared to stomachs from the vehicle-treated control group Based on the physiopathology of the IR experimental model our results are in concordance with Chen et al

(2012) and Wang et al (2013) in which they observed that punicalagin

and punicalin both polyphenols presents in Terminalia catappa wereable to attenuate oxidative stress and hypoxia-induced apoptosis

These results therefore con1047297rm the gastroprotective effect of this fraction and encourage the continuation of the studies of theeffects of FrAq treatment and the characterization of its mechan-isms of action

Studies already performed with the hydroalcoholic extract of Terminalia catappa and other species of this genus such as

Terminalia chebula and Terminalia fagifolia have demonstrated theantisecretory effect of these species (Kumar et al 2014 Mishraet al 2013 Nunes et al 2014) Unlike these studies our resultshave shown that the oral and intraduodenal treatment with FrAq(25 mgkg) does not promote the antiulcerogenic effect in anantisecretory manner However our result also illustrated thatoral treatment with FrAq was able to signi1047297cantly increase thegastric volume in relation to the control group treated with vehicle(530 mL and 374 mL respectively) Our hypothesis was that theFrAq elicits the increase of gastric juice because of the amount of adherent gastric mucus

The success of the pharmacological treatment for gastric ulcersrelies on the augmentation of the protective factors of the gastricmucosa such as the mucus barrier (Al-Jiboury and Kaunitz 2012Brzozowska et al 2013) The adherent gastric mucus is the 1047297rst lineof defense against acid and serves as a barrier against self-digestionthe functional integrity of the gastric mucosa depends on thisbarrier (Wallace 2008) Our study reveals a signi1047297cant increase inthe amount of adherent mucus in the animals treated with FrAqthus justifying the increase in gastric volume by treatment with thisfraction This study involving the FrAq is highly signi1047297cant in theongoing search for an antiulcerogenic therapy as the prolonged useof antisecretory drugs such as proton-pump inhibitors and H2

blockers can provoke serious side effects (Ozdil et al 2013 Eomet al 2011)

In addition to the increased production of the mucus barrierseveral studies have demonstrated a complex defense system invol-ving the gastric mucosa which include the regulation of gastricmucosal blood 1047298ow the formation of sulfhydryl compounds NO andendogenous PGs (Brzozowski et al 2000 Pawlik et al 2001)

NO is considered to be one of the most important defensiveendogenous agents in the gastric mucosa and plays a physiologicalrole in the homeostasis of the gastrointestinal tract (Brzozowskiet al 2006) Together with the endogenous prostaglandins NO alsopreserves the gastric mucosa integrity and the inhibition of NOrelease can result in disturbances in the gastrointestinal motilityblood 1047298ow and acid secretion (Brzozowski et al 2006 Khalifaet al 2002 Wallace and Ma 2001) In addition to the increase ingastric mucus our results show that the restoration of the endo-genous NO represents an additional mechanism responsible for thegastroprotective effects of FrAq as a NO-synthase inhibitor elicits asigni1047297cant increase in gastric lesion in stomachs previously treatedwith the fraction The gastroprotective effect of the FrAq through

endogenous NO agrees with the protective effect of this fractionagainst IR in rats by inhibiting the excessive formation of ROS

Our study is also in concordance with those performed byPandya et al (2013) in which they evaluated the status of theethanolic extract of Terminalia catappa (at dose of 200 mgkg) andillustrated an augmentation of the antioxidant defense systemagainst Ehrlich carcinoma Kinoshita et al (2007) also describedthe antioxidant and hepatoprotective actions from the aqueousextract of the leaves of Terminalia catappa As well as NO theincrease in gastric SH content is important for the limiting theproduction of oxygen-derived free radicals (Genestra 2007)Our study showed that the pre-treatment of animals with NEM(a sulfhydryl inhibitor) did not change the gastroprotective effectof the FrAq ( p4005) Therefore our results exclude the involve-

ment of SH content in the gastroprotective effect of FrAq

Fig 6 Representative zymography results of the gastric ulcers from the differentgroups illustrating MMP-2 and MMP-9 activity Treatment for 7 days ndash vehicle (line 1)lansoprazole (line 2) aqueous fraction (FrAq) obtained from leaves of Terminalia

catappa at dose of 25 mgkg (line 3) and sham (line 4) Treatment for 14 days ndash vehicle(line 5) lansoprazole (line 6) aqueous fraction (FrAq) obtained from leaves of Terminalia catappa at dose of 25 mgkg (line 7) and sham (line 8) Gelatinolytic bandsof 92 72 64 and 57 kDa were observed which correspond to active-MMP-9pro-enzyme intermediate and active-MMP-2 respectively

Fig 7 Effect of treatment with the vehicle lansoprazole (30 mgkg) or the aqueousfraction (FrAq) obtained from Terminalia catappa (FrAq) on the gelatinolytic activityof MMP-9 (black column) and MMP-2 (white column) in the gastric mucosa after 7

(panel a) and 14 days (panel b) of treatment Values show the ratio of the IOD fromthe treatment groups to the control group (vehicle) IOD value

L Pinheiro Silva et al Journal of Ethnopharmacology 159 (2015) 285ndash 295 291

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Among the humoral factors in the gastric mucosa endogenousPGs play an important role in the protective effect by stimulatingthe secretion of mucus maintaining the local blood 1047298ow andincreasing the resistance of epithelial cells to potential damage bycytotoxins (Takeuchi 2010) Our results illustrate that the admin-istration of a non-selective COX inhibitor (indomethacin) comple-tely abolished the gastroprotective action of the FrAq This resulthighlights the relevance of PGs in the antiulcer action of thisfraction and agreement with the previously results of fraction inincrease in the amount of adherent mucus According to Takeuchi(2010) one of the mechanisms underlying the action of PGE2 isexactly the increase in mucus secretion that augments the pro-tective factors on the gastric mucosa

Based on the results regarding the gastroprotective effect of theFrAq against gastric injury induced by different ulcerogenic agentswe con1047297rmed that the action of this fraction was mediated by the

activation of defensive mucosa-protective factors such as increasedmucus production NO pathways and endogenous PGs However itis desirable for any new antiulcer drug that the preventive effect isalso accompanied by ulcer healing effects

Antiulcer drugs such as H2-receptor antagonists fail in promot-ing the healing of gastric ulcers when the regenerated mucosapresents with poor histological maturity and ulcer relapse is notprevented by cimetidine (Arakawa et al 2012)

This study also determined the effect of the FrAq on the healingof gastric ulcers induced by acetic acid in rats treated for 7 or 14consecutive days The acetic acid induced a deep necrotic lesioninvolving the entire mucosal depth and penetrating through themuscularis mucosae Ulcer healing is a dynamic process of 1047297llingmucosal defects with proliferating and migrating epithelial cells

and connective tissue which results in the reconstruction of the

mucosal architecture (Okabe and Amagase 2005) Chronic treat-ment with FrAq (25 mgkg) demonstrated a dramatic reduction inthe ulcerative lesion area by treatment for 7 days (80) which wasmore effective than 14 days of treatment (37) This resultdemonstrated the FrAq (25 mgkg) accelerates the healing within7 days of treatment and the healing effect remains after treatmentof 14 days when compared to control group or lansoprazole group

The injection of acetic acid into gastric mucosa induces thedevelopment of deep gastric ulceration and gastric mucosal damagedirectly associated with ECM degradation in which the zinc-dependent matrix metalloproteinases (MMPs) play a crucial role

In several animal studies of gastric ulcer attention has focusedon the role of MMPs mainly MMP-2 and MMP-9 (Li et al 2013)Wound formation and the following healing are dynamic processesof ECM remodeling that are mainly in1047298uenced by MMP-2 andMMP-9 (Gyenge et al 2013)

According to Yeh et al (2012) MMP-9 is the protease mostsigni1047297cantly involved in the degradation of the basement membraneand is associated with pathological states that include in1047298ammationand cancer Li et al (2013) described enhanced expression of MMP-9in the margin of the ulcer in patients with this disease Their studysuggested that the presence of MMP-9 at the margin of the ulcer maybe indicative of in1047298ammation and poor wound healing Ulcerogenicagents such as indomethacin exhibited 12-fold higher pro-MMP-9activity and ethanol exhibited 22-fold higher pro-MMP-9 activity inrat gastric tissues relative to untreated tissues (Mei et al 2013) Ourresults showed MMP-9 activity only after treatments with the vehicleand lansoprazole (7 days) These results determined that the presenceof MMP-9 activity at the gastric mucosa in groups treated with the

vehicle was related with the absence of healing in ulcers of the gastricmucosa Our results also show that the seemingly healing macro-scopic ulcer observed by treating animals with lansoprazole (7 days)may not represent a good wound healing because the presence of MMP-9 activity in gastric damage further demonstrates the persis-tence of the in1047298ammatory process at the gastric mucosa Our resultsshown that treatment of the acetic acid induced chronic ulcers withFrAq (25 mgkg) for 7 and 14 days inhibited the MMP-9 activity Ourresults could be strengthened by the study from Yeh et al (2012) inwhich extract from Terminalia catappa leaves exerts an antimetasticeffect by attenuating MMP-9 mediated in1047298ammation in hepatocellu-lar carcinoma In addition to our results obtained regarding MMP-9our results also reported high MMP-2 activity in gastric ulcers of thecontrol group treated with vehicle (7 and 14 days) and lower MMP-2

activity was observed after the treatment of animals with

Table 2

Effects of the aqueous fraction (FrAq) obtained from leaves of Terminalia catappa (25 mgkg) and lansoprazole (30 mgkg) administered orally for 14 consecutive days onselected toxicological parameters (nfrac145ndash6)

Treatment (po)

Vehicle Lansoprazole FrAq

Organ weights (g)Heart 3857048 3887032 3677013

Lung 4497064 5347143 4207040Kidney 5147063 4917018 4917007Liver 10107123 9997028 9707017Spleen 2697028 2837024 2677027

Biochemical parameterGlucose 136907700 136407580 151007850Creatinine 0567002 0567003 0537004Urea 38107112 39407535 39607423γ-GT 0967017 2027055 1247025AST 157117134 1812071751 1906071450ALT 44337251 48007504 4317311

Results are expressed as the means7SEM obtained from different groups The units for the biochemical parameters of serum are UL (ALT ndash alanine aminotransferaseAST ndash aspartate aminotransferase γndashGT ndash gamma-glutamyl transpeptidase) and mgdL (urea and creatinine)

Table 3

Identi1047297cation of the substances obtained in the aqueous fraction (FrAq) from theleaves of Terminalia catappa by FIA-ESI-IT-MSn

[MndashH] MSn ions Identi1047297cation

1083 781 [M-152-152-H] Punicalagin (1)

601 [M-152-152-180-H]

781 601 [M-180-H] Punicalin (2)

601 409 [M-191-H] Gallagic acid (3)

301 257 [M-44-H] Ellagic acid (4)

229 [M-44-28-H]

Adapted from Mininel et al (2014)

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lansoprazole and FrAq Recent studies have highlighted the functionof MMP-2 in the healing process of rat gastric ulcers induced by aceticacid (Gyenge et al 2013) but the function of MMP-2 is not wellunderstood

The antiulcerogenic actions demonstrated by FrAq could beattributed to the phenolic compounds present in this fractionMininel et al (2014) analyzed mass spectra in a full-scan of theextract and this fraction and showing similarity to each otherhighlighted by the precursor ions m z 1083 (punicalagin) m z 781(pulicalin) m z 601 (gallagic acid) and m z 301 attributed to ellagicacid In these studies the chromatogram from HPLC-PDA of the

aqueous fraction of Terminalia catappa con1047297rmed the majority of the

compounds punicalagin (anomers α and β) and punicalin (anomers αand β) are similar to the hydroalcoholic extract studied by Mininelet al (2014) Ellagic acid (EA) is one of the naturally occurringpolyphenols found in the FrAq from Terminalia catappa leavesNumerous pharmacological studies have suggested that EA providesmucosal protective action in the stomach against ethanol or ische-miandashreperfusion injury (Iino et al 2001 Iino et al 2002) and severalgastroprotective mechanisms are attributed to this compound Forexample Chatterjee et al (2012) showed that EA enhanced prosta-glandins when compared with the ulcerated untreated group

In our 1047297nal experimental series we evaluated the subacute

toxicological parameters from groups that received vehicle

500 1000 1500 2000

mz

0

5

10

15

20

25

30

35

40

45

50

55

60

6570

75

80

85

90

95

100108339

7814554115

10131395449

11329163927

13498731207

139719156207

171229

R e l a t i v e A b u n d a n c e

Fig 8 First-order mass spectrum of the aqueous fraction (FrAq) obtained from Terminalia catappa (FrAq) at the negative mode Range of ions with m z 100ndash2000 Da

T_130129165016 1-1000 RT060-096 AV70 NL271E2

F ITMS - c ESI Full ms2 108300cid3000 [29500-120000]

300 400 500 600 700 800 900 1000 1100 1200

mz

0

5

10

15

20

25

30

35

40

45

50

55

6065

70

75

80

85

90

95

100

R e l a t i v e A b u n d a n c

e

Fig 9 Mass spectrum of the second order (MS2) of the precursor ion m z 1083 from the aqueous fraction (FrAq) obtained in the negative mode of leaves from Terminalia

catappa Range of ions with mz 300-1200 Da

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lansoprazole or the FrAq over 14 days Important toxicity para-meters including changes in body weight mortality and weight of the vital organs were determined as shown in Table 2 Treatmentwith FrAq for 14 days with 25 mgkg did not signi1047297cantly alter thebody weight of animals compared to the vehicle control over the14-days treatment period (data not shown) and the averageweights of the major organs of the FrAq-treated subjects werecomparable to those of the control group Table 2 also shows thatFrAq did not affect several biochemical parameters Treatment didnot signi1047297cantly alter the body weight the weight of vital organsand biochemical parameters of serum However Mininel et al(2014) evaluated the mutagenicity of the hydroalcoholic extractfrom the leaves of Terminalia catappa with the Ames test andillustrated that this extract exhibited mutagenic activity in vitro athigh concentrations leading them to recommend caution whenusing this compound for medicinal purposes

This study also evaluated the activity of aqueous fraction (FrAq)against Helicobacter pylori and determination of minimal inhibitoryconcentration (MIC) of 1250 mgmL considered an important anti-microbial activity The literature do not show a consensus in relationto MIC values obtained for natural products Aligannis et al (2001)consider MICs with values equal to or less than 500 mgmL as potentinhibitors MICs between 600 and 1500 mgmL and MIC moderateinhibitors as above 1600 mgmL as weak inhibitors Webster et al(2008) established as another satisfactory MIC value equal to or lessthan 1000 mgmL In this sense the result reported in this investiga-tion demonstrated the inhibitory potential of Terminalia catappaagainst this important bacteria responsible to gastric ulcer

5 Conclusions

We determined that the FrAq from Terminalia catappa leaveshas an important anti-Helicobacter pylori activity excellent pre-ventive and curative effects on acute and chronically inducedgastric ulcers The mechanisms involved in the gastroprotectionare related to the NO pathway an increase in the mucus and theendogenous prostaglandins and this fraction was able to healulcers through the inhibition of MMP-9 and MMP-2 activities

Acknowledgments

This study was supported by the Biota-FAPESP project (Fundaccedilatildeo

de Amparo agrave Pesquisa do Estado de Satildeo Paulo) CNPq (Conselho

Nacional de Desenvolvimento Cientiacute1047297co e Tecnoloacutegico) and CAPES(Coordenaccedilatildeo de Aperfeiccediloamento Pessoal de Niacutevel Superior)

References

Aligannis N Kalpotzakis E Mitaku S Chinou IB 2001 Composition andantimicrobial activity of the essential oils of two Origanum species Journal of

Agricultural and Food Chemistry 49 4168ndash

4170Al-Jiboury H Kaunitz JD 2012 Gastroduodenal mucosal defense CurrentOpinion of Gastroenterology 28 594ndash601

Arakawa T Watanabe T Tanigawa T Tominaga K Fujiwara Y Morimoto K2012 Quality of ulcer healing in gastrointestinal tract its pathophysiology andclinical relevance World Journal of Gastroenterology 18 4811ndash4822

Arrieta J Benitez J Flores E Castillo C Navarrete A 2003 Puri1047297cation of gastroprotective triterpenoids from stem bark of Amphipterygium adstringensroles of prostaglandins sulfhydryls nitric oxide and capsaicin neurons PlantaMedica 69 905ndash909

Bradford MM 1976 A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye bindingAnalytical Biochemistry 72 248ndash254

Brzozowska I Strzalka M Drozdowicz D Konturek SJ Brzozowski T 2013Mechanisms of esophageal protection gastroprotection and ulcer healing bymelatonin Implications for the therapeutic use of melatonin in gastroesopha-geal re1047298ux disease (GERD) and peptic ulcer disease Current PharmaceuticalDesign httpdxdoi org1021741381612819666131119110258 (Epub ahead of print)

Brzozowski T Konturek PC Konturek SJ Pajdo R Schuppan D Drozdowicz DPtak A Pawlik M Nakamura T Hahn EG 2000 Involvement of cycloox-ygenase (COX)-2 products in acceleration of ulcer healing by gastrin andhepatocyte growth factor Journal of Physiology and Pharmacology 51 751ndash773

Brzozowski T Konturek PC Sliwowski Z Pajdo R Drozdowicz D Kwiecien SBurnat G Konturek SJ Pawlik WW 2006 Prostaglandincyclooxygenasepathway in ghrelin-induced gastroprotection against ischemia-reperfusioninjury Journal of Pharmacology and Experimental Therapeutics 319 477ndash487

Camargo MC Garciacutea A Riquelme A Otero W Camargo CA Garciacutea THCandia R Bruce MG Rabkin CS 2014 The problem of Helicobacter pyloriresistance to antibiotics a systematic review in Latin America Clinical andSystematic Reviews 109 485ndash495

Chatterjee A Chatterjee S Das S Saha A Chattopadhyay S Bandyopadhyay SK2012 Ellagic acid facilitates indomethacin-induced gastric ulcer healing via COX-2 up-regulation Acta Biochimica et Biophysica Sinica 44 565ndash576

Chen PS Li JH 2005 Chemopreventive effect of punicalagin a novel tannincomponent isolated from Terminalia catappa on H-ras-transformed NIH3T3cells Toxicology Letter 163 44ndash53

Chen PS Li JH Liu TY Lin TC 2000 Folk medicine Terminalia catappa and its

major tannin component punicalagin are effective against bleomycin-inducedgenotoxicity in Chinese hamster ovary cells Cancer Letter 152 115ndash122

Chen B Tuuli MG Longtine MS Shin JS Lawrence R Inder T Michael ND2012 Pomegranate juice and punicalagin attenuate oxidative stress andapoptosis in human placenta and in human placental trophoblasts American

Journal of Physiology and Endocrinology Metabolism 302 E1142ndashE1152CLSI Manual Clinical and Laboratory Standards Institute 2006 Methods for

dilution antimicrobial susceptibility tests for bacteria that grow aerobicallyapproved standards 6ordf ed Performance standards for antimicrobial suscept-ibility testing Sixteenth informational supplement M100-S16 (tab 2J) Clinicaland Laboratory Standards Institute Wayne PA

De Foneska A Kaunitz JD 2010 Gastroduodenal mucosal defense CurrentOpinion Gastroenterology 26 604ndash610

de Carvalho KI Bonamin F Dos Santos RC Peacuterico LL Beserra FP de Sousa DPFilho JM da Rocha LR Hiruma-Lima CA 2014 Geraniol-a 1047298avoring agentwith multifunctional effects in protecting the gastric and duodenal mucosaNaunyn Schmiedebergs Archieves of Pharmacology 387 355ndash365

Eom CS Park SM Myung SK Yun JM Ahn JS 2011 Use of acid-suppressive

drugs and risk of fracture a meta-analysis of observational studies AnnalsFamily Medicine 9 257ndash267Fan YM Xu LZ Gao J Wang Y Tang XH Zhao XN Zhang ZX 2004

Phytochemical and antiin1047298ammatory studies on Terminalia catappa Fitoterapia75 253ndash260

Fyhrquist P Mwasumbi L Haeggstroumlm CA Vuorela H Hiltunen R Vuorela P2002 Ethnobotanical and antimicrobial investigation on some species of Terminalia and Combretum (Combretaceae) growing in Tanzania Journal of Ethnopharmacology 79 169ndash177

Genestra M 2007 Oxyl radicals redox-sensitive signalling cascades and antiox-idants Cell Signalling 19 1807ndash1819

Germoseacuten-Robineau L 2014 Farmacopea Vegetal Carbentildea CICY editorial YucataacutenMexico p 360

Gyenge M Amagase K Kunimi S Matsuoka R Takeuchi K 2013 Roles of pro-angiogenic and anti-angiogenic factors as well as matrix metalloproteinases inhealing of NSAID-induced small intestinal ulcers in rats Life Science 93441ndash447

Iino T Nakahara K Miki W Kiso Y Ogawa Y Kato S Takeuchi K 2001 Lessdamaging effect of whisky in rat stomachs in comparison with pure ethanol

Digestion 64 (214ndash221) 2001

1 - α e β punicalagin

2 - α e βpunicalin

00 50 100 150 200 250 300 350 400

Retention Time [min]

0

1000000

2000000

I n t e n

s i t y [ micro V ]

T catappa FrAq - CH5

Fig 10 Chromatogram from HPLCndashPDA of the aqueous fraction of the leaves of Terminalia catappa Hydro Column 1047298ow 1 mLmin gradient method 5ndash60 MeOH40 min HPLCndashPDA (Jascos) 270 nm

L Pinheiro Silva et al Journal of Ethnopharmacology 159 (2015) 285ndash 295294

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underlying mechanisms of the gastroprotective and healing effectsof the aqueous fraction (FrAq) obtained from the leaves have yet tobe evaluated

In the present study FrAq showed a marked gastroprotectiveeffect evidenced by the dramatic inhibition of the gastric damageproduced by ethanol Ethanol induced erosion ulcerative lesionsand petechial bleeding in the mucosa of the stomach in humansand an ethanol-induced gastric ulcer model is commonly used tostudy both the pathogenesis and new therapeutics for the treat-ment of gastric ulcer disease (Oyagi et al 2010)

Our results have shown the effective gastroprotective effect of theaqueous fraction obtained from the leaves of Terminalia catappa

A previous study by Nunes et al (2012) evaluated the ethanolic

extract of bark from this species against gastric ulcers induced byethanol Aside from using a different part of plant (the bark insteadleaves) this study has demonstrated gastroprotection at a dose 10times higher than our study (250 mgkg vs 25 mgkg) Our studiesalso highlighted the use of the renewable part of this plant (leaves)that would enable the management of this plant for the futureproduction of a phytotherapeutic against gastric ulcer

Gastric ulcer induced by ethanol is commonly associated withreduced mucosal blood 1047298ow and ischemia that causes deleteriouseffects on the gastric mucosa mainly in the aging gastric mucosa(Tarnawski et al 2014) Ischemia weakens the gastric mucosal barrierand increases the acid back-diffusion predisposing the gastric mucosa

to damage (Rao and Vijayakumar 2007) The restoration of blood 1047298ow

Fig 4 The ulcerative lesion area (ULA-mm2) for gastric ulcers induced by ethanol in rats pretreated with L -NAME (panel a) with vehicle or together with carbenoxolone(100 mgkg) or the aqueous fraction (FrAq) obtained from leaves of Terminalia catappa (25 mgkg) pretreated with N -ethylmaleimide (panel b) with vehicle or together withcarbenoxolone (100 mgkg) and FrAq (25 mgkg) or pretreated with INDO ndash indomethacin (panel c) with vehicle or together with carbenoxolone (100 mgkg) or FrAq(25 mgkg) The results are reported as the mean7SEM Statistical signi1047297cance was determined by ANOVA followed by Dunnetts test n po005 nn po001 nnn po0001compared to the corresponding vehicle group o005 po001 compared to the corresponding NEMthornvehicle L -Namethornvehicle or INDOthornvehicle NS ndash no signi1047297cantdifferences

0

2

4

6

8

10

Vehicle Lansoprazole

30 mgkg

FrAq

25 mgkg

U L A ( m m sup2 )

0

5

10

15

20

Vehicle Lansoprazole

30 mgkg

FrAq

25 mgkg

U L A ( m m sup2 )

Fig 5 Effect of the oral administration of the aqueous fraction (FrAq) obtained from the leaves of Terminalia catappa (25 mgkg) on the healing of ulcers produced by theintroduction of acetic acid solution into the stomachs of rats The ulceration was scored on the 7th day (panel a) and 14th day (panel b) after surgery The results are reported

as the mean7

SEM Statistical signi1047297

cance was determined by ANOVA followed by Dunnetts test

nn

po

001

nnn

po

0001 compared to the corresponding vehicle group

L Pinheiro Silva et al Journal of Ethnopharmacology 159 (2015) 285ndash 295290

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(reperfusion) after a period of ischemia initiates a cascade of changesincluding the release of local ROS in1047298ammatory responses tissuedamage by fragmenting cellular DNA and an increase in the adhesionof neutrophils to endothelial cells a phenomenon known as ische-miandashreperfusion (IR) (Smith et al 1996 De Foneska and Kaunitz2010)

The current study shows that oral treatment with FrAq (25 mgkg)signi1047297cantly decreased the extent of ulcerative lesions in the gastrictissue submitted to IR injury compared to stomachs from the vehicle-treated control group Based on the physiopathology of the IR experimental model our results are in concordance with Chen et al

(2012) and Wang et al (2013) in which they observed that punicalagin

and punicalin both polyphenols presents in Terminalia catappa wereable to attenuate oxidative stress and hypoxia-induced apoptosis

These results therefore con1047297rm the gastroprotective effect of this fraction and encourage the continuation of the studies of theeffects of FrAq treatment and the characterization of its mechan-isms of action

Studies already performed with the hydroalcoholic extract of Terminalia catappa and other species of this genus such as

Terminalia chebula and Terminalia fagifolia have demonstrated theantisecretory effect of these species (Kumar et al 2014 Mishraet al 2013 Nunes et al 2014) Unlike these studies our resultshave shown that the oral and intraduodenal treatment with FrAq(25 mgkg) does not promote the antiulcerogenic effect in anantisecretory manner However our result also illustrated thatoral treatment with FrAq was able to signi1047297cantly increase thegastric volume in relation to the control group treated with vehicle(530 mL and 374 mL respectively) Our hypothesis was that theFrAq elicits the increase of gastric juice because of the amount of adherent gastric mucus

The success of the pharmacological treatment for gastric ulcersrelies on the augmentation of the protective factors of the gastricmucosa such as the mucus barrier (Al-Jiboury and Kaunitz 2012Brzozowska et al 2013) The adherent gastric mucus is the 1047297rst lineof defense against acid and serves as a barrier against self-digestionthe functional integrity of the gastric mucosa depends on thisbarrier (Wallace 2008) Our study reveals a signi1047297cant increase inthe amount of adherent mucus in the animals treated with FrAqthus justifying the increase in gastric volume by treatment with thisfraction This study involving the FrAq is highly signi1047297cant in theongoing search for an antiulcerogenic therapy as the prolonged useof antisecretory drugs such as proton-pump inhibitors and H2

blockers can provoke serious side effects (Ozdil et al 2013 Eomet al 2011)

In addition to the increased production of the mucus barrierseveral studies have demonstrated a complex defense system invol-ving the gastric mucosa which include the regulation of gastricmucosal blood 1047298ow the formation of sulfhydryl compounds NO andendogenous PGs (Brzozowski et al 2000 Pawlik et al 2001)

NO is considered to be one of the most important defensiveendogenous agents in the gastric mucosa and plays a physiologicalrole in the homeostasis of the gastrointestinal tract (Brzozowskiet al 2006) Together with the endogenous prostaglandins NO alsopreserves the gastric mucosa integrity and the inhibition of NOrelease can result in disturbances in the gastrointestinal motilityblood 1047298ow and acid secretion (Brzozowski et al 2006 Khalifaet al 2002 Wallace and Ma 2001) In addition to the increase ingastric mucus our results show that the restoration of the endo-genous NO represents an additional mechanism responsible for thegastroprotective effects of FrAq as a NO-synthase inhibitor elicits asigni1047297cant increase in gastric lesion in stomachs previously treatedwith the fraction The gastroprotective effect of the FrAq through

endogenous NO agrees with the protective effect of this fractionagainst IR in rats by inhibiting the excessive formation of ROS

Our study is also in concordance with those performed byPandya et al (2013) in which they evaluated the status of theethanolic extract of Terminalia catappa (at dose of 200 mgkg) andillustrated an augmentation of the antioxidant defense systemagainst Ehrlich carcinoma Kinoshita et al (2007) also describedthe antioxidant and hepatoprotective actions from the aqueousextract of the leaves of Terminalia catappa As well as NO theincrease in gastric SH content is important for the limiting theproduction of oxygen-derived free radicals (Genestra 2007)Our study showed that the pre-treatment of animals with NEM(a sulfhydryl inhibitor) did not change the gastroprotective effectof the FrAq ( p4005) Therefore our results exclude the involve-

ment of SH content in the gastroprotective effect of FrAq

Fig 6 Representative zymography results of the gastric ulcers from the differentgroups illustrating MMP-2 and MMP-9 activity Treatment for 7 days ndash vehicle (line 1)lansoprazole (line 2) aqueous fraction (FrAq) obtained from leaves of Terminalia

catappa at dose of 25 mgkg (line 3) and sham (line 4) Treatment for 14 days ndash vehicle(line 5) lansoprazole (line 6) aqueous fraction (FrAq) obtained from leaves of Terminalia catappa at dose of 25 mgkg (line 7) and sham (line 8) Gelatinolytic bandsof 92 72 64 and 57 kDa were observed which correspond to active-MMP-9pro-enzyme intermediate and active-MMP-2 respectively

Fig 7 Effect of treatment with the vehicle lansoprazole (30 mgkg) or the aqueousfraction (FrAq) obtained from Terminalia catappa (FrAq) on the gelatinolytic activityof MMP-9 (black column) and MMP-2 (white column) in the gastric mucosa after 7

(panel a) and 14 days (panel b) of treatment Values show the ratio of the IOD fromthe treatment groups to the control group (vehicle) IOD value

L Pinheiro Silva et al Journal of Ethnopharmacology 159 (2015) 285ndash 295 291

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Among the humoral factors in the gastric mucosa endogenousPGs play an important role in the protective effect by stimulatingthe secretion of mucus maintaining the local blood 1047298ow andincreasing the resistance of epithelial cells to potential damage bycytotoxins (Takeuchi 2010) Our results illustrate that the admin-istration of a non-selective COX inhibitor (indomethacin) comple-tely abolished the gastroprotective action of the FrAq This resulthighlights the relevance of PGs in the antiulcer action of thisfraction and agreement with the previously results of fraction inincrease in the amount of adherent mucus According to Takeuchi(2010) one of the mechanisms underlying the action of PGE2 isexactly the increase in mucus secretion that augments the pro-tective factors on the gastric mucosa

Based on the results regarding the gastroprotective effect of theFrAq against gastric injury induced by different ulcerogenic agentswe con1047297rmed that the action of this fraction was mediated by the

activation of defensive mucosa-protective factors such as increasedmucus production NO pathways and endogenous PGs However itis desirable for any new antiulcer drug that the preventive effect isalso accompanied by ulcer healing effects

Antiulcer drugs such as H2-receptor antagonists fail in promot-ing the healing of gastric ulcers when the regenerated mucosapresents with poor histological maturity and ulcer relapse is notprevented by cimetidine (Arakawa et al 2012)

This study also determined the effect of the FrAq on the healingof gastric ulcers induced by acetic acid in rats treated for 7 or 14consecutive days The acetic acid induced a deep necrotic lesioninvolving the entire mucosal depth and penetrating through themuscularis mucosae Ulcer healing is a dynamic process of 1047297llingmucosal defects with proliferating and migrating epithelial cells

and connective tissue which results in the reconstruction of the

mucosal architecture (Okabe and Amagase 2005) Chronic treat-ment with FrAq (25 mgkg) demonstrated a dramatic reduction inthe ulcerative lesion area by treatment for 7 days (80) which wasmore effective than 14 days of treatment (37) This resultdemonstrated the FrAq (25 mgkg) accelerates the healing within7 days of treatment and the healing effect remains after treatmentof 14 days when compared to control group or lansoprazole group

The injection of acetic acid into gastric mucosa induces thedevelopment of deep gastric ulceration and gastric mucosal damagedirectly associated with ECM degradation in which the zinc-dependent matrix metalloproteinases (MMPs) play a crucial role

In several animal studies of gastric ulcer attention has focusedon the role of MMPs mainly MMP-2 and MMP-9 (Li et al 2013)Wound formation and the following healing are dynamic processesof ECM remodeling that are mainly in1047298uenced by MMP-2 andMMP-9 (Gyenge et al 2013)

According to Yeh et al (2012) MMP-9 is the protease mostsigni1047297cantly involved in the degradation of the basement membraneand is associated with pathological states that include in1047298ammationand cancer Li et al (2013) described enhanced expression of MMP-9in the margin of the ulcer in patients with this disease Their studysuggested that the presence of MMP-9 at the margin of the ulcer maybe indicative of in1047298ammation and poor wound healing Ulcerogenicagents such as indomethacin exhibited 12-fold higher pro-MMP-9activity and ethanol exhibited 22-fold higher pro-MMP-9 activity inrat gastric tissues relative to untreated tissues (Mei et al 2013) Ourresults showed MMP-9 activity only after treatments with the vehicleand lansoprazole (7 days) These results determined that the presenceof MMP-9 activity at the gastric mucosa in groups treated with the

vehicle was related with the absence of healing in ulcers of the gastricmucosa Our results also show that the seemingly healing macro-scopic ulcer observed by treating animals with lansoprazole (7 days)may not represent a good wound healing because the presence of MMP-9 activity in gastric damage further demonstrates the persis-tence of the in1047298ammatory process at the gastric mucosa Our resultsshown that treatment of the acetic acid induced chronic ulcers withFrAq (25 mgkg) for 7 and 14 days inhibited the MMP-9 activity Ourresults could be strengthened by the study from Yeh et al (2012) inwhich extract from Terminalia catappa leaves exerts an antimetasticeffect by attenuating MMP-9 mediated in1047298ammation in hepatocellu-lar carcinoma In addition to our results obtained regarding MMP-9our results also reported high MMP-2 activity in gastric ulcers of thecontrol group treated with vehicle (7 and 14 days) and lower MMP-2

activity was observed after the treatment of animals with

Table 2

Effects of the aqueous fraction (FrAq) obtained from leaves of Terminalia catappa (25 mgkg) and lansoprazole (30 mgkg) administered orally for 14 consecutive days onselected toxicological parameters (nfrac145ndash6)

Treatment (po)

Vehicle Lansoprazole FrAq

Organ weights (g)Heart 3857048 3887032 3677013

Lung 4497064 5347143 4207040Kidney 5147063 4917018 4917007Liver 10107123 9997028 9707017Spleen 2697028 2837024 2677027

Biochemical parameterGlucose 136907700 136407580 151007850Creatinine 0567002 0567003 0537004Urea 38107112 39407535 39607423γ-GT 0967017 2027055 1247025AST 157117134 1812071751 1906071450ALT 44337251 48007504 4317311

Results are expressed as the means7SEM obtained from different groups The units for the biochemical parameters of serum are UL (ALT ndash alanine aminotransferaseAST ndash aspartate aminotransferase γndashGT ndash gamma-glutamyl transpeptidase) and mgdL (urea and creatinine)

Table 3

Identi1047297cation of the substances obtained in the aqueous fraction (FrAq) from theleaves of Terminalia catappa by FIA-ESI-IT-MSn

[MndashH] MSn ions Identi1047297cation

1083 781 [M-152-152-H] Punicalagin (1)

601 [M-152-152-180-H]

781 601 [M-180-H] Punicalin (2)

601 409 [M-191-H] Gallagic acid (3)

301 257 [M-44-H] Ellagic acid (4)

229 [M-44-28-H]

Adapted from Mininel et al (2014)

L Pinheiro Silva et al Journal of Ethnopharmacology 159 (2015) 285ndash 295292

7262019 1-s20-S0378874114008095-main

httpslidepdfcomreaderfull1-s20-s0378874114008095-main 911

lansoprazole and FrAq Recent studies have highlighted the functionof MMP-2 in the healing process of rat gastric ulcers induced by aceticacid (Gyenge et al 2013) but the function of MMP-2 is not wellunderstood

The antiulcerogenic actions demonstrated by FrAq could beattributed to the phenolic compounds present in this fractionMininel et al (2014) analyzed mass spectra in a full-scan of theextract and this fraction and showing similarity to each otherhighlighted by the precursor ions m z 1083 (punicalagin) m z 781(pulicalin) m z 601 (gallagic acid) and m z 301 attributed to ellagicacid In these studies the chromatogram from HPLC-PDA of the

aqueous fraction of Terminalia catappa con1047297rmed the majority of the

compounds punicalagin (anomers α and β) and punicalin (anomers αand β) are similar to the hydroalcoholic extract studied by Mininelet al (2014) Ellagic acid (EA) is one of the naturally occurringpolyphenols found in the FrAq from Terminalia catappa leavesNumerous pharmacological studies have suggested that EA providesmucosal protective action in the stomach against ethanol or ische-miandashreperfusion injury (Iino et al 2001 Iino et al 2002) and severalgastroprotective mechanisms are attributed to this compound Forexample Chatterjee et al (2012) showed that EA enhanced prosta-glandins when compared with the ulcerated untreated group

In our 1047297nal experimental series we evaluated the subacute

toxicological parameters from groups that received vehicle

500 1000 1500 2000

mz

0

5

10

15

20

25

30

35

40

45

50

55

60

6570

75

80

85

90

95

100108339

7814554115

10131395449

11329163927

13498731207

139719156207

171229

R e l a t i v e A b u n d a n c e

Fig 8 First-order mass spectrum of the aqueous fraction (FrAq) obtained from Terminalia catappa (FrAq) at the negative mode Range of ions with m z 100ndash2000 Da

T_130129165016 1-1000 RT060-096 AV70 NL271E2

F ITMS - c ESI Full ms2 108300cid3000 [29500-120000]

300 400 500 600 700 800 900 1000 1100 1200

mz

0

5

10

15

20

25

30

35

40

45

50

55

6065

70

75

80

85

90

95

100

R e l a t i v e A b u n d a n c

e

Fig 9 Mass spectrum of the second order (MS2) of the precursor ion m z 1083 from the aqueous fraction (FrAq) obtained in the negative mode of leaves from Terminalia

catappa Range of ions with mz 300-1200 Da

L Pinheiro Silva et al Journal of Ethnopharmacology 159 (2015) 285ndash 295 293

7262019 1-s20-S0378874114008095-main

httpslidepdfcomreaderfull1-s20-s0378874114008095-main 1011

lansoprazole or the FrAq over 14 days Important toxicity para-meters including changes in body weight mortality and weight of the vital organs were determined as shown in Table 2 Treatmentwith FrAq for 14 days with 25 mgkg did not signi1047297cantly alter thebody weight of animals compared to the vehicle control over the14-days treatment period (data not shown) and the averageweights of the major organs of the FrAq-treated subjects werecomparable to those of the control group Table 2 also shows thatFrAq did not affect several biochemical parameters Treatment didnot signi1047297cantly alter the body weight the weight of vital organsand biochemical parameters of serum However Mininel et al(2014) evaluated the mutagenicity of the hydroalcoholic extractfrom the leaves of Terminalia catappa with the Ames test andillustrated that this extract exhibited mutagenic activity in vitro athigh concentrations leading them to recommend caution whenusing this compound for medicinal purposes

This study also evaluated the activity of aqueous fraction (FrAq)against Helicobacter pylori and determination of minimal inhibitoryconcentration (MIC) of 1250 mgmL considered an important anti-microbial activity The literature do not show a consensus in relationto MIC values obtained for natural products Aligannis et al (2001)consider MICs with values equal to or less than 500 mgmL as potentinhibitors MICs between 600 and 1500 mgmL and MIC moderateinhibitors as above 1600 mgmL as weak inhibitors Webster et al(2008) established as another satisfactory MIC value equal to or lessthan 1000 mgmL In this sense the result reported in this investiga-tion demonstrated the inhibitory potential of Terminalia catappaagainst this important bacteria responsible to gastric ulcer

5 Conclusions

We determined that the FrAq from Terminalia catappa leaveshas an important anti-Helicobacter pylori activity excellent pre-ventive and curative effects on acute and chronically inducedgastric ulcers The mechanisms involved in the gastroprotectionare related to the NO pathway an increase in the mucus and theendogenous prostaglandins and this fraction was able to healulcers through the inhibition of MMP-9 and MMP-2 activities

Acknowledgments

This study was supported by the Biota-FAPESP project (Fundaccedilatildeo

de Amparo agrave Pesquisa do Estado de Satildeo Paulo) CNPq (Conselho

Nacional de Desenvolvimento Cientiacute1047297co e Tecnoloacutegico) and CAPES(Coordenaccedilatildeo de Aperfeiccediloamento Pessoal de Niacutevel Superior)

References

Aligannis N Kalpotzakis E Mitaku S Chinou IB 2001 Composition andantimicrobial activity of the essential oils of two Origanum species Journal of

Agricultural and Food Chemistry 49 4168ndash

4170Al-Jiboury H Kaunitz JD 2012 Gastroduodenal mucosal defense CurrentOpinion of Gastroenterology 28 594ndash601

Arakawa T Watanabe T Tanigawa T Tominaga K Fujiwara Y Morimoto K2012 Quality of ulcer healing in gastrointestinal tract its pathophysiology andclinical relevance World Journal of Gastroenterology 18 4811ndash4822

Arrieta J Benitez J Flores E Castillo C Navarrete A 2003 Puri1047297cation of gastroprotective triterpenoids from stem bark of Amphipterygium adstringensroles of prostaglandins sulfhydryls nitric oxide and capsaicin neurons PlantaMedica 69 905ndash909

Bradford MM 1976 A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye bindingAnalytical Biochemistry 72 248ndash254

Brzozowska I Strzalka M Drozdowicz D Konturek SJ Brzozowski T 2013Mechanisms of esophageal protection gastroprotection and ulcer healing bymelatonin Implications for the therapeutic use of melatonin in gastroesopha-geal re1047298ux disease (GERD) and peptic ulcer disease Current PharmaceuticalDesign httpdxdoi org1021741381612819666131119110258 (Epub ahead of print)

Brzozowski T Konturek PC Konturek SJ Pajdo R Schuppan D Drozdowicz DPtak A Pawlik M Nakamura T Hahn EG 2000 Involvement of cycloox-ygenase (COX)-2 products in acceleration of ulcer healing by gastrin andhepatocyte growth factor Journal of Physiology and Pharmacology 51 751ndash773

Brzozowski T Konturek PC Sliwowski Z Pajdo R Drozdowicz D Kwiecien SBurnat G Konturek SJ Pawlik WW 2006 Prostaglandincyclooxygenasepathway in ghrelin-induced gastroprotection against ischemia-reperfusioninjury Journal of Pharmacology and Experimental Therapeutics 319 477ndash487

Camargo MC Garciacutea A Riquelme A Otero W Camargo CA Garciacutea THCandia R Bruce MG Rabkin CS 2014 The problem of Helicobacter pyloriresistance to antibiotics a systematic review in Latin America Clinical andSystematic Reviews 109 485ndash495

Chatterjee A Chatterjee S Das S Saha A Chattopadhyay S Bandyopadhyay SK2012 Ellagic acid facilitates indomethacin-induced gastric ulcer healing via COX-2 up-regulation Acta Biochimica et Biophysica Sinica 44 565ndash576

Chen PS Li JH 2005 Chemopreventive effect of punicalagin a novel tannincomponent isolated from Terminalia catappa on H-ras-transformed NIH3T3cells Toxicology Letter 163 44ndash53

Chen PS Li JH Liu TY Lin TC 2000 Folk medicine Terminalia catappa and its

major tannin component punicalagin are effective against bleomycin-inducedgenotoxicity in Chinese hamster ovary cells Cancer Letter 152 115ndash122

Chen B Tuuli MG Longtine MS Shin JS Lawrence R Inder T Michael ND2012 Pomegranate juice and punicalagin attenuate oxidative stress andapoptosis in human placenta and in human placental trophoblasts American

Journal of Physiology and Endocrinology Metabolism 302 E1142ndashE1152CLSI Manual Clinical and Laboratory Standards Institute 2006 Methods for

dilution antimicrobial susceptibility tests for bacteria that grow aerobicallyapproved standards 6ordf ed Performance standards for antimicrobial suscept-ibility testing Sixteenth informational supplement M100-S16 (tab 2J) Clinicaland Laboratory Standards Institute Wayne PA

De Foneska A Kaunitz JD 2010 Gastroduodenal mucosal defense CurrentOpinion Gastroenterology 26 604ndash610

de Carvalho KI Bonamin F Dos Santos RC Peacuterico LL Beserra FP de Sousa DPFilho JM da Rocha LR Hiruma-Lima CA 2014 Geraniol-a 1047298avoring agentwith multifunctional effects in protecting the gastric and duodenal mucosaNaunyn Schmiedebergs Archieves of Pharmacology 387 355ndash365

Eom CS Park SM Myung SK Yun JM Ahn JS 2011 Use of acid-suppressive

drugs and risk of fracture a meta-analysis of observational studies AnnalsFamily Medicine 9 257ndash267Fan YM Xu LZ Gao J Wang Y Tang XH Zhao XN Zhang ZX 2004

Phytochemical and antiin1047298ammatory studies on Terminalia catappa Fitoterapia75 253ndash260

Fyhrquist P Mwasumbi L Haeggstroumlm CA Vuorela H Hiltunen R Vuorela P2002 Ethnobotanical and antimicrobial investigation on some species of Terminalia and Combretum (Combretaceae) growing in Tanzania Journal of Ethnopharmacology 79 169ndash177

Genestra M 2007 Oxyl radicals redox-sensitive signalling cascades and antiox-idants Cell Signalling 19 1807ndash1819

Germoseacuten-Robineau L 2014 Farmacopea Vegetal Carbentildea CICY editorial YucataacutenMexico p 360

Gyenge M Amagase K Kunimi S Matsuoka R Takeuchi K 2013 Roles of pro-angiogenic and anti-angiogenic factors as well as matrix metalloproteinases inhealing of NSAID-induced small intestinal ulcers in rats Life Science 93441ndash447

Iino T Nakahara K Miki W Kiso Y Ogawa Y Kato S Takeuchi K 2001 Lessdamaging effect of whisky in rat stomachs in comparison with pure ethanol

Digestion 64 (214ndash221) 2001

1 - α e β punicalagin

2 - α e βpunicalin

00 50 100 150 200 250 300 350 400

Retention Time [min]

0

1000000

2000000

I n t e n

s i t y [ micro V ]

T catappa FrAq - CH5

Fig 10 Chromatogram from HPLCndashPDA of the aqueous fraction of the leaves of Terminalia catappa Hydro Column 1047298ow 1 mLmin gradient method 5ndash60 MeOH40 min HPLCndashPDA (Jascos) 270 nm

L Pinheiro Silva et al Journal of Ethnopharmacology 159 (2015) 285ndash 295294

7262019 1-s20-S0378874114008095-main

httpslidepdfcomreaderfull1-s20-s0378874114008095-main 711

(reperfusion) after a period of ischemia initiates a cascade of changesincluding the release of local ROS in1047298ammatory responses tissuedamage by fragmenting cellular DNA and an increase in the adhesionof neutrophils to endothelial cells a phenomenon known as ische-miandashreperfusion (IR) (Smith et al 1996 De Foneska and Kaunitz2010)

The current study shows that oral treatment with FrAq (25 mgkg)signi1047297cantly decreased the extent of ulcerative lesions in the gastrictissue submitted to IR injury compared to stomachs from the vehicle-treated control group Based on the physiopathology of the IR experimental model our results are in concordance with Chen et al

(2012) and Wang et al (2013) in which they observed that punicalagin

and punicalin both polyphenols presents in Terminalia catappa wereable to attenuate oxidative stress and hypoxia-induced apoptosis

These results therefore con1047297rm the gastroprotective effect of this fraction and encourage the continuation of the studies of theeffects of FrAq treatment and the characterization of its mechan-isms of action

Studies already performed with the hydroalcoholic extract of Terminalia catappa and other species of this genus such as

Terminalia chebula and Terminalia fagifolia have demonstrated theantisecretory effect of these species (Kumar et al 2014 Mishraet al 2013 Nunes et al 2014) Unlike these studies our resultshave shown that the oral and intraduodenal treatment with FrAq(25 mgkg) does not promote the antiulcerogenic effect in anantisecretory manner However our result also illustrated thatoral treatment with FrAq was able to signi1047297cantly increase thegastric volume in relation to the control group treated with vehicle(530 mL and 374 mL respectively) Our hypothesis was that theFrAq elicits the increase of gastric juice because of the amount of adherent gastric mucus

The success of the pharmacological treatment for gastric ulcersrelies on the augmentation of the protective factors of the gastricmucosa such as the mucus barrier (Al-Jiboury and Kaunitz 2012Brzozowska et al 2013) The adherent gastric mucus is the 1047297rst lineof defense against acid and serves as a barrier against self-digestionthe functional integrity of the gastric mucosa depends on thisbarrier (Wallace 2008) Our study reveals a signi1047297cant increase inthe amount of adherent mucus in the animals treated with FrAqthus justifying the increase in gastric volume by treatment with thisfraction This study involving the FrAq is highly signi1047297cant in theongoing search for an antiulcerogenic therapy as the prolonged useof antisecretory drugs such as proton-pump inhibitors and H2

blockers can provoke serious side effects (Ozdil et al 2013 Eomet al 2011)

In addition to the increased production of the mucus barrierseveral studies have demonstrated a complex defense system invol-ving the gastric mucosa which include the regulation of gastricmucosal blood 1047298ow the formation of sulfhydryl compounds NO andendogenous PGs (Brzozowski et al 2000 Pawlik et al 2001)

NO is considered to be one of the most important defensiveendogenous agents in the gastric mucosa and plays a physiologicalrole in the homeostasis of the gastrointestinal tract (Brzozowskiet al 2006) Together with the endogenous prostaglandins NO alsopreserves the gastric mucosa integrity and the inhibition of NOrelease can result in disturbances in the gastrointestinal motilityblood 1047298ow and acid secretion (Brzozowski et al 2006 Khalifaet al 2002 Wallace and Ma 2001) In addition to the increase ingastric mucus our results show that the restoration of the endo-genous NO represents an additional mechanism responsible for thegastroprotective effects of FrAq as a NO-synthase inhibitor elicits asigni1047297cant increase in gastric lesion in stomachs previously treatedwith the fraction The gastroprotective effect of the FrAq through

endogenous NO agrees with the protective effect of this fractionagainst IR in rats by inhibiting the excessive formation of ROS

Our study is also in concordance with those performed byPandya et al (2013) in which they evaluated the status of theethanolic extract of Terminalia catappa (at dose of 200 mgkg) andillustrated an augmentation of the antioxidant defense systemagainst Ehrlich carcinoma Kinoshita et al (2007) also describedthe antioxidant and hepatoprotective actions from the aqueousextract of the leaves of Terminalia catappa As well as NO theincrease in gastric SH content is important for the limiting theproduction of oxygen-derived free radicals (Genestra 2007)Our study showed that the pre-treatment of animals with NEM(a sulfhydryl inhibitor) did not change the gastroprotective effectof the FrAq ( p4005) Therefore our results exclude the involve-

ment of SH content in the gastroprotective effect of FrAq

Fig 6 Representative zymography results of the gastric ulcers from the differentgroups illustrating MMP-2 and MMP-9 activity Treatment for 7 days ndash vehicle (line 1)lansoprazole (line 2) aqueous fraction (FrAq) obtained from leaves of Terminalia

catappa at dose of 25 mgkg (line 3) and sham (line 4) Treatment for 14 days ndash vehicle(line 5) lansoprazole (line 6) aqueous fraction (FrAq) obtained from leaves of Terminalia catappa at dose of 25 mgkg (line 7) and sham (line 8) Gelatinolytic bandsof 92 72 64 and 57 kDa were observed which correspond to active-MMP-9pro-enzyme intermediate and active-MMP-2 respectively

Fig 7 Effect of treatment with the vehicle lansoprazole (30 mgkg) or the aqueousfraction (FrAq) obtained from Terminalia catappa (FrAq) on the gelatinolytic activityof MMP-9 (black column) and MMP-2 (white column) in the gastric mucosa after 7

(panel a) and 14 days (panel b) of treatment Values show the ratio of the IOD fromthe treatment groups to the control group (vehicle) IOD value

L Pinheiro Silva et al Journal of Ethnopharmacology 159 (2015) 285ndash 295 291

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Among the humoral factors in the gastric mucosa endogenousPGs play an important role in the protective effect by stimulatingthe secretion of mucus maintaining the local blood 1047298ow andincreasing the resistance of epithelial cells to potential damage bycytotoxins (Takeuchi 2010) Our results illustrate that the admin-istration of a non-selective COX inhibitor (indomethacin) comple-tely abolished the gastroprotective action of the FrAq This resulthighlights the relevance of PGs in the antiulcer action of thisfraction and agreement with the previously results of fraction inincrease in the amount of adherent mucus According to Takeuchi(2010) one of the mechanisms underlying the action of PGE2 isexactly the increase in mucus secretion that augments the pro-tective factors on the gastric mucosa

Based on the results regarding the gastroprotective effect of theFrAq against gastric injury induced by different ulcerogenic agentswe con1047297rmed that the action of this fraction was mediated by the

activation of defensive mucosa-protective factors such as increasedmucus production NO pathways and endogenous PGs However itis desirable for any new antiulcer drug that the preventive effect isalso accompanied by ulcer healing effects

Antiulcer drugs such as H2-receptor antagonists fail in promot-ing the healing of gastric ulcers when the regenerated mucosapresents with poor histological maturity and ulcer relapse is notprevented by cimetidine (Arakawa et al 2012)

This study also determined the effect of the FrAq on the healingof gastric ulcers induced by acetic acid in rats treated for 7 or 14consecutive days The acetic acid induced a deep necrotic lesioninvolving the entire mucosal depth and penetrating through themuscularis mucosae Ulcer healing is a dynamic process of 1047297llingmucosal defects with proliferating and migrating epithelial cells

and connective tissue which results in the reconstruction of the

mucosal architecture (Okabe and Amagase 2005) Chronic treat-ment with FrAq (25 mgkg) demonstrated a dramatic reduction inthe ulcerative lesion area by treatment for 7 days (80) which wasmore effective than 14 days of treatment (37) This resultdemonstrated the FrAq (25 mgkg) accelerates the healing within7 days of treatment and the healing effect remains after treatmentof 14 days when compared to control group or lansoprazole group

The injection of acetic acid into gastric mucosa induces thedevelopment of deep gastric ulceration and gastric mucosal damagedirectly associated with ECM degradation in which the zinc-dependent matrix metalloproteinases (MMPs) play a crucial role

In several animal studies of gastric ulcer attention has focusedon the role of MMPs mainly MMP-2 and MMP-9 (Li et al 2013)Wound formation and the following healing are dynamic processesof ECM remodeling that are mainly in1047298uenced by MMP-2 andMMP-9 (Gyenge et al 2013)

According to Yeh et al (2012) MMP-9 is the protease mostsigni1047297cantly involved in the degradation of the basement membraneand is associated with pathological states that include in1047298ammationand cancer Li et al (2013) described enhanced expression of MMP-9in the margin of the ulcer in patients with this disease Their studysuggested that the presence of MMP-9 at the margin of the ulcer maybe indicative of in1047298ammation and poor wound healing Ulcerogenicagents such as indomethacin exhibited 12-fold higher pro-MMP-9activity and ethanol exhibited 22-fold higher pro-MMP-9 activity inrat gastric tissues relative to untreated tissues (Mei et al 2013) Ourresults showed MMP-9 activity only after treatments with the vehicleand lansoprazole (7 days) These results determined that the presenceof MMP-9 activity at the gastric mucosa in groups treated with the

vehicle was related with the absence of healing in ulcers of the gastricmucosa Our results also show that the seemingly healing macro-scopic ulcer observed by treating animals with lansoprazole (7 days)may not represent a good wound healing because the presence of MMP-9 activity in gastric damage further demonstrates the persis-tence of the in1047298ammatory process at the gastric mucosa Our resultsshown that treatment of the acetic acid induced chronic ulcers withFrAq (25 mgkg) for 7 and 14 days inhibited the MMP-9 activity Ourresults could be strengthened by the study from Yeh et al (2012) inwhich extract from Terminalia catappa leaves exerts an antimetasticeffect by attenuating MMP-9 mediated in1047298ammation in hepatocellu-lar carcinoma In addition to our results obtained regarding MMP-9our results also reported high MMP-2 activity in gastric ulcers of thecontrol group treated with vehicle (7 and 14 days) and lower MMP-2

activity was observed after the treatment of animals with

Table 2

Effects of the aqueous fraction (FrAq) obtained from leaves of Terminalia catappa (25 mgkg) and lansoprazole (30 mgkg) administered orally for 14 consecutive days onselected toxicological parameters (nfrac145ndash6)

Treatment (po)

Vehicle Lansoprazole FrAq

Organ weights (g)Heart 3857048 3887032 3677013

Lung 4497064 5347143 4207040Kidney 5147063 4917018 4917007Liver 10107123 9997028 9707017Spleen 2697028 2837024 2677027

Biochemical parameterGlucose 136907700 136407580 151007850Creatinine 0567002 0567003 0537004Urea 38107112 39407535 39607423γ-GT 0967017 2027055 1247025AST 157117134 1812071751 1906071450ALT 44337251 48007504 4317311

Results are expressed as the means7SEM obtained from different groups The units for the biochemical parameters of serum are UL (ALT ndash alanine aminotransferaseAST ndash aspartate aminotransferase γndashGT ndash gamma-glutamyl transpeptidase) and mgdL (urea and creatinine)

Table 3

Identi1047297cation of the substances obtained in the aqueous fraction (FrAq) from theleaves of Terminalia catappa by FIA-ESI-IT-MSn

[MndashH] MSn ions Identi1047297cation

1083 781 [M-152-152-H] Punicalagin (1)

601 [M-152-152-180-H]

781 601 [M-180-H] Punicalin (2)

601 409 [M-191-H] Gallagic acid (3)

301 257 [M-44-H] Ellagic acid (4)

229 [M-44-28-H]

Adapted from Mininel et al (2014)

L Pinheiro Silva et al Journal of Ethnopharmacology 159 (2015) 285ndash 295292

7262019 1-s20-S0378874114008095-main

httpslidepdfcomreaderfull1-s20-s0378874114008095-main 911

lansoprazole and FrAq Recent studies have highlighted the functionof MMP-2 in the healing process of rat gastric ulcers induced by aceticacid (Gyenge et al 2013) but the function of MMP-2 is not wellunderstood

The antiulcerogenic actions demonstrated by FrAq could beattributed to the phenolic compounds present in this fractionMininel et al (2014) analyzed mass spectra in a full-scan of theextract and this fraction and showing similarity to each otherhighlighted by the precursor ions m z 1083 (punicalagin) m z 781(pulicalin) m z 601 (gallagic acid) and m z 301 attributed to ellagicacid In these studies the chromatogram from HPLC-PDA of the

aqueous fraction of Terminalia catappa con1047297rmed the majority of the

compounds punicalagin (anomers α and β) and punicalin (anomers αand β) are similar to the hydroalcoholic extract studied by Mininelet al (2014) Ellagic acid (EA) is one of the naturally occurringpolyphenols found in the FrAq from Terminalia catappa leavesNumerous pharmacological studies have suggested that EA providesmucosal protective action in the stomach against ethanol or ische-miandashreperfusion injury (Iino et al 2001 Iino et al 2002) and severalgastroprotective mechanisms are attributed to this compound Forexample Chatterjee et al (2012) showed that EA enhanced prosta-glandins when compared with the ulcerated untreated group

In our 1047297nal experimental series we evaluated the subacute

toxicological parameters from groups that received vehicle

500 1000 1500 2000

mz

0

5

10

15

20

25

30

35

40

45

50

55

60

6570

75

80

85

90

95

100108339

7814554115

10131395449

11329163927

13498731207

139719156207

171229

R e l a t i v e A b u n d a n c e

Fig 8 First-order mass spectrum of the aqueous fraction (FrAq) obtained from Terminalia catappa (FrAq) at the negative mode Range of ions with m z 100ndash2000 Da

T_130129165016 1-1000 RT060-096 AV70 NL271E2

F ITMS - c ESI Full ms2 108300cid3000 [29500-120000]

300 400 500 600 700 800 900 1000 1100 1200

mz

0

5

10

15

20

25

30

35

40

45

50

55

6065

70

75

80

85

90

95

100

R e l a t i v e A b u n d a n c

e

Fig 9 Mass spectrum of the second order (MS2) of the precursor ion m z 1083 from the aqueous fraction (FrAq) obtained in the negative mode of leaves from Terminalia

catappa Range of ions with mz 300-1200 Da

L Pinheiro Silva et al Journal of Ethnopharmacology 159 (2015) 285ndash 295 293

7262019 1-s20-S0378874114008095-main

httpslidepdfcomreaderfull1-s20-s0378874114008095-main 1011

lansoprazole or the FrAq over 14 days Important toxicity para-meters including changes in body weight mortality and weight of the vital organs were determined as shown in Table 2 Treatmentwith FrAq for 14 days with 25 mgkg did not signi1047297cantly alter thebody weight of animals compared to the vehicle control over the14-days treatment period (data not shown) and the averageweights of the major organs of the FrAq-treated subjects werecomparable to those of the control group Table 2 also shows thatFrAq did not affect several biochemical parameters Treatment didnot signi1047297cantly alter the body weight the weight of vital organsand biochemical parameters of serum However Mininel et al(2014) evaluated the mutagenicity of the hydroalcoholic extractfrom the leaves of Terminalia catappa with the Ames test andillustrated that this extract exhibited mutagenic activity in vitro athigh concentrations leading them to recommend caution whenusing this compound for medicinal purposes

This study also evaluated the activity of aqueous fraction (FrAq)against Helicobacter pylori and determination of minimal inhibitoryconcentration (MIC) of 1250 mgmL considered an important anti-microbial activity The literature do not show a consensus in relationto MIC values obtained for natural products Aligannis et al (2001)consider MICs with values equal to or less than 500 mgmL as potentinhibitors MICs between 600 and 1500 mgmL and MIC moderateinhibitors as above 1600 mgmL as weak inhibitors Webster et al(2008) established as another satisfactory MIC value equal to or lessthan 1000 mgmL In this sense the result reported in this investiga-tion demonstrated the inhibitory potential of Terminalia catappaagainst this important bacteria responsible to gastric ulcer

5 Conclusions

We determined that the FrAq from Terminalia catappa leaveshas an important anti-Helicobacter pylori activity excellent pre-ventive and curative effects on acute and chronically inducedgastric ulcers The mechanisms involved in the gastroprotectionare related to the NO pathway an increase in the mucus and theendogenous prostaglandins and this fraction was able to healulcers through the inhibition of MMP-9 and MMP-2 activities

Acknowledgments

This study was supported by the Biota-FAPESP project (Fundaccedilatildeo

de Amparo agrave Pesquisa do Estado de Satildeo Paulo) CNPq (Conselho

Nacional de Desenvolvimento Cientiacute1047297co e Tecnoloacutegico) and CAPES(Coordenaccedilatildeo de Aperfeiccediloamento Pessoal de Niacutevel Superior)

References

Aligannis N Kalpotzakis E Mitaku S Chinou IB 2001 Composition andantimicrobial activity of the essential oils of two Origanum species Journal of

Agricultural and Food Chemistry 49 4168ndash

4170Al-Jiboury H Kaunitz JD 2012 Gastroduodenal mucosal defense CurrentOpinion of Gastroenterology 28 594ndash601

Arakawa T Watanabe T Tanigawa T Tominaga K Fujiwara Y Morimoto K2012 Quality of ulcer healing in gastrointestinal tract its pathophysiology andclinical relevance World Journal of Gastroenterology 18 4811ndash4822

Arrieta J Benitez J Flores E Castillo C Navarrete A 2003 Puri1047297cation of gastroprotective triterpenoids from stem bark of Amphipterygium adstringensroles of prostaglandins sulfhydryls nitric oxide and capsaicin neurons PlantaMedica 69 905ndash909

Bradford MM 1976 A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye bindingAnalytical Biochemistry 72 248ndash254

Brzozowska I Strzalka M Drozdowicz D Konturek SJ Brzozowski T 2013Mechanisms of esophageal protection gastroprotection and ulcer healing bymelatonin Implications for the therapeutic use of melatonin in gastroesopha-geal re1047298ux disease (GERD) and peptic ulcer disease Current PharmaceuticalDesign httpdxdoi org1021741381612819666131119110258 (Epub ahead of print)

Brzozowski T Konturek PC Konturek SJ Pajdo R Schuppan D Drozdowicz DPtak A Pawlik M Nakamura T Hahn EG 2000 Involvement of cycloox-ygenase (COX)-2 products in acceleration of ulcer healing by gastrin andhepatocyte growth factor Journal of Physiology and Pharmacology 51 751ndash773

Brzozowski T Konturek PC Sliwowski Z Pajdo R Drozdowicz D Kwiecien SBurnat G Konturek SJ Pawlik WW 2006 Prostaglandincyclooxygenasepathway in ghrelin-induced gastroprotection against ischemia-reperfusioninjury Journal of Pharmacology and Experimental Therapeutics 319 477ndash487

Camargo MC Garciacutea A Riquelme A Otero W Camargo CA Garciacutea THCandia R Bruce MG Rabkin CS 2014 The problem of Helicobacter pyloriresistance to antibiotics a systematic review in Latin America Clinical andSystematic Reviews 109 485ndash495

Chatterjee A Chatterjee S Das S Saha A Chattopadhyay S Bandyopadhyay SK2012 Ellagic acid facilitates indomethacin-induced gastric ulcer healing via COX-2 up-regulation Acta Biochimica et Biophysica Sinica 44 565ndash576

Chen PS Li JH 2005 Chemopreventive effect of punicalagin a novel tannincomponent isolated from Terminalia catappa on H-ras-transformed NIH3T3cells Toxicology Letter 163 44ndash53

Chen PS Li JH Liu TY Lin TC 2000 Folk medicine Terminalia catappa and its

major tannin component punicalagin are effective against bleomycin-inducedgenotoxicity in Chinese hamster ovary cells Cancer Letter 152 115ndash122

Chen B Tuuli MG Longtine MS Shin JS Lawrence R Inder T Michael ND2012 Pomegranate juice and punicalagin attenuate oxidative stress andapoptosis in human placenta and in human placental trophoblasts American

Journal of Physiology and Endocrinology Metabolism 302 E1142ndashE1152CLSI Manual Clinical and Laboratory Standards Institute 2006 Methods for

dilution antimicrobial susceptibility tests for bacteria that grow aerobicallyapproved standards 6ordf ed Performance standards for antimicrobial suscept-ibility testing Sixteenth informational supplement M100-S16 (tab 2J) Clinicaland Laboratory Standards Institute Wayne PA

De Foneska A Kaunitz JD 2010 Gastroduodenal mucosal defense CurrentOpinion Gastroenterology 26 604ndash610

de Carvalho KI Bonamin F Dos Santos RC Peacuterico LL Beserra FP de Sousa DPFilho JM da Rocha LR Hiruma-Lima CA 2014 Geraniol-a 1047298avoring agentwith multifunctional effects in protecting the gastric and duodenal mucosaNaunyn Schmiedebergs Archieves of Pharmacology 387 355ndash365

Eom CS Park SM Myung SK Yun JM Ahn JS 2011 Use of acid-suppressive

drugs and risk of fracture a meta-analysis of observational studies AnnalsFamily Medicine 9 257ndash267Fan YM Xu LZ Gao J Wang Y Tang XH Zhao XN Zhang ZX 2004

Phytochemical and antiin1047298ammatory studies on Terminalia catappa Fitoterapia75 253ndash260

Fyhrquist P Mwasumbi L Haeggstroumlm CA Vuorela H Hiltunen R Vuorela P2002 Ethnobotanical and antimicrobial investigation on some species of Terminalia and Combretum (Combretaceae) growing in Tanzania Journal of Ethnopharmacology 79 169ndash177

Genestra M 2007 Oxyl radicals redox-sensitive signalling cascades and antiox-idants Cell Signalling 19 1807ndash1819

Germoseacuten-Robineau L 2014 Farmacopea Vegetal Carbentildea CICY editorial YucataacutenMexico p 360

Gyenge M Amagase K Kunimi S Matsuoka R Takeuchi K 2013 Roles of pro-angiogenic and anti-angiogenic factors as well as matrix metalloproteinases inhealing of NSAID-induced small intestinal ulcers in rats Life Science 93441ndash447

Iino T Nakahara K Miki W Kiso Y Ogawa Y Kato S Takeuchi K 2001 Lessdamaging effect of whisky in rat stomachs in comparison with pure ethanol

Digestion 64 (214ndash221) 2001

1 - α e β punicalagin

2 - α e βpunicalin

00 50 100 150 200 250 300 350 400

Retention Time [min]

0

1000000

2000000

I n t e n

s i t y [ micro V ]

T catappa FrAq - CH5

Fig 10 Chromatogram from HPLCndashPDA of the aqueous fraction of the leaves of Terminalia catappa Hydro Column 1047298ow 1 mLmin gradient method 5ndash60 MeOH40 min HPLCndashPDA (Jascos) 270 nm

L Pinheiro Silva et al Journal of Ethnopharmacology 159 (2015) 285ndash 295294

7262019 1-s20-S0378874114008095-main

httpslidepdfcomreaderfull1-s20-s0378874114008095-main 811

Among the humoral factors in the gastric mucosa endogenousPGs play an important role in the protective effect by stimulatingthe secretion of mucus maintaining the local blood 1047298ow andincreasing the resistance of epithelial cells to potential damage bycytotoxins (Takeuchi 2010) Our results illustrate that the admin-istration of a non-selective COX inhibitor (indomethacin) comple-tely abolished the gastroprotective action of the FrAq This resulthighlights the relevance of PGs in the antiulcer action of thisfraction and agreement with the previously results of fraction inincrease in the amount of adherent mucus According to Takeuchi(2010) one of the mechanisms underlying the action of PGE2 isexactly the increase in mucus secretion that augments the pro-tective factors on the gastric mucosa

Based on the results regarding the gastroprotective effect of theFrAq against gastric injury induced by different ulcerogenic agentswe con1047297rmed that the action of this fraction was mediated by the

activation of defensive mucosa-protective factors such as increasedmucus production NO pathways and endogenous PGs However itis desirable for any new antiulcer drug that the preventive effect isalso accompanied by ulcer healing effects

Antiulcer drugs such as H2-receptor antagonists fail in promot-ing the healing of gastric ulcers when the regenerated mucosapresents with poor histological maturity and ulcer relapse is notprevented by cimetidine (Arakawa et al 2012)

This study also determined the effect of the FrAq on the healingof gastric ulcers induced by acetic acid in rats treated for 7 or 14consecutive days The acetic acid induced a deep necrotic lesioninvolving the entire mucosal depth and penetrating through themuscularis mucosae Ulcer healing is a dynamic process of 1047297llingmucosal defects with proliferating and migrating epithelial cells

and connective tissue which results in the reconstruction of the

mucosal architecture (Okabe and Amagase 2005) Chronic treat-ment with FrAq (25 mgkg) demonstrated a dramatic reduction inthe ulcerative lesion area by treatment for 7 days (80) which wasmore effective than 14 days of treatment (37) This resultdemonstrated the FrAq (25 mgkg) accelerates the healing within7 days of treatment and the healing effect remains after treatmentof 14 days when compared to control group or lansoprazole group

The injection of acetic acid into gastric mucosa induces thedevelopment of deep gastric ulceration and gastric mucosal damagedirectly associated with ECM degradation in which the zinc-dependent matrix metalloproteinases (MMPs) play a crucial role

In several animal studies of gastric ulcer attention has focusedon the role of MMPs mainly MMP-2 and MMP-9 (Li et al 2013)Wound formation and the following healing are dynamic processesof ECM remodeling that are mainly in1047298uenced by MMP-2 andMMP-9 (Gyenge et al 2013)

According to Yeh et al (2012) MMP-9 is the protease mostsigni1047297cantly involved in the degradation of the basement membraneand is associated with pathological states that include in1047298ammationand cancer Li et al (2013) described enhanced expression of MMP-9in the margin of the ulcer in patients with this disease Their studysuggested that the presence of MMP-9 at the margin of the ulcer maybe indicative of in1047298ammation and poor wound healing Ulcerogenicagents such as indomethacin exhibited 12-fold higher pro-MMP-9activity and ethanol exhibited 22-fold higher pro-MMP-9 activity inrat gastric tissues relative to untreated tissues (Mei et al 2013) Ourresults showed MMP-9 activity only after treatments with the vehicleand lansoprazole (7 days) These results determined that the presenceof MMP-9 activity at the gastric mucosa in groups treated with the

vehicle was related with the absence of healing in ulcers of the gastricmucosa Our results also show that the seemingly healing macro-scopic ulcer observed by treating animals with lansoprazole (7 days)may not represent a good wound healing because the presence of MMP-9 activity in gastric damage further demonstrates the persis-tence of the in1047298ammatory process at the gastric mucosa Our resultsshown that treatment of the acetic acid induced chronic ulcers withFrAq (25 mgkg) for 7 and 14 days inhibited the MMP-9 activity Ourresults could be strengthened by the study from Yeh et al (2012) inwhich extract from Terminalia catappa leaves exerts an antimetasticeffect by attenuating MMP-9 mediated in1047298ammation in hepatocellu-lar carcinoma In addition to our results obtained regarding MMP-9our results also reported high MMP-2 activity in gastric ulcers of thecontrol group treated with vehicle (7 and 14 days) and lower MMP-2

activity was observed after the treatment of animals with

Table 2

Effects of the aqueous fraction (FrAq) obtained from leaves of Terminalia catappa (25 mgkg) and lansoprazole (30 mgkg) administered orally for 14 consecutive days onselected toxicological parameters (nfrac145ndash6)

Treatment (po)

Vehicle Lansoprazole FrAq

Organ weights (g)Heart 3857048 3887032 3677013

Lung 4497064 5347143 4207040Kidney 5147063 4917018 4917007Liver 10107123 9997028 9707017Spleen 2697028 2837024 2677027

Biochemical parameterGlucose 136907700 136407580 151007850Creatinine 0567002 0567003 0537004Urea 38107112 39407535 39607423γ-GT 0967017 2027055 1247025AST 157117134 1812071751 1906071450ALT 44337251 48007504 4317311

Results are expressed as the means7SEM obtained from different groups The units for the biochemical parameters of serum are UL (ALT ndash alanine aminotransferaseAST ndash aspartate aminotransferase γndashGT ndash gamma-glutamyl transpeptidase) and mgdL (urea and creatinine)

Table 3

Identi1047297cation of the substances obtained in the aqueous fraction (FrAq) from theleaves of Terminalia catappa by FIA-ESI-IT-MSn

[MndashH] MSn ions Identi1047297cation

1083 781 [M-152-152-H] Punicalagin (1)

601 [M-152-152-180-H]

781 601 [M-180-H] Punicalin (2)

601 409 [M-191-H] Gallagic acid (3)

301 257 [M-44-H] Ellagic acid (4)

229 [M-44-28-H]

Adapted from Mininel et al (2014)

L Pinheiro Silva et al Journal of Ethnopharmacology 159 (2015) 285ndash 295292

7262019 1-s20-S0378874114008095-main

httpslidepdfcomreaderfull1-s20-s0378874114008095-main 911

lansoprazole and FrAq Recent studies have highlighted the functionof MMP-2 in the healing process of rat gastric ulcers induced by aceticacid (Gyenge et al 2013) but the function of MMP-2 is not wellunderstood

The antiulcerogenic actions demonstrated by FrAq could beattributed to the phenolic compounds present in this fractionMininel et al (2014) analyzed mass spectra in a full-scan of theextract and this fraction and showing similarity to each otherhighlighted by the precursor ions m z 1083 (punicalagin) m z 781(pulicalin) m z 601 (gallagic acid) and m z 301 attributed to ellagicacid In these studies the chromatogram from HPLC-PDA of the

aqueous fraction of Terminalia catappa con1047297rmed the majority of the

compounds punicalagin (anomers α and β) and punicalin (anomers αand β) are similar to the hydroalcoholic extract studied by Mininelet al (2014) Ellagic acid (EA) is one of the naturally occurringpolyphenols found in the FrAq from Terminalia catappa leavesNumerous pharmacological studies have suggested that EA providesmucosal protective action in the stomach against ethanol or ische-miandashreperfusion injury (Iino et al 2001 Iino et al 2002) and severalgastroprotective mechanisms are attributed to this compound Forexample Chatterjee et al (2012) showed that EA enhanced prosta-glandins when compared with the ulcerated untreated group

In our 1047297nal experimental series we evaluated the subacute

toxicological parameters from groups that received vehicle

500 1000 1500 2000

mz

0

5

10

15

20

25

30

35

40

45

50

55

60

6570

75

80

85

90

95

100108339

7814554115

10131395449

11329163927

13498731207

139719156207

171229

R e l a t i v e A b u n d a n c e

Fig 8 First-order mass spectrum of the aqueous fraction (FrAq) obtained from Terminalia catappa (FrAq) at the negative mode Range of ions with m z 100ndash2000 Da

T_130129165016 1-1000 RT060-096 AV70 NL271E2

F ITMS - c ESI Full ms2 108300cid3000 [29500-120000]

300 400 500 600 700 800 900 1000 1100 1200

mz

0

5

10

15

20

25

30

35

40

45

50

55

6065

70

75

80

85

90

95

100

R e l a t i v e A b u n d a n c

e

Fig 9 Mass spectrum of the second order (MS2) of the precursor ion m z 1083 from the aqueous fraction (FrAq) obtained in the negative mode of leaves from Terminalia

catappa Range of ions with mz 300-1200 Da

L Pinheiro Silva et al Journal of Ethnopharmacology 159 (2015) 285ndash 295 293

7262019 1-s20-S0378874114008095-main

httpslidepdfcomreaderfull1-s20-s0378874114008095-main 1011

lansoprazole or the FrAq over 14 days Important toxicity para-meters including changes in body weight mortality and weight of the vital organs were determined as shown in Table 2 Treatmentwith FrAq for 14 days with 25 mgkg did not signi1047297cantly alter thebody weight of animals compared to the vehicle control over the14-days treatment period (data not shown) and the averageweights of the major organs of the FrAq-treated subjects werecomparable to those of the control group Table 2 also shows thatFrAq did not affect several biochemical parameters Treatment didnot signi1047297cantly alter the body weight the weight of vital organsand biochemical parameters of serum However Mininel et al(2014) evaluated the mutagenicity of the hydroalcoholic extractfrom the leaves of Terminalia catappa with the Ames test andillustrated that this extract exhibited mutagenic activity in vitro athigh concentrations leading them to recommend caution whenusing this compound for medicinal purposes

This study also evaluated the activity of aqueous fraction (FrAq)against Helicobacter pylori and determination of minimal inhibitoryconcentration (MIC) of 1250 mgmL considered an important anti-microbial activity The literature do not show a consensus in relationto MIC values obtained for natural products Aligannis et al (2001)consider MICs with values equal to or less than 500 mgmL as potentinhibitors MICs between 600 and 1500 mgmL and MIC moderateinhibitors as above 1600 mgmL as weak inhibitors Webster et al(2008) established as another satisfactory MIC value equal to or lessthan 1000 mgmL In this sense the result reported in this investiga-tion demonstrated the inhibitory potential of Terminalia catappaagainst this important bacteria responsible to gastric ulcer

5 Conclusions

We determined that the FrAq from Terminalia catappa leaveshas an important anti-Helicobacter pylori activity excellent pre-ventive and curative effects on acute and chronically inducedgastric ulcers The mechanisms involved in the gastroprotectionare related to the NO pathway an increase in the mucus and theendogenous prostaglandins and this fraction was able to healulcers through the inhibition of MMP-9 and MMP-2 activities

Acknowledgments

This study was supported by the Biota-FAPESP project (Fundaccedilatildeo

de Amparo agrave Pesquisa do Estado de Satildeo Paulo) CNPq (Conselho

Nacional de Desenvolvimento Cientiacute1047297co e Tecnoloacutegico) and CAPES(Coordenaccedilatildeo de Aperfeiccediloamento Pessoal de Niacutevel Superior)

References

Aligannis N Kalpotzakis E Mitaku S Chinou IB 2001 Composition andantimicrobial activity of the essential oils of two Origanum species Journal of

Agricultural and Food Chemistry 49 4168ndash

4170Al-Jiboury H Kaunitz JD 2012 Gastroduodenal mucosal defense CurrentOpinion of Gastroenterology 28 594ndash601

Arakawa T Watanabe T Tanigawa T Tominaga K Fujiwara Y Morimoto K2012 Quality of ulcer healing in gastrointestinal tract its pathophysiology andclinical relevance World Journal of Gastroenterology 18 4811ndash4822

Arrieta J Benitez J Flores E Castillo C Navarrete A 2003 Puri1047297cation of gastroprotective triterpenoids from stem bark of Amphipterygium adstringensroles of prostaglandins sulfhydryls nitric oxide and capsaicin neurons PlantaMedica 69 905ndash909

Bradford MM 1976 A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye bindingAnalytical Biochemistry 72 248ndash254

Brzozowska I Strzalka M Drozdowicz D Konturek SJ Brzozowski T 2013Mechanisms of esophageal protection gastroprotection and ulcer healing bymelatonin Implications for the therapeutic use of melatonin in gastroesopha-geal re1047298ux disease (GERD) and peptic ulcer disease Current PharmaceuticalDesign httpdxdoi org1021741381612819666131119110258 (Epub ahead of print)

Brzozowski T Konturek PC Konturek SJ Pajdo R Schuppan D Drozdowicz DPtak A Pawlik M Nakamura T Hahn EG 2000 Involvement of cycloox-ygenase (COX)-2 products in acceleration of ulcer healing by gastrin andhepatocyte growth factor Journal of Physiology and Pharmacology 51 751ndash773

Brzozowski T Konturek PC Sliwowski Z Pajdo R Drozdowicz D Kwiecien SBurnat G Konturek SJ Pawlik WW 2006 Prostaglandincyclooxygenasepathway in ghrelin-induced gastroprotection against ischemia-reperfusioninjury Journal of Pharmacology and Experimental Therapeutics 319 477ndash487

Camargo MC Garciacutea A Riquelme A Otero W Camargo CA Garciacutea THCandia R Bruce MG Rabkin CS 2014 The problem of Helicobacter pyloriresistance to antibiotics a systematic review in Latin America Clinical andSystematic Reviews 109 485ndash495

Chatterjee A Chatterjee S Das S Saha A Chattopadhyay S Bandyopadhyay SK2012 Ellagic acid facilitates indomethacin-induced gastric ulcer healing via COX-2 up-regulation Acta Biochimica et Biophysica Sinica 44 565ndash576

Chen PS Li JH 2005 Chemopreventive effect of punicalagin a novel tannincomponent isolated from Terminalia catappa on H-ras-transformed NIH3T3cells Toxicology Letter 163 44ndash53

Chen PS Li JH Liu TY Lin TC 2000 Folk medicine Terminalia catappa and its

major tannin component punicalagin are effective against bleomycin-inducedgenotoxicity in Chinese hamster ovary cells Cancer Letter 152 115ndash122

Chen B Tuuli MG Longtine MS Shin JS Lawrence R Inder T Michael ND2012 Pomegranate juice and punicalagin attenuate oxidative stress andapoptosis in human placenta and in human placental trophoblasts American

Journal of Physiology and Endocrinology Metabolism 302 E1142ndashE1152CLSI Manual Clinical and Laboratory Standards Institute 2006 Methods for

dilution antimicrobial susceptibility tests for bacteria that grow aerobicallyapproved standards 6ordf ed Performance standards for antimicrobial suscept-ibility testing Sixteenth informational supplement M100-S16 (tab 2J) Clinicaland Laboratory Standards Institute Wayne PA

De Foneska A Kaunitz JD 2010 Gastroduodenal mucosal defense CurrentOpinion Gastroenterology 26 604ndash610

de Carvalho KI Bonamin F Dos Santos RC Peacuterico LL Beserra FP de Sousa DPFilho JM da Rocha LR Hiruma-Lima CA 2014 Geraniol-a 1047298avoring agentwith multifunctional effects in protecting the gastric and duodenal mucosaNaunyn Schmiedebergs Archieves of Pharmacology 387 355ndash365

Eom CS Park SM Myung SK Yun JM Ahn JS 2011 Use of acid-suppressive

drugs and risk of fracture a meta-analysis of observational studies AnnalsFamily Medicine 9 257ndash267Fan YM Xu LZ Gao J Wang Y Tang XH Zhao XN Zhang ZX 2004

Phytochemical and antiin1047298ammatory studies on Terminalia catappa Fitoterapia75 253ndash260

Fyhrquist P Mwasumbi L Haeggstroumlm CA Vuorela H Hiltunen R Vuorela P2002 Ethnobotanical and antimicrobial investigation on some species of Terminalia and Combretum (Combretaceae) growing in Tanzania Journal of Ethnopharmacology 79 169ndash177

Genestra M 2007 Oxyl radicals redox-sensitive signalling cascades and antiox-idants Cell Signalling 19 1807ndash1819

Germoseacuten-Robineau L 2014 Farmacopea Vegetal Carbentildea CICY editorial YucataacutenMexico p 360

Gyenge M Amagase K Kunimi S Matsuoka R Takeuchi K 2013 Roles of pro-angiogenic and anti-angiogenic factors as well as matrix metalloproteinases inhealing of NSAID-induced small intestinal ulcers in rats Life Science 93441ndash447

Iino T Nakahara K Miki W Kiso Y Ogawa Y Kato S Takeuchi K 2001 Lessdamaging effect of whisky in rat stomachs in comparison with pure ethanol

Digestion 64 (214ndash221) 2001

1 - α e β punicalagin

2 - α e βpunicalin

00 50 100 150 200 250 300 350 400

Retention Time [min]

0

1000000

2000000

I n t e n

s i t y [ micro V ]

T catappa FrAq - CH5

Fig 10 Chromatogram from HPLCndashPDA of the aqueous fraction of the leaves of Terminalia catappa Hydro Column 1047298ow 1 mLmin gradient method 5ndash60 MeOH40 min HPLCndashPDA (Jascos) 270 nm

L Pinheiro Silva et al Journal of Ethnopharmacology 159 (2015) 285ndash 295294

7262019 1-s20-S0378874114008095-main

httpslidepdfcomreaderfull1-s20-s0378874114008095-main 911

lansoprazole and FrAq Recent studies have highlighted the functionof MMP-2 in the healing process of rat gastric ulcers induced by aceticacid (Gyenge et al 2013) but the function of MMP-2 is not wellunderstood

The antiulcerogenic actions demonstrated by FrAq could beattributed to the phenolic compounds present in this fractionMininel et al (2014) analyzed mass spectra in a full-scan of theextract and this fraction and showing similarity to each otherhighlighted by the precursor ions m z 1083 (punicalagin) m z 781(pulicalin) m z 601 (gallagic acid) and m z 301 attributed to ellagicacid In these studies the chromatogram from HPLC-PDA of the

aqueous fraction of Terminalia catappa con1047297rmed the majority of the

compounds punicalagin (anomers α and β) and punicalin (anomers αand β) are similar to the hydroalcoholic extract studied by Mininelet al (2014) Ellagic acid (EA) is one of the naturally occurringpolyphenols found in the FrAq from Terminalia catappa leavesNumerous pharmacological studies have suggested that EA providesmucosal protective action in the stomach against ethanol or ische-miandashreperfusion injury (Iino et al 2001 Iino et al 2002) and severalgastroprotective mechanisms are attributed to this compound Forexample Chatterjee et al (2012) showed that EA enhanced prosta-glandins when compared with the ulcerated untreated group

In our 1047297nal experimental series we evaluated the subacute

toxicological parameters from groups that received vehicle

500 1000 1500 2000

mz

0

5

10

15

20

25

30

35

40

45

50

55

60

6570

75

80

85

90

95

100108339

7814554115

10131395449

11329163927

13498731207

139719156207

171229

R e l a t i v e A b u n d a n c e

Fig 8 First-order mass spectrum of the aqueous fraction (FrAq) obtained from Terminalia catappa (FrAq) at the negative mode Range of ions with m z 100ndash2000 Da

T_130129165016 1-1000 RT060-096 AV70 NL271E2

F ITMS - c ESI Full ms2 108300cid3000 [29500-120000]

300 400 500 600 700 800 900 1000 1100 1200

mz

0

5

10

15

20

25

30

35

40

45

50

55

6065

70

75

80

85

90

95

100

R e l a t i v e A b u n d a n c

e

Fig 9 Mass spectrum of the second order (MS2) of the precursor ion m z 1083 from the aqueous fraction (FrAq) obtained in the negative mode of leaves from Terminalia

catappa Range of ions with mz 300-1200 Da

L Pinheiro Silva et al Journal of Ethnopharmacology 159 (2015) 285ndash 295 293

7262019 1-s20-S0378874114008095-main

httpslidepdfcomreaderfull1-s20-s0378874114008095-main 1011

lansoprazole or the FrAq over 14 days Important toxicity para-meters including changes in body weight mortality and weight of the vital organs were determined as shown in Table 2 Treatmentwith FrAq for 14 days with 25 mgkg did not signi1047297cantly alter thebody weight of animals compared to the vehicle control over the14-days treatment period (data not shown) and the averageweights of the major organs of the FrAq-treated subjects werecomparable to those of the control group Table 2 also shows thatFrAq did not affect several biochemical parameters Treatment didnot signi1047297cantly alter the body weight the weight of vital organsand biochemical parameters of serum However Mininel et al(2014) evaluated the mutagenicity of the hydroalcoholic extractfrom the leaves of Terminalia catappa with the Ames test andillustrated that this extract exhibited mutagenic activity in vitro athigh concentrations leading them to recommend caution whenusing this compound for medicinal purposes

This study also evaluated the activity of aqueous fraction (FrAq)against Helicobacter pylori and determination of minimal inhibitoryconcentration (MIC) of 1250 mgmL considered an important anti-microbial activity The literature do not show a consensus in relationto MIC values obtained for natural products Aligannis et al (2001)consider MICs with values equal to or less than 500 mgmL as potentinhibitors MICs between 600 and 1500 mgmL and MIC moderateinhibitors as above 1600 mgmL as weak inhibitors Webster et al(2008) established as another satisfactory MIC value equal to or lessthan 1000 mgmL In this sense the result reported in this investiga-tion demonstrated the inhibitory potential of Terminalia catappaagainst this important bacteria responsible to gastric ulcer

5 Conclusions

We determined that the FrAq from Terminalia catappa leaveshas an important anti-Helicobacter pylori activity excellent pre-ventive and curative effects on acute and chronically inducedgastric ulcers The mechanisms involved in the gastroprotectionare related to the NO pathway an increase in the mucus and theendogenous prostaglandins and this fraction was able to healulcers through the inhibition of MMP-9 and MMP-2 activities

Acknowledgments

This study was supported by the Biota-FAPESP project (Fundaccedilatildeo

de Amparo agrave Pesquisa do Estado de Satildeo Paulo) CNPq (Conselho

Nacional de Desenvolvimento Cientiacute1047297co e Tecnoloacutegico) and CAPES(Coordenaccedilatildeo de Aperfeiccediloamento Pessoal de Niacutevel Superior)

References

Aligannis N Kalpotzakis E Mitaku S Chinou IB 2001 Composition andantimicrobial activity of the essential oils of two Origanum species Journal of

Agricultural and Food Chemistry 49 4168ndash

4170Al-Jiboury H Kaunitz JD 2012 Gastroduodenal mucosal defense CurrentOpinion of Gastroenterology 28 594ndash601

Arakawa T Watanabe T Tanigawa T Tominaga K Fujiwara Y Morimoto K2012 Quality of ulcer healing in gastrointestinal tract its pathophysiology andclinical relevance World Journal of Gastroenterology 18 4811ndash4822

Arrieta J Benitez J Flores E Castillo C Navarrete A 2003 Puri1047297cation of gastroprotective triterpenoids from stem bark of Amphipterygium adstringensroles of prostaglandins sulfhydryls nitric oxide and capsaicin neurons PlantaMedica 69 905ndash909

Bradford MM 1976 A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye bindingAnalytical Biochemistry 72 248ndash254

Brzozowska I Strzalka M Drozdowicz D Konturek SJ Brzozowski T 2013Mechanisms of esophageal protection gastroprotection and ulcer healing bymelatonin Implications for the therapeutic use of melatonin in gastroesopha-geal re1047298ux disease (GERD) and peptic ulcer disease Current PharmaceuticalDesign httpdxdoi org1021741381612819666131119110258 (Epub ahead of print)

Brzozowski T Konturek PC Konturek SJ Pajdo R Schuppan D Drozdowicz DPtak A Pawlik M Nakamura T Hahn EG 2000 Involvement of cycloox-ygenase (COX)-2 products in acceleration of ulcer healing by gastrin andhepatocyte growth factor Journal of Physiology and Pharmacology 51 751ndash773

Brzozowski T Konturek PC Sliwowski Z Pajdo R Drozdowicz D Kwiecien SBurnat G Konturek SJ Pawlik WW 2006 Prostaglandincyclooxygenasepathway in ghrelin-induced gastroprotection against ischemia-reperfusioninjury Journal of Pharmacology and Experimental Therapeutics 319 477ndash487

Camargo MC Garciacutea A Riquelme A Otero W Camargo CA Garciacutea THCandia R Bruce MG Rabkin CS 2014 The problem of Helicobacter pyloriresistance to antibiotics a systematic review in Latin America Clinical andSystematic Reviews 109 485ndash495

Chatterjee A Chatterjee S Das S Saha A Chattopadhyay S Bandyopadhyay SK2012 Ellagic acid facilitates indomethacin-induced gastric ulcer healing via COX-2 up-regulation Acta Biochimica et Biophysica Sinica 44 565ndash576

Chen PS Li JH 2005 Chemopreventive effect of punicalagin a novel tannincomponent isolated from Terminalia catappa on H-ras-transformed NIH3T3cells Toxicology Letter 163 44ndash53

Chen PS Li JH Liu TY Lin TC 2000 Folk medicine Terminalia catappa and its

major tannin component punicalagin are effective against bleomycin-inducedgenotoxicity in Chinese hamster ovary cells Cancer Letter 152 115ndash122

Chen B Tuuli MG Longtine MS Shin JS Lawrence R Inder T Michael ND2012 Pomegranate juice and punicalagin attenuate oxidative stress andapoptosis in human placenta and in human placental trophoblasts American

Journal of Physiology and Endocrinology Metabolism 302 E1142ndashE1152CLSI Manual Clinical and Laboratory Standards Institute 2006 Methods for

dilution antimicrobial susceptibility tests for bacteria that grow aerobicallyapproved standards 6ordf ed Performance standards for antimicrobial suscept-ibility testing Sixteenth informational supplement M100-S16 (tab 2J) Clinicaland Laboratory Standards Institute Wayne PA

De Foneska A Kaunitz JD 2010 Gastroduodenal mucosal defense CurrentOpinion Gastroenterology 26 604ndash610

de Carvalho KI Bonamin F Dos Santos RC Peacuterico LL Beserra FP de Sousa DPFilho JM da Rocha LR Hiruma-Lima CA 2014 Geraniol-a 1047298avoring agentwith multifunctional effects in protecting the gastric and duodenal mucosaNaunyn Schmiedebergs Archieves of Pharmacology 387 355ndash365

Eom CS Park SM Myung SK Yun JM Ahn JS 2011 Use of acid-suppressive

drugs and risk of fracture a meta-analysis of observational studies AnnalsFamily Medicine 9 257ndash267Fan YM Xu LZ Gao J Wang Y Tang XH Zhao XN Zhang ZX 2004

Phytochemical and antiin1047298ammatory studies on Terminalia catappa Fitoterapia75 253ndash260

Fyhrquist P Mwasumbi L Haeggstroumlm CA Vuorela H Hiltunen R Vuorela P2002 Ethnobotanical and antimicrobial investigation on some species of Terminalia and Combretum (Combretaceae) growing in Tanzania Journal of Ethnopharmacology 79 169ndash177

Genestra M 2007 Oxyl radicals redox-sensitive signalling cascades and antiox-idants Cell Signalling 19 1807ndash1819

Germoseacuten-Robineau L 2014 Farmacopea Vegetal Carbentildea CICY editorial YucataacutenMexico p 360

Gyenge M Amagase K Kunimi S Matsuoka R Takeuchi K 2013 Roles of pro-angiogenic and anti-angiogenic factors as well as matrix metalloproteinases inhealing of NSAID-induced small intestinal ulcers in rats Life Science 93441ndash447

Iino T Nakahara K Miki W Kiso Y Ogawa Y Kato S Takeuchi K 2001 Lessdamaging effect of whisky in rat stomachs in comparison with pure ethanol

Digestion 64 (214ndash221) 2001

1 - α e β punicalagin

2 - α e βpunicalin

00 50 100 150 200 250 300 350 400

Retention Time [min]

0

1000000

2000000

I n t e n

s i t y [ micro V ]

T catappa FrAq - CH5

Fig 10 Chromatogram from HPLCndashPDA of the aqueous fraction of the leaves of Terminalia catappa Hydro Column 1047298ow 1 mLmin gradient method 5ndash60 MeOH40 min HPLCndashPDA (Jascos) 270 nm

L Pinheiro Silva et al Journal of Ethnopharmacology 159 (2015) 285ndash 295294

7262019 1-s20-S0378874114008095-main

httpslidepdfcomreaderfull1-s20-s0378874114008095-main 1011

lansoprazole or the FrAq over 14 days Important toxicity para-meters including changes in body weight mortality and weight of the vital organs were determined as shown in Table 2 Treatmentwith FrAq for 14 days with 25 mgkg did not signi1047297cantly alter thebody weight of animals compared to the vehicle control over the14-days treatment period (data not shown) and the averageweights of the major organs of the FrAq-treated subjects werecomparable to those of the control group Table 2 also shows thatFrAq did not affect several biochemical parameters Treatment didnot signi1047297cantly alter the body weight the weight of vital organsand biochemical parameters of serum However Mininel et al(2014) evaluated the mutagenicity of the hydroalcoholic extractfrom the leaves of Terminalia catappa with the Ames test andillustrated that this extract exhibited mutagenic activity in vitro athigh concentrations leading them to recommend caution whenusing this compound for medicinal purposes

This study also evaluated the activity of aqueous fraction (FrAq)against Helicobacter pylori and determination of minimal inhibitoryconcentration (MIC) of 1250 mgmL considered an important anti-microbial activity The literature do not show a consensus in relationto MIC values obtained for natural products Aligannis et al (2001)consider MICs with values equal to or less than 500 mgmL as potentinhibitors MICs between 600 and 1500 mgmL and MIC moderateinhibitors as above 1600 mgmL as weak inhibitors Webster et al(2008) established as another satisfactory MIC value equal to or lessthan 1000 mgmL In this sense the result reported in this investiga-tion demonstrated the inhibitory potential of Terminalia catappaagainst this important bacteria responsible to gastric ulcer

5 Conclusions

We determined that the FrAq from Terminalia catappa leaveshas an important anti-Helicobacter pylori activity excellent pre-ventive and curative effects on acute and chronically inducedgastric ulcers The mechanisms involved in the gastroprotectionare related to the NO pathway an increase in the mucus and theendogenous prostaglandins and this fraction was able to healulcers through the inhibition of MMP-9 and MMP-2 activities

Acknowledgments

This study was supported by the Biota-FAPESP project (Fundaccedilatildeo

de Amparo agrave Pesquisa do Estado de Satildeo Paulo) CNPq (Conselho

Nacional de Desenvolvimento Cientiacute1047297co e Tecnoloacutegico) and CAPES(Coordenaccedilatildeo de Aperfeiccediloamento Pessoal de Niacutevel Superior)

References

Aligannis N Kalpotzakis E Mitaku S Chinou IB 2001 Composition andantimicrobial activity of the essential oils of two Origanum species Journal of

Agricultural and Food Chemistry 49 4168ndash

4170Al-Jiboury H Kaunitz JD 2012 Gastroduodenal mucosal defense CurrentOpinion of Gastroenterology 28 594ndash601

Arakawa T Watanabe T Tanigawa T Tominaga K Fujiwara Y Morimoto K2012 Quality of ulcer healing in gastrointestinal tract its pathophysiology andclinical relevance World Journal of Gastroenterology 18 4811ndash4822

Arrieta J Benitez J Flores E Castillo C Navarrete A 2003 Puri1047297cation of gastroprotective triterpenoids from stem bark of Amphipterygium adstringensroles of prostaglandins sulfhydryls nitric oxide and capsaicin neurons PlantaMedica 69 905ndash909

Bradford MM 1976 A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye bindingAnalytical Biochemistry 72 248ndash254

Brzozowska I Strzalka M Drozdowicz D Konturek SJ Brzozowski T 2013Mechanisms of esophageal protection gastroprotection and ulcer healing bymelatonin Implications for the therapeutic use of melatonin in gastroesopha-geal re1047298ux disease (GERD) and peptic ulcer disease Current PharmaceuticalDesign httpdxdoi org1021741381612819666131119110258 (Epub ahead of print)

Brzozowski T Konturek PC Konturek SJ Pajdo R Schuppan D Drozdowicz DPtak A Pawlik M Nakamura T Hahn EG 2000 Involvement of cycloox-ygenase (COX)-2 products in acceleration of ulcer healing by gastrin andhepatocyte growth factor Journal of Physiology and Pharmacology 51 751ndash773

Brzozowski T Konturek PC Sliwowski Z Pajdo R Drozdowicz D Kwiecien SBurnat G Konturek SJ Pawlik WW 2006 Prostaglandincyclooxygenasepathway in ghrelin-induced gastroprotection against ischemia-reperfusioninjury Journal of Pharmacology and Experimental Therapeutics 319 477ndash487

Camargo MC Garciacutea A Riquelme A Otero W Camargo CA Garciacutea THCandia R Bruce MG Rabkin CS 2014 The problem of Helicobacter pyloriresistance to antibiotics a systematic review in Latin America Clinical andSystematic Reviews 109 485ndash495

Chatterjee A Chatterjee S Das S Saha A Chattopadhyay S Bandyopadhyay SK2012 Ellagic acid facilitates indomethacin-induced gastric ulcer healing via COX-2 up-regulation Acta Biochimica et Biophysica Sinica 44 565ndash576

Chen PS Li JH 2005 Chemopreventive effect of punicalagin a novel tannincomponent isolated from Terminalia catappa on H-ras-transformed NIH3T3cells Toxicology Letter 163 44ndash53

Chen PS Li JH Liu TY Lin TC 2000 Folk medicine Terminalia catappa and its

major tannin component punicalagin are effective against bleomycin-inducedgenotoxicity in Chinese hamster ovary cells Cancer Letter 152 115ndash122

Chen B Tuuli MG Longtine MS Shin JS Lawrence R Inder T Michael ND2012 Pomegranate juice and punicalagin attenuate oxidative stress andapoptosis in human placenta and in human placental trophoblasts American

Journal of Physiology and Endocrinology Metabolism 302 E1142ndashE1152CLSI Manual Clinical and Laboratory Standards Institute 2006 Methods for

dilution antimicrobial susceptibility tests for bacteria that grow aerobicallyapproved standards 6ordf ed Performance standards for antimicrobial suscept-ibility testing Sixteenth informational supplement M100-S16 (tab 2J) Clinicaland Laboratory Standards Institute Wayne PA

De Foneska A Kaunitz JD 2010 Gastroduodenal mucosal defense CurrentOpinion Gastroenterology 26 604ndash610

de Carvalho KI Bonamin F Dos Santos RC Peacuterico LL Beserra FP de Sousa DPFilho JM da Rocha LR Hiruma-Lima CA 2014 Geraniol-a 1047298avoring agentwith multifunctional effects in protecting the gastric and duodenal mucosaNaunyn Schmiedebergs Archieves of Pharmacology 387 355ndash365

Eom CS Park SM Myung SK Yun JM Ahn JS 2011 Use of acid-suppressive

drugs and risk of fracture a meta-analysis of observational studies AnnalsFamily Medicine 9 257ndash267Fan YM Xu LZ Gao J Wang Y Tang XH Zhao XN Zhang ZX 2004

Phytochemical and antiin1047298ammatory studies on Terminalia catappa Fitoterapia75 253ndash260

Fyhrquist P Mwasumbi L Haeggstroumlm CA Vuorela H Hiltunen R Vuorela P2002 Ethnobotanical and antimicrobial investigation on some species of Terminalia and Combretum (Combretaceae) growing in Tanzania Journal of Ethnopharmacology 79 169ndash177

Genestra M 2007 Oxyl radicals redox-sensitive signalling cascades and antiox-idants Cell Signalling 19 1807ndash1819

Germoseacuten-Robineau L 2014 Farmacopea Vegetal Carbentildea CICY editorial YucataacutenMexico p 360

Gyenge M Amagase K Kunimi S Matsuoka R Takeuchi K 2013 Roles of pro-angiogenic and anti-angiogenic factors as well as matrix metalloproteinases inhealing of NSAID-induced small intestinal ulcers in rats Life Science 93441ndash447

Iino T Nakahara K Miki W Kiso Y Ogawa Y Kato S Takeuchi K 2001 Lessdamaging effect of whisky in rat stomachs in comparison with pure ethanol

Digestion 64 (214ndash221) 2001

1 - α e β punicalagin

2 - α e βpunicalin

00 50 100 150 200 250 300 350 400

Retention Time [min]

0

1000000

2000000

I n t e n

s i t y [ micro V ]

T catappa FrAq - CH5

Fig 10 Chromatogram from HPLCndashPDA of the aqueous fraction of the leaves of Terminalia catappa Hydro Column 1047298ow 1 mLmin gradient method 5ndash60 MeOH40 min HPLCndashPDA (Jascos) 270 nm

L Pinheiro Silva et al Journal of Ethnopharmacology 159 (2015) 285ndash 295294

7262019 1-s20-S0378874114008095-main

httpslidepdfcomreaderfull1-s20-s0378874114008095-main 1111

Iino T Tashima K Umeda M Ogawa Y Kato S Takata K Takeuchi K 2002Effect of ellagic acid on gastric damage induced in ischemic rat stomachsfollowing ammonia or reperfusion Life Science 70 1139ndash1150

Khalifa MA Hassan MKA Ashour OM Heeba G 2002 Evaluation of theantiulcer activity of pibutidine hydrochloride (IT-066) the new histamineH2-receptor antagonist in cold-restraint stressand ethanol-induced ulcermodels in rats Al Azhar Medical Journal 31 33ndash47

Kinoshita S Inoue Y Nakama S Ichiba T Aniya Y 2007 Antioxidant andhepatoprotective actions of medicinal herb Terminalia catappa L from OkinawaIsland and its tannin corilagin Phytomedicine 14 755ndash762

Kumar BG Kumari D RAjeshwar G Umadevi V Kotla NG 2014 Antiulceractivity of ethanolic extract of Terminalia catappa leaves against gastric ulcersby pyrolic ligation induced model in rats International Journal of Pharmaceu-tical Sciences and Drug Research 6 38ndash40

Laine L Takeuchi K Tarnawski A 2008 Gastric mucosal defense and cytoprotec-tion bench to bedside Gastroenterology 135 41ndash60

Lin CC Chen YL Lin JM Ujiie T 1997 Evaluation of the antioxidant andhepatoprotective activity of Terminalia catappa The American Journal of Chinese Medicine 25 (153ndash161) 1997

Li SL Zhao JR Ren XY Xie JP Ma QZ Rong QH 2013 Increased expressionof matrix metalloproteinase-9 associated with gastric ulcer recurrence World

Journal of Gastroenterology 19 4590ndash4595Lin CC Hsu YF Lin TC 1999 Effects of punicalagin and punicalin on

carrageenan-induced in1047298ammation in rats The American Journal of ChineseMedicine 27 371ndash376

Masuda T Yonemori S Oyama Y Takeda Y Tanaka T Andoh T Shinohara A Nakata M 1999 Evaluation of the antioxidant activity of environmentalplants activity of the leaf extracts from seashore plants Journal of AgricultureFood and Chemistry 47 1749ndash1754

Mei XT Xu DH Xu SK Zheng YP Xu S B 2013 Zinc(II)-curcumin acceleratesthe healing of acetic acid-induced chronic gastric ulcers in rats by decreasingoxidative stress and downregulation of matrix metalloproteinase-9 FoodChemistry and Toxicology 60 448ndash454

Megraud F Coenen S Versporten A Kist M Lopez-Brea M Hirschl AMAndersen LP Goossens H Glupczynski Y 2013 Helicobacter pylori resis-tance to antibiotics in Europe and its relationship to antibiotic consumptionGut 62 34ndash42

Mininel FJ Leonardo Junior CS Espanha LG Resende FA Varanda EA LeiteCQ Vilegas W Dos Santos LC 2014 Characterization and quanti1047297cationof compounds in the hydroalcoholic extract of the leaves from Terminaliacatappa Linn (Combretaceae) and their mutagenic activity Evidence-BasedComplementary and Alternative Medicine httpdxdoiorg1011552014676902 (Epub ahead of print)

Mishra V Agrawal M Onasanwo SA Madhur G Rastogi P Pandey HP Palit GNarender T 2013 Anti-secretory and cyto-protective effects of chebulinic acidisolated from the fruits of Terminalia chebula on gastric ulcers Phytomedicine 20506ndash511

Morimoto Y Shimohara K Oshima S Sukamoto T 1991 Effects of the new anti-ulcer agent KB-5492 on experimental gastric mucosal lesions and gastricmucosal defensive factors compared to those of teprenone and cimetidine

Japanese Journal of Pharmacology 57 495ndash505Nagappa AN Thakurdesai PA Venkat Rao N Singh J 2003 Antidiabetic activity

of Terminalia catappa Linn fruits Journal of Ethnopharmacology 88 45ndash50Nunes PH Martins M do C Oliveira R de C Chaves MH Sousa EA Leite JR

Veacuteras LM Almeida FR 2014 Gastric antiulcerogenic and hypokinetic activitiesof Terminalia fagifolia Mart Zucc (Combretaceae) Biomed Research International httpdxdoiorg101155201426174 ([Epub ahead of print])

Nunes AF Viana VSL Brito Junior EC Rabelo RS Nunes Filho DM Nunes PHMMartins MCC 2012 Antiulcerogenic activity of ethanol extract of the bark fromTerminalia catappa in gastric ulcer model induced by ethanol in Rattus norgegicusPharmacologyonline 9 98ndash101

Okabe S Roth JL Pfeiffer CJ 1971 A method for experimental penetratinggastric and duodenal ulcers in rats Observations on normal healing American

Journal of Digestive Diseases 16 277ndash284Okabe S Amagase K 2005 An overview of acetic acid ulcer modelsmdashthe history

and state of the art of peptic ulcer research Biological and Pharmaceutical

Bulletin 28 1321ndash1341Olfert ED Cross BM McWilliam AA 1993 Guide to the Care and Use of Experimental

Animals Canadian Council on Animal Care Ottawa Ontario pp 1ndash213

Oyagi A Ogawa K Kakino M Hara H 2010 Protective effects of a gastro-intestinal agent containing Korean red ginseng on gastric ulcer models in miceBMC Complementary and Alternative Medicine httpdxdoiorg1011861472-6882-10-45 (Published online August 18 2010)

Ozdil K Kahraman R Sahin A Calhan T Gozden EH Akyuz U Erer BSokmen MH 2013 Bone density in proton pump inhibitors users a prospec-tive study Rheumatology International 33 2255ndash2260

Pandya NB Tigari P Dupadahalli K Kamurthy H Nadendla RR 2013Antitumor and antioxidant status of Terminalia catappa against Ehrlich ascitescarcinoma in Swiss albino mice Indian Journal of Pharmacology 45 464ndash469

Pawlik M Ptak A Pajdo R Konturek PC Brzozowski T Konturek SJ 2001

Sensory nerves and calcitonin gene related peptide in the effect of ischemicpreconditioning on acute and chronic gastric lesions induced by ischemia-reperfusion Journal of Physiology and Pharmacology 52 569ndash581

Parreira P Duarte MF Reis CA Martins MC 2014 Helicobacter pylori infectiona brief overview on alternative natural treatments to conventional therapyCritical Reviews in Microbiology 10 1ndash12

Rafatullah S Tariq M Al-Yahya MA Mossa JS Ageel AM 1990 Evaluation of turmeric (Curcuma longa) for gastric and duodenal antiulcer activity in rats

Journal of Ethnopharmacology 29 25ndash34Rao CV Vijayakumar M 2007 Protective effect of (thorn)-catechin against gastric

mucosal injury induced by ischaemia-reperfusion in rats Journal of Pharmacyand Pharmacology 59 1103ndash1107

Ratnasooriya WD Dharmasiri MG 2000 Effects of Terminalia catappa seeds onsexual behaviour and fertility of male rats Asian Journal of Andrology 2 213ndash219

Shay H Komarov SA Fels SE Meraze D Gruentein M Siplet H 1945A simple method for the uniform production of gastric ulceration in the ratGastroenterology 5 43ndash61

Smith GS Mercer DW Cross JM Barreto JC Miller TA 1996 Gastric injury

induced by ethanol and ischemia-reperfusion in the rat Differing roles for lipidperoxidation and oxygen radicals Digestive Diseases and Sciences 41 1157ndash1164Takeuchi K 2010 Prostaglandin EP receptors and their roles in mucosal protection and

ulcer healing in the gastrointestinal tract Advances Clinical Chemistry 51 121ndash144Tang X Gao J Wang Y Fan YM Xu LZ Zhao XN Xu Q Qian ZM 2006 Effective

protection of Terminalia catappa L leaves from damage induced by carbontetrachloride in liver mitochondria Journal of Nutritional Biochemistry 17 177ndash178

Tarnawski AS Ahluwalia A Jones MK 2014 Increased susceptibility of aginggastric mucosa to injury the mechanisms and clinical implications World

Journal of Gastroenterology 20 4467ndash4482Takeuchi H Trang VT Morimoto N Nishida Y Matsumura Y Sugiura T 2014

Natural products and food components with anti-Helicobacter pylori activitiesWorld Journal of Gastroenterology 20 8971ndash8978

Thomson LAJ Evans B 2006 Terminalia catappa (tropical almond) 22 InElevitch CR (Ed) Species pro1047297les for paci1047297c Island agro1047298orestry permanentagriculture resources (PAR) 2006 langhttpwwwtraditionaltreeorgrang

Ueda S Yoshikawa T Takahashi S Ichikawa H Yasuda M Oyamada HTanigawa T Sugino S Kondo M 1989 Role of free radicals and lipidperoxidation in gastric mucosal injury induced by ischemia-reperfusion in rats

Scandinavian Journal of Gastroenterology 162 55ndash58Wagner H 2011 Synergy research approaching a new generation of phytophar-maceuticals Fitoterapia 82 34ndash37

Wallace JL 2008 Prostaglandins NSAIDs and gastric mucosal protection whydoesnt the stomach digest itself Physiological Review 88 1547ndash1565

Wallace JL Ma L 2001 In1047298ammatory mediators in gastrointestinal defense andinjury Experimental Biology and Medicine 226 1003ndash1015

Wang Y Zhang H Liang H Yuan Q 2013 Puri1047297cation antioxidant activity andprotein-precipitating capacity of punicalin from pomegranate husk FoodChemistry 138 437ndash443

Webster D Taschereau P Belland RJ Sand Rennie CRP 2008 Antifungalactivity of medicinal plant extracts preliminary screening studies Journal of Ethnopharmacology 115 140ndash146

Yeh CB Hsieh MJ Hsieh YS Chien MH Lin PY Chiou HL Yang SF 2012Terminalia catappa exerts antimetastatic effects on hepatocellular carcinomathrough transcriptional inhibition of matrix metalloproteinase-9 by modulatingNF-κB and AP-1 Activity Evidence-Based Complementary and Alternative Medi-cine httpdxdoiorg1011552012595292 (Published online 2012 November 8)

Yeh CB Yu YL Lin CW Chiou HL Hsieh MJ Yang SF 2014 Terminaliacatappa attenuates urokinase-type plasminogen activator expression throughErk pathways in Hepatocellular carcinoma BMC Complementary and Alter-native Medicine 14 141 httpdxdoiorg1011861472-6882-14-141

L Pinheiro Silva et al Journal of Ethnopharmacology 159 (2015) 285ndash 295 295