Journal of Ethnopharmacology ()

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Contents lists available at ScienceDirect
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journal homepage: www.elsevier.com/locate/jep
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Research Paper
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Lasa Pinheiro Silva a, Clio Damacena de Angelis a, Flavia Bonamin a, Hlio Kushima a,
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Francisco Jos Mininel b, Lourdes Campaner dos Santos b, Flavia Karina Delella c,
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Sergio Luis Felisbino c, Wagner Vilegas d, Lucia Regina Machado da Rocha a,
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Matheus Aparecido dos Santos Ramos f, Tais Maria Bauab f,
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e,n,1
, Clelia Akiko Hiruma-Lima a,n,1
22 Q1 Walber Toma
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Univ. Estadual Paulista-UNESP Departamento de Fisiologia, Instituto de Biocincias, CEP 18618-970 Botucatu, SP, Brazil
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Univ. Estadual Paulista-UNESP Departamento de Qumica Orgnica, Instituto de Qumica, CEP 14800-900, Araraquara, SP, Brazil
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Univ. Estadual Paulista-UNESP Departamento de Morfologia, Instituto de Biocincias, CEP 18618-970, Botucatu, SP, Brazil
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Univ. Estadual Paulista-UNESP Campus Experimental do Litoral Paulista, CEP 11330-900 So Vicente, SP, Brazil
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Universidade Santa Ceclia Ps-Graduao em Sustentabilidade de Ecossistemas Costeiros e Marinhos, Rua Oswaldo Cruz, 266,
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Boqueiro, CEP 11045907 Santos, SP, Brazil
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Univ. Estadual Paulista-UNESP Departamento de Cincias Biolgicas, Faculdade de Cincias Farmacuticas, CEP 14801-902, Araraquara, SP, Brazil
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art ic l e i nf o
a b s t r a c t
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Article history:
Ethnopharmacological relevance: Terminalia catappa L. (Combretaceae) is a medicinal plant listed as a
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Received 22 August 2014
pharmacopeia vegetable from Caribbean to treat gastritis. The objective of this study was to evaluate the Q7
Received in revised form
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gastroprotective and healing effect of the aqueous fraction (FrAq) obtained from the leaves of Terminalia
3 November 2014
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catappa and to determine the antiulcer mechanism of action in experimental rodent models and its
Accepted 13 November 2014
activity to Helicobacter pylori.
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Material and methods: In rodents, the FrAq was challenged by different necrotizing agents, such as
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Keywords:
absolute ethanol and ischemiareperfusion injury. The antiulcer mechanism of action of FrAq was
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Terminalia catappa L.
assessed and the healing effects of the fraction after seven and 14 days of treatment was evaluated by
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Combretaceae
matrix metalloproteinase activity (MMP-2 and MMP-9). The toxicological effect of subacute treatment
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Gastric healing action
with FrAq during 14 days of treatment was also analyzed. The anti-Helicobacter pylori activity was
Helicobacter pylori
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determined by microdilution. The phytochemical study of the fraction was analyzed by experiments with
MMP-9
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FIA-ESI-IT-MSn (Direct Flow Analysis-ionization Electrospray Ion Trap Tandem Mass Spectrometry) and
MMP-2
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high performance liquid chromatography (HPLC) coupled to a photodiode array (PDA).
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Results: Oral treatment with FrAq (25 mg/kg) signicantly decreased the number of ulcerative lesions
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induced by ethanol and ischemia/reperfusion injury. The action of FrAq was mediated by the activation of
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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 signicantly
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reduced the lesion area (80% and 37%, respectively) compared to the negative control group. Analyses of
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MMP-9 and MMP-2 activity from gastric mucosa conrmed the accelerated gastric healing effect of FrAq.
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This extract also presented considerable activity against Helicobacter pylori. The mass spectrum and
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MS/MS of the aqueous fraction indicates the existence of many different phenolic compounds, including
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punicalagin, punicalin, and gallagic acid, among others.
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Conclusions: We concluded that FrAq from Terminalia catappa leaves has excellent preventive and
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curative effects on acute and chronic induced gastric ulcers and showed an important prole against
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Helicobacter pylori.
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& 2014 Published by Elsevier Ireland Ltd.
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Corresponding authors. Tel.: 55 14 38800312; fax: 55 1438153744.
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E-mail addresses: walbertoma@gmail.com (W. Toma), hiruma@ibb.unesp.br (C. Akiko Hiruma-Lima).
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Contributed equally to the supervision of this study.
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http://dx.doi.org/10.1016/j.jep.2014.11.025
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0378-8741/& 2014 Published by Elsevier Ireland Ltd.
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Journal of Ethnopharmacology

Terminalia catappa L.: A medicinal plant from the Caribbean

pharmacopeia with anti-Helicobacter pylori and antiulcer action
in experimental rodent models

Please cite this article as: Pinheiro Silva, L., et al., Terminalia catappa L.: A medicinal plant from the Caribbean.... Journal of
Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.11.025i

L. Pinheiro Silva et al. / Journal of Ethnopharmacology ()

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1. Introduction

2.2. Chemicals and reagents

Terminalia catappa is a species of the family Combretaceae and

popularly known in Brazil as amendoeira, amendoeira-da-praia,
amendoeira-da-ndia, cuca, guarda-sol, castanheira da ndia,
castanhola and chapu-de-Sol. This plant is widely distributed
in countries with tropical and subtropical climates, especially in
coastal regions due to the plant's ability to easily adapt to salinity
and winds (Thomson and Evans, 2006). In Asian countries, the
leaves of this species are commonly used for the treatment of
dermatitis, hepatitis, diarrhea and pyresis (Chen et al., 2000). This
plant was also listed in Pharmacopeia vegetables of the Caribbean,
where the leaves of this plant are used in a decoction for gastritis
and urinary infection (Germosn-Robineau, 2014). The literature
also shows that the polar extract from different parts (leaves, fruits
and bark) of Terminalia catappa have shown the following biological
activities: antimicrobial, antifungal (Fyhrquist et al., 2002), antioxidant (Masuda et al., 1999; Chen and Li, 2005; Pandya et al., 2013),
antimetastatic (Yeh et al., 2012, 2014), anti-inammatory (Fan et al.,
2004; Lin et al., 1999), hepatoprotective (Lin et al., 1997; Chen et al.,
2000; Tang et al., 2006; Chen and Li, 2005), mutagenic (Mininel
et al., 2014), aphrodisiac (Ratnasooriya and Dharmasiri, 2000) and
antidiabetic (Nagappa et al., 2003). Nunes et al. (2012) described the
gastroprotective effect of the ethanolic extract obtained from bark
of this species and Kumar et al. (2014) previously reported the
antisecretory effect of the ethanolic extract from leaves, although
the mechanism responsible for this preventive effect remains
unknown, thus the use of this specie against gastric bacteria is
relevant. Infection caused to Helicobacter pylori is considered the
most prevalent cause of gastric diseases such as ulcers, dyspepsia
and stomach cancer (Camargo et al., 2014). The drug therapy available for the treatment of diseases caused by this microorganism has
limitation such as the high rate of resistance to conventional drugs
and inappropriate patient treatment, as presented to numerous side
effects (Megraud et al., 2013). In this sense, the use of natural
products as an alternative to control this bacterium has shown to be
satisfactory, what drives the improvement of investigations of medicinal plants as adjuvant or new antimicrobial drugs (Parreira et al.,
2014; Takeuchi et al., 2014).
The aims of this present work were to characterize the antiHelicobacter pylori activity and antiulcer effect of the aqueous
fraction obtained from leaves of Terminalia catappa and determine
the mechanism of action for this medicinal species.

HPLCgrade methanol (MeOH) and acetonitrile were purchased

from J.T. Baker (Baker-Mallinckrodt, Phillipsburg, NJ, USA). HPLC
grade water (18 M cm) was obtained using a direct Milli-Q purication system (Millipore Co., Bedford, MA, USA). Sep-Pak RP18
cartridges (500 mg/mL) for solid-phase extraction (SPE) were purchased from Phenomenex Co. (Torrance, CA, USA).

2. Material and methods

2.1. Preparation of the aqueous fraction
The leaves of Terminalia catappa were collected in January
(2010) by Msc. Lasa Pinheiro Silva in Santos/SP, Brazil. The specimen was identied by Msc. Paulo Salles Penteado Sampaio, and
the voucher specimen was deposited to the Herbarium at the
Universidade Santa Ceclia, Santos, SP, Brazil, under the register
M. Tomaz 01, for future reference. The leaves from Terminalia
catappa (378.54 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 ux of 2.0 mL/min/kg. The hydroalcoholic extract
submitted to the rotary evaporator (45 1C) resulted in 33.1 g of
product (a yield of 8.75%). This extract was partitioned into three
fractions: a hexane fraction FrHex (7.09 g, 24.96%), an ethyl
acetate fraction FrEtOAc (12.22 g, 43.02%) and an aqueous
fraction FrAq (8.43 g, 26.16%). As the commonly used formulation of this medicinal plant is a decoction, we chose to utilize the
FrAq in the experimental pharmacological protocols.

2.3. Apparatus
The mass spectrometry experiments were performed on LCQ
Fleet equipment (Thermo Scientics) equipped with the dispersal of
the directly introduced sample via ow injection analysis (FIA). The
studied matrix was analyzed by electrospray ionization (ESI), and
multiple stages of fragmentation (MS2, MS3, MSn) were performed at
an ion trap (IT) interface. The negative mode was selected for the
generation and analysis of the mass spectra for the rst order (MS)
and for the remaining multi-stage experiments under the following
conditions: capillary voltage,  25 V; voltage spray,  5 kV; capillary
temperature, 275 1C. A carrier gas (N2) with a ow of 8 arbitrary
units (A.U.) was used, and the collision gas was helium (He). The
track acquisition was 1002000 m/z. Xcalibur version 1.3 software
(Thermo Finigans) was used to acquire and process the data.
For the FIA-ESI-IT-MSn assay, 10 mg of the aqueous fraction was
dissolved in 1 mL of MeOH:H2O (1:1, v/v) after using an ultrasonic
bath for 5 min. The samples were then ltered through a 0.22 mm
PTFE lter, and aliquots of 20 mL were directly injected into the
FIA-ESI-IT-MSn system.
For the HPLCPDA a clean-up step was performed to remove any
contaminants; the solution was puried by solid phase extraction
(SPE) using Phenomenex Strata C18 cartridges (500 mg of stationary
phase) that were previously activated with 5 mL of MeOH and
equilibrated with 5 mL of MeOH:H2O (1:1, v/v). The dried aqueous
fraction was diluted to 10 mg/mL in HPLC solvent. A 20 mL aliquot
was injected directly into the HPLCPDA with detection at 270 nm.
The identication of the different compounds in the chromatographic prole of the aqueous fraction was done by comparing their
retention times (tr) and the UV spectra with those isolates previously described in the literature (Mininel et al., 2014).
2.4. Animals
Male Wistar rats weighing 180250 g were obtained from breeding at the Biotrio Central at the Universidade Estadual Jlio de
Mesquita Filho (UNESP), Botucatu-SP. Animals were fed with a
certied Nuvilab CR-diet, with free access to tap water and were
housed on a 12 h light/dark cycle at 6071% humidity and a
temperature of 2272 1C. The UNESP Institutional Animal Care and
Use Committee, following the recommendations of the Canadian
Council on Animal Care (Olfert et al., 1993), approved all of the
employed protocols under number 18/05.
2.5. Evaluation of the gastroprotection activity of the FrAq
2.5.1. Gastric ulcer induced by absolute ethanol
The rats were divided into ve treatment groups (n 714)
and fasted for 12 h prior to receiving an oral dose of the vehicle
(saline 10 mL/kg), carbenoxolone (100 mg/kg) and FrAq (12.5, 25
and 100 mg/kg). After 60 min, all groups were orally treated with
1 mL of absolute ethanol for the gastric ulcer induction. One hour
later, animals were killed and their stomachs excised. The incision
into each stomach was performed along the greater curvature and
examined for linear hemorrhagic lesions in the glandular region
(Morimoto et al., 1991). Then, the stomachs were photographed and

Please cite this article as: Pinheiro Silva, L., et al., Terminalia catappa L.: A medicinal plant from the Caribbean.... Journal of
Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.11.025i

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L. Pinheiro Silva et al. / Journal of Ethnopharmacology ()

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the extent of the lesions was measured by ulcerative lesion area

(U.L.A.) in mm2 by the program AVSoft BioView Spectras.
2.5.2. Gastric ulcer induced by ischemiareperfusion
Ischemiareperfusion damage was produced in rats by a
method proposed by Ueda et al. (1989). Rats (n 79) were orally
administered saline (10 mL/kg), lansoprazole (30 mg/kg) and FrAq
(the lower effective dose of 25 mg/kg). Thirty minutes later, the
animals were anaesthetized by intramuscular injection of ketamine (50 mg/kg) and xylazine (10 mg/kg). The left side of the
abdomen was shaved, and an incision was made. Briey, the celiac
artery was dissected, freed of excess fat and clamped for 30 min
(ischemia phase) using a micro-bulldog clamp. Re-oxygenation
was allowed by removing the clamp for 60 min (reperfusion
phase). At the end of this period, the animals were killed and
the stomachs were excised and opened along the great curvature
for the detection of the U.L.A.
2.6. Determination of mechanisms of action from FrAq
2.6.1. Gastric secretion in lesions induced by pylorus ligature
The method of Shay et al. (1945) was used with modication. Rats
(n 68) were fasted for 12 h and, immediately after pylorus ligature,
saline (10 mL/kg), cimetidine (100 mg/kg) or FrAq (25 mg/kg) was
administered intraduodenally or orally. The rats were killed 4 h later,
their abdomens were opened and the stomachs removed. The gastric
content was collected to determine the total amount of gastric-juice
acid (mL). Distilled water was added, and the resultant solution was
centrifuged at 3000g for 10 min. The hydrogen ion concentrations
(mEq/mL/4 h) were recorded in the gastric secretion by adjusting the
supernatant volume by titration to pH 7.0 with 0.01 N NaOH.
2.6.2. Determination of the gastric mucus content
This assay was done as described by Rafatullah et al. (1990)
with modication. After a 12 h fast, rats (n 6) received saline
(10 mL/kg), carbenoxolone (100 mg/kg) and FrAq from Terminalia
catappa (25 mg/kg) orally. The pylorus was ligated thirty minutes
after treatment. The animals were killed 4 h after pylorus ligation
and the glandular portion of the stomachs was removed and
weighed. Each segment was immediately immersed in 10 mL of
0.1% Alcian blue solution (0.16 M sucrose/0.05 M sodium acetate,
pH 5.8) for 2 h, followed by two successive rinses with 10 mL of
0.25 M sucrose (the rst for 15 min and the second for 45 min) to
remove the excess dye. Each stomach was then transferred to
0.5 M magnesium chloride solution for 2 h. Four milliliters of the
dye solution was then vigorously shaken with an equal volume of
ether, the resulting emulsion was centrifuged at 2000g, and the
absorbance of the aqueous layer was measured at 580 nm. The
amount of blue dye extracted per gram of wet glandular tissue
was then calculated from a standard curve of dye prepared in a
sucroseacetate solution.
2.6.3. Determination of the role of nitric oxide (NO), prostaglandin
(PGE) and sulfhydryl compounds (SH) in gastric protection
Male rats (n 5) were divided into nine groups and pretreated with either saline, L-NAME (N-nitro-L-arginine methyl ester,
70 mg/kg) an inhibitor of NO synthesis, INDO (indomethacin,
30 mg/kg) an inhibitor of PGE or NEM (N-ethylmaleimide,
10 mg/kg) a blocker of SH compounds (Arrieta et al., 2003).
Thirty minutes after the pretreatment, the animals were administered (p.o.) saline (10 mL/kg), carbenoxolone (100 mg/kg) and FrAq
(25 mg/kg). After 60 min, all groups received 1 mL absolute
ethanol to induce gastric ulcers. One hour after receiving ethanol
the rats were killed for the determination of gastric lesions.

2.7. Effect of FrAq healing acetic acid-induced gastric lesions

The experiment was performed according to the method described by Okabe et al. (1971). Six groups (n56) of male Wistar rats
were fasted for 12 h before this experiment. Under anesthesia, a
laparotomy was done in all animals through a midline epigastric
incision. A plastic 4.2 mm internal diameter tube was rmly applied
to 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 bathed
with saline (20 1C) to avoid adherence to the external surface of the
ulcerated region and then the abdomen was then closed and all
the animals were fed normally. This process resulted in a chronic
ulceration of the mucosa and submucosa, with an approximate ulcer
area of 13.8 mm2. FrAq (25 mg/kg) from Terminalia catappa, lansoprazole (30 mg/kg) or saline (10 mL/kg) were administered for the
determination of the healing effects by the subacute treatment
during 7 and 14 days. All treatments were done orally once a day
beginning one day after surgery. One day after the last drug
administration, the rats were killed and the stomachs were removed.
The gastric lesions were evaluated by examining the inner gastric
surface with a dissecting magnifying glass.
2.7.1. Extraction of total protein and zymography
Tissue with gastric ulcer from each experimental group described
previously was used to extract total protein. The extraction was
carried out following the ratio of 30 mg of tissue: 0.1 mL of 50 mM
TrisHCl solution, pH 7.5, containing 0.25% Triton X-100, 10 mM
CaCl2 and protease inhibitor cocktail (P-8849 Sigma-Aldrich,
St Louis, MO, USA) by crushing with the Polytron type homogenizer.
The homogenate was centrifuged at 4000g for 20 min at 4 1C. The
supernatant was collected and protein content was quantied by the
Bradford (1976).
Samples of extracted proteins (28 g) of the gastric ulcer from
different experimental groups treated during 7 and 14 days were
subjected to electrophoresis under non-reducing conditions on 8%
polyacrylamide gel copolymerized with 0.1% puried gelatin
(Sigma-Aldrich Co. LLC. St. Louis, MO, U.S.A.). After electrophoresis,
the gels were subjected to two washes of 15 min in a solution of
2.5% Triton X-100 to remove SDS, and two washes of 5 min in
50 mM pH 8.0 TrisHCl buffer. Subsequently, gels were incubated
in 50 mM TrisHCl buffer, pH 8.0, containing 5 mM of CaCl2 for
22 h at 37 1C. Finally, the gels were stained with Coomassie
Brilliant Blue R-250 (Sigma-Aldrich Co. LLC. St. Louis, MO, U.S.A.).
The relative molecular weight of the bands was determined
according to the molecular weight standard (Precision Plus Protein, BIO-RAD) used in electrophoresis. The bands obtained
through zymography were scanned and analyzed by densitometry.
The 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 from
each experimental group was pooled together for extraction. The
zymography with pooled samples was repeated three time. Values
were plotted in a histogram showing the ratio of the IOD of the
treated groups to the control group IOD.
2.7.2. Toxicological evaluation
Some toxicological parameters were also obtained from three
groups of animals subjected to the healing gastric ulcer model and
treated orally during 14 consecutive days once a day with: FrAq
(25 mg/kg) from Terminalia catappa leaves, lansoprazole (30 mg/kg)
and saline (10 mL/kg). Body weight was recorded daily throughout
the experimental period, and the macroscopic analyses and weight of
vital organs (liver, kidneys, heart, spleen and lungs) were compared

Please cite this article as: Pinheiro Silva, L., et al., Terminalia catappa L.: A medicinal plant from the Caribbean.... Journal of
Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.11.025i

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L. Pinheiro Silva et al. / Journal of Ethnopharmacology ()

400
n.s.

300
2

between either the FrAq or lansoprazole and the vehicle-treated

groups to evaluate subacute toxicity. On the day after the last drug
administration, the rats from each treatment group were killed, and
their blood was collected. The blood samples were then centrifuged
(3000g for 10 min), and the serum obtained was frozen at  20 1C
until biochemical analysis. Serum biochemical parameters, including
glucose, urea, creatinine, -glutamyl transpeptidase (-GT), aspartate
aminotransferase (AST) and alanine aminotransferase (ALT), were
measured using an automated biochemical analyzer (SBA-200,
Companhia Equipadora de Laboratrios Modernos, So Paulo, Brazil).

**

***

200

100
2.8. Minimum inhibitory concentration (MIC)
We used Helicobacter pylori ATCC 43504 using a microdilution
technique following by CLSI (2006) with modications to determine
the minimal inhibitory concentration (MIC) values. Helicobacter pylori
was inoculated on Mueller-Hinton agar plates containing 5% sheep
blood and incubated at 36 1C for 72 h, in 10% CO2 atmosphere.
Inoculates were prepared in the same medium at a density adjusted
to a 2.0 McFarland turbidity standard. The working suspension for
microorganism was diluted 1:10 and a 100 mL volume was added to
each 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 2%
DMSO, ranging from 0.5 to 1000 mg/mL were obtained when a 100 mL
volume of the fraction was transferred to the rst well of each row,
and serial 2-fold dilutions were performed. Amoxicillin was used as
reference antimicrobial compound and the MICs were recorded after
incubation of the microplates at 36 1C for 72 h in a 10% CO2 atmosphere. The MICs were recorded as the lowest concentration at
which no growth was observed. This record was facilitated by
addition of 20 mL of resazurin solution (100 mg/mL) as revelator to
each of well and incubation until 2 h. A pink color indicated bacterial
growth and a blue color indicated a non-bacterial growth.

***
0
Vehicle Carbenoxolone
100 mg/kg

12.5

25

100

FrAq (mg/kg)

Fig. 1. Effect of pretreatment with the aqueous fraction (FrAq) obtained from the
leaves of Terminalia catappa on ethanol-induced gastric ulcers in rats. The animals
orally received saline solution (vehicle), carbenoxolone or FrAq. The results are
expressed as the mean 7 S.E.M. (n 714), and statistical signicance was determined by one-way analysis of variance (ANOVA) followed by Dunnett's test
(nn p o0.01, nnn p o0.001 and n.s. no signicant differences).

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ULA (mm)

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ULA (mm )

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2.9. Statistical analysis

The results were expressed as the mean 7standard error of the
mean (S.E.M.) of the parameters obtained. Statistical comparisons
were done by one-way analysis of variance (ANOVA) followed by
Dunnett's or Tukey's post hoc test, with the level of signicance set
at np o0.05; nnp o0.01 and nnnp o0.001.

0
Vehicle

Lansoprazole
30 mg/kg

FrAq
25 mg/kg

3. Results

Fig. 2. Effect of pretreatment with the aqueous fraction (FrAq) obtained from the
leaves of Terminalia catappa on ischemiareperfusion induced gastric ulcers in rats.
The animals orally received saline solution (vehicle), lansoprazole or FrAq. The
results are expressed as the mean 7 S.E.M. (n 79), and statistical signicance was
determined by one-way analysis of variance (ANOVA) followed by Dunnett's test,
with the level of signicance set at nnp o 0.01 and nnnp o0.001.

In the present study, we evaluated the protective effect of FrAq

obtained from the leaves of Terminalia catappa against the gastric
mucosa damage induced by absolute ethanol. To establish a dose
response prole for the antiulcer activity of FrAq, we used varying
doses of FrAq (12.5, 25 and 100 mg/kg body weight) to identify the
lowest dose that could elicit an optimal gastroprotective effect
(Fig. 1). The oral administration of ethanol rapidly penetrated the
gastric mucosa and caused membrane damage, erosion, and hemorrhagic ulcerations. The pre-treatment of rats with FrAq signicantly
inhibited the formation of gastric lesions by 41% (25 mg/kg) and 36%
(100 mg/kg) compared to vehicle-treated control group (po0.001).
A lower dose of FrAq (12.5 mg/kg) evaluated in this study did not
show a gastroprotective effect against ethanol (p40.05). We also
observed no signicant differences in the gastroprotective effect of
FrAq at doses of 25 or 100 mg/kg (p40.05). Thus, the subsequent
experiments with FrAq were carried out the lower dose of 25 mg/kg
administered orally.
This study evaluated the ability of FrAq to protect the gastric
mucosa of rats from oxidative damage induced during an I/R procedure (Fig. 2). We observed that oral treatment with FrAq (25 mg/kg) as

well as lansoprazole (30 mg/kg) signicantly decreased the extent of

ulcerative lesions by 33% and 71%, respectively, when compared to
stomachs from the vehicle-treated control group (po0.01).
After conrming the gastroprotective effect of FrAq against
lesions induced by ethanol and I/R, the next step was to evaluate
the antiulcer mechanisms of action for this fraction. We studied the
effect of FrAq on gastric juice parameters to evaluate their possible
anti-secretory action by the pylorus ligation procedure (Table 1). We
observed that the administration of FrAq by an intraduodenal or
oral route was not able to change the H concentration of gastric
juice when compared to vehicle-treated animals (p40.05). This
result excluded the possibility of the antisecretory effect of FrAq.
However, this assay also revealed that the oral treatment with FrAq
was able to signicantly increase gastric volume (42%). This effect
could represent that FrAq was able to induce an increase in the
amount of adherent gastric mucus. Next, we evaluated the effects of
the oral administration of FrAq on this gastric mucus in pyloric
ligature rats (Fig. 3). We observed that oral treatment with FrAq
caused an increase (43%) in the amount of adherent gastric mucus
compared to the vehicle-treated group (po0.05), conrming our

Please cite this article as: Pinheiro Silva, L., et al., Terminalia catappa L.: A medicinal plant from the Caribbean.... Journal of
Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.11.025i

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Table 1
Effects of the aqueous fraction (FrAq) from the leaves of Terminalia catappa (25 mg/kg)
administered through the intraduodenal (i.d.) or oral (p.o.) route on the biochemical
parameters of gastric juice obtained from pylorus-ligature rats (n68).
Treatment

Route

Dose (mg/kg)

Gastric juice (mL)

[H ] (Eq/mL/4 h)

Vehicle
Cimetidine
FrAq

i.d.

100
25

7.02 7 2.25
4.29 7 1.19nn
8.02 7 2.18

6.99 7 1.84
2.777 1.30nn
7.34 7 0.65

Vehicle
Cimetidine
FrAq

p.o.

100
25

3.747 1.01
2.63 7 0.67
5.30 7 1.67 n

5.25 7 1.51
1.94 7 0.87nn
6.337 0.43

Data represents the means7 S.E.M. The asterisks denote the signicance levels,
when compared with the control group.
n

po 0.05.
p o0.01 by one-way ANOVA followed by Dunnett's test.

nn

n.s.

3000

Alcian blue
(mg/g wet tissue)

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64
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2000

1000

0
Vehicle

Carbenoxolone
100 mg/kg

FrAq
25 mg/kg

Fig. 3. Effects of the aqueous fraction (FrAq) obtained from the leaves of Terminalia
catappa (25 mg/kg) on the production of adherent gastric mucus (measured as the
amount of bound Alcian blue) in pylorus-ligated rats relative to the control values
(n 6). The results are the mean7 S.E.M. Statistical signicance was determined by
ANOVA followed by Dunnett's t test (np o0.05; nnn p o0.01 and n.s. no signicant
differences).

hypothesis. As expected, carbenoxolone (used as positive control)

enhanced the mucus production and there was no statistical
difference between the groups treated with carbenoxolone and
FrAq (p40.05).
In addition to mucus as defense mechanism elicited by FrAq,
other factors involved with the strengthening of gastric mucosa were
also investigated, such as NO (nitric oxide), sulfhydryl (SH) compounds and PGs, which are factors that maintain mucosal integrity.
Our results (Fig. 4a) shown that the pretreatment of animals with
NEM (sulfhydryl inhibitor) did not change the gastroprotective effect
of FrAq (p40.05) compared to the vehicle-pretreated FrAq group.
Therefore, this result excluded the involvement of sulfhydryl groups
in the gastroprotective effect of FrAq. However, the group of animals
pretreated with L-NAME (a NO-synthase inhibitor) and treated with
FrAq display a signicant increase in gastric lesions (Fig. 4b). A similar
picture emerged when the effect of PGs was studied. Fig. 4c shows
that administration of indomethacin markedly increased the size of
the ulcerative lesion and completely abolished the previously
observed protective effect of FrAq. Our results illustrated that beside
the increase in gastric mucus barrier induced by FrAq, the gastroprotective effect of this fraction also involved the action of NO and
endogenous PGs levels. After determining the factors involved with
FrAq action, the possible healing effect of FrAq was evaluated with
the 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 to

accelerate the healing of chronic gastric ulcers in rats. At the dose
of 25 mg/kg FrAq, 7 and 14 days of treatment signicantly decreased
the area of lesion in 80% and 37%, respectively when compared to the
control group treated with vehicle (po0.01). In the group treated
with lansoprazole at 30 mg/kg, the healing effect was also observed
with 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 with
vehicle, lansoprazole and FrAq for 7 or 14 days. Our results show that
MMP-9 activity was present only after treatment during 7 days in
stomachs from the vehicle and lansoprazole-group. We also observed
that this MMP-9 activity was absence for all treatments during 14
days. In contrast, MMP-2 activity was present in all treated groups
including the sham group (without gastric lesion). The treatment of
animals that exhibited major MMP-2 activity after 7 and 14 days was
compared to the animals treated with vehicle. Lower activity of
MMP-2 was observed after treatment of the animals with lansoprazole and FrAq for 7 days (Fig. 7a,  40 and 26%, respectively, in relation to the vehicle) and 14 days (Fig. 7b,  54 and 73%, respectively,
in relation to the vehicle).
Other important data obtained from this test based on the
treatment of animals with FrAq for 7 and 14 consecutive days was
the absence of death in these groups and also absence body
weight changes (data not shown), and some organ weights and
biochemical parameters of serum (Table 2). These results, therefore, ensure that based on the parameters used in this study, the
FrAq at doses of 25 mg/kg does not cause signicant toxic effects
after 7 or 14 consecutive days of treatment.
This study also evaluated the activity of FrAq against Helicobacter pylori and presented minimal inhibitory concentration
(MIC) of 125.0 mg/mL considered a accentuated activity and the
positive control (amoxicillin) presented MIC of 15.0 mg/mL.
The analysis of mass spectra of FrAq (Fig. 9) showed the same
compounds identied in the hydroalcoholic extract studied by
Mininel et al., (2014) as shown in Table 3. The spectra (Fig. 8) show
the ions with m/z 1083 (punicalagin); m/z 601 attributed to
gallagic acid and at m/z 301 attributed to ellagic acid, illustrating
the similarity with the hydroalcoholic extract (Mininel et al.,
2014). The fragmentation of the second order (MS2) ion at m/z
1083 (punicalagin) leads to ions at m/z 781 (punicalin) and the ion
with m/z 601 (gallagic acid) as shown in Fig. 9. The chromatographic prole of the water fraction is also shown in Fig. 10. In the
chromatogram, two peaks are highlighted, eluting at tr 17.2 min
and tr 23.3 min, indicating the presence of the major compound
punicalagin ( and anomers). Two peaks with tr 28.0 min and tr.
30.1 min (region 2) were observed, corresponding to the compound punicalin ( and anomers), derived from punicalagin.

4. Discussion
The development of gastric ulcers is a complex and multifactorial process including bacterial infections, the increase of acid
secretion, generation of reactive oxygen species (ROS), inhibition
of the endogenous PGs, and the degradation of the extracellular
matrix (ECM) (Laine et al., 2008; Mei et al., 2013). Research during
the last decade has offered new insights in the preventative
therapy and the healing of gastric ulcers and the synergistic
efciency of a multi-target approach based on individual mechanisms of action could be the new perspective for treatment of this
disease (Wagner, 2011; de Carvalho et al., 2014).
Terminalia catappa, is a medicinal plant widely distributed in
tropical and subtropical regions and it has been previously reported
that this species exhibits a variety of biological effects. However, the

Please cite this article as: Pinheiro Silva, L., et al., Terminalia catappa L.: A medicinal plant from the Caribbean.... Journal of
Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.11.025i

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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 mg/kg) or the aqueous fraction (FrAq) obtained from leaves of Terminalia catappa (25 mg/kg), pretreated with N-ethylmaleimide (panel b) with vehicle or together with
carbenoxolone (100 mg/kg) and FrAq (25 mg/kg), or pretreated with INDO indomethacin (panel c) with vehicle or together with carbenoxolone (100 mg/kg) or FrAq
(25 mg/kg). The results are reported as the mean7 S.E.M. Statistical signicance was determined by ANOVA followed by Dunnett's test. np o0.05; nnp o 0.01; nnnp o 0.001
compared to the corresponding vehicle group. # o 0.05; ##p o 0.01 compared to the corresponding NEM vehicle, L-Name vehicle or INDO vehicle. N.S. no signicant
differences.

10

20

8
ULA (mm)

15
ULA (mm)

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10
5

***

***

**

***

2
0

0
Vehicle

Lansoprazole
30 mg/kg

FrAq
25 mg/kg

Vehicle

Lansoprazole
30 mg/kg

FrAq
25 mg/kg

Fig. 5. Effect of the oral administration of the aqueous fraction (FrAq) obtained from the leaves of Terminalia catappa (25 mg/kg) on the healing of ulcers produced by the
introduction 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 S.E.M. Statistical signicance was determined by ANOVA followed by Dunnett's test. nnp o 0.01;nnnpo 0.001 compared to the corresponding vehicle group.

underlying mechanisms of the gastroprotective and healing effects

of the aqueous fraction (FrAq) obtained from the leaves have yet to
be evaluated.
In the present study, FrAq showed a marked gastroprotective
effect, evidenced by the dramatic inhibition of the gastric damage
produced by ethanol. Ethanol induced erosion, ulcerative lesions,
and petechial bleeding in the mucosa of the stomach in humans
and an ethanol-induced gastric ulcer model is commonly used to
study both the pathogenesis and new therapeutics for the treatment of gastric ulcer disease (Oyagi et al., 2010).
Our results have shown the effective gastroprotective effect of the
aqueous 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 by

ethanol. Aside from using a different part of plant (the bark instead
leaves), this study has demonstrated gastroprotection at a dose 10
times higher than our study (250 mg/kg vs. 25 mg/kg). Our studies
also highlighted the use of the renewable part of this plant (leaves)
that would enable the management of this plant for the future
production of a phytotherapeutic against gastric ulcer.
Gastric ulcer induced by ethanol is commonly associated with
reduced mucosal blood ow and ischemia that causes deleterious
effects on the gastric mucosa, mainly in the aging gastric mucosa
(Tarnawski et al., 2014). Ischemia weakens the gastric mucosal barrier
and increases the acid back-diffusion, predisposing the gastric mucosa
to damage (Rao and Vijayakumar, 2007). The restoration of blood ow

Please cite this article as: Pinheiro Silva, L., et al., Terminalia catappa L.: A medicinal plant from the Caribbean.... Journal of
Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.11.025i

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Fig. 6. Representative zymography results of the gastric ulcers from the different
groups illustrating MMP-2 and MMP-9 activity. Treatment for 7 days vehicle (line 1),
lansoprazole (line 2), aqueous fraction (FrAq) obtained from leaves of Terminalia
catappa at dose of 25 mg/kg (line 3) and sham (line 4). Treatment for 14 days vehicle
(line 5), lansoprazole (line 6), aqueous fraction (FrAq) obtained from leaves of
Terminalia catappa at dose of 25 mg/kg (line 7) and sham (line 8). Gelatinolytic bands
of 92, 72, 64 and 57 kDa were observed, which correspond to active-MMP-9,
pro-enzyme, intermediate and active-MMP-2, respectively.

Fig. 7. Effect of treatment with the vehicle, lansoprazole (30 mg/kg) or the aqueous
fraction (FrAq) obtained from Terminalia catappa (FrAq) on the gelatinolytic activity
of 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 from
the treatment groups to the control group (vehicle) IOD value.

(reperfusion) after a period of ischemia initiates a cascade of changes,

including the release of local ROS, inammatory responses, tissue
damage by fragmenting cellular DNA and an increase in the adhesion
of neutrophils to endothelial cells, a phenomenon known as ischemiareperfusion (I/R) (Smith et al., 1996; De Foneska and Kaunitz
2010).
The current study shows that oral treatment with FrAq (25 mg/kg)
signicantly decreased the extent of ulcerative lesions in the gastric
tissue submitted to I/R injury compared to stomachs from the vehicletreated control group. Based on the physiopathology of the I/R
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, were

able to attenuate oxidative stress and hypoxia-induced apoptosis.
These results therefore conrm the gastroprotective effect of
this fraction and encourage the continuation of the studies of the
effects of FrAq treatment and the characterization of its mechanisms 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 the
antisecretory effect of these species (Kumar et al., 2014; Mishra
et al., 2013; Nunes et al., 2014). Unlike these studies, our results
have shown that the oral and intraduodenal treatment with FrAq
(25 mg/kg) does not promote the antiulcerogenic effect in an
antisecretory manner. However, our result also illustrated that
oral treatment with FrAq was able to signicantly increase the
gastric volume in relation to the control group treated with vehicle
(5.30 mL and 3.74 mL, respectively). Our hypothesis was that the
FrAq elicits the increase of gastric juice because of the amount of
adherent gastric mucus.
The success of the pharmacological treatment for gastric ulcers
relies on the augmentation of the protective factors of the gastric
mucosa such as the mucus barrier (Al-Jiboury and Kaunitz, 2012;
Brzozowska et al., 2013). The adherent gastric mucus is the rst line
of defense against acid and serves as a barrier against self-digestion;
the functional integrity of the gastric mucosa depends on this
barrier (Wallace, 2008). Our study reveals a signicant increase in
the amount of adherent mucus in the animals treated with FrAq,
thus justifying the increase in gastric volume by treatment with this
fraction. This study involving the FrAq is highly signicant in the
ongoing search for an antiulcerogenic therapy, as the prolonged use
of antisecretory drugs such as proton-pump inhibitors and H2
blockers can provoke serious side effects (Ozdil et al., 2013; Eom
et al., 2011).
In addition to the increased production of the mucus barrier,
several studies have demonstrated a complex defense system involving the gastric mucosa, which include the regulation of gastric
mucosal blood ow, the formation of sulfhydryl compounds, NO and
endogenous PGs (Brzozowski et al., 2000; Pawlik et al., 2001).
NO is considered to be one of the most important defensive
endogenous agents in the gastric mucosa and plays a physiological
role in the homeostasis of the gastrointestinal tract (Brzozowski
et al., 2006). Together with the endogenous prostaglandins, NO also
preserves the gastric mucosa integrity and the inhibition of NO
release can result in disturbances in the gastrointestinal motility,
blood ow, and acid secretion (Brzozowski et al., 2006; Khalifa
et al., 2002; Wallace and Ma, 2001). In addition to the increase in
gastric mucus, our results show that the restoration of the endogenous NO represents an additional mechanism responsible for the
gastroprotective effects of FrAq, as a NO-synthase inhibitor elicits a
signicant increase in gastric lesion in stomachs previously treated
with the fraction. The gastroprotective effect of the FrAq through
endogenous NO agrees with the protective effect of this fraction
against I/R in rats by inhibiting the excessive formation of ROS.
Our study is also in concordance with those performed by
Pandya et al. (2013) in which they evaluated the status of the
ethanolic extract of Terminalia catappa (at dose of 200 mg/kg) and
illustrated an augmentation of the antioxidant defense system
against Ehrlich carcinoma. Kinoshita et al. (2007) also described
the antioxidant and hepatoprotective actions from the aqueous
extract of the leaves of Terminalia catappa. As well as NO, the
increase in gastric SH content is important for the limiting the
production 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 effect
of the FrAq (p 40.05). Therefore, our results exclude the involvement of SH content in the gastroprotective effect of FrAq.

Please cite this article as: Pinheiro Silva, L., et al., Terminalia catappa L.: A medicinal plant from the Caribbean.... Journal of
Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.11.025i

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Table 2
Effects of the aqueous fraction (FrAq) obtained from leaves of Terminalia catappa (25 mg/kg) and lansoprazole (30 mg/kg) administered orally for 14 consecutive days on
selected toxicological parameters (n 56).
Treatment (p.o.)
Vehicle
Organ weights (g)
Heart
Lung
Kidney
Liver
Spleen

Lansoprazole

3.85 7 0.48
4.497 0.64
5.147 0.63
10.107 1.23
2.69 7 0.28

Biochemical parameter
Glucose
Creatinine
Urea
-GT
AST
ALT

136.90 7 7.00
0.56 7 0.02
38.107 1.12
0.96 7 0.17
157.117 13.4
44.337 2.51

3.88 7 0.32
5.34 7 1.43
4.917 0.18
9.99 7 0.28
2.83 7 0.24
136.40 7 5.80
0.56 7 0.03
39.40 7 5.35
2.02 7 0.55
181.20 7 17.51
48.007 5.04

FrAq

3.677 0.13
4.20 7 0.40
4.917 0.07
9.707 0.17
2.677 0.27
151.007 8.50
0.53 7 0.04
39.60 7 4.23
1.247 0.25
190.60 7 14.50
43.17 3.11

Results are expressed as the means 7S.E.M. obtained from different groups. The units for the biochemical parameters of serum are U/L (ALT alanine aminotransferase,
AST aspartate aminotransferase, GT gamma-glutamyl transpeptidase) and mg/dL (urea and creatinine).

Table 3
Identication of the substances obtained in the aqueous fraction (FrAq) from the
leaves of Terminalia catappa by FIA-ESI-IT-MSn.
[MH] 
1083
781
601
301

MSn ions
781
601
601
409
257
229

Identication


[M-152-152-H]
[M-152-152-180-H] 
[M-180-H] 
[M-191-H] 
[M-44-H] 
[M-44-28-H] 

Punicalagin (1)
Punicalin (2)
Gallagic acid (3)
Ellagic acid (4)

Adapted from Mininel et al. (2014).

Among the humoral factors in the gastric mucosa, endogenous

PGs play an important role in the protective effect by stimulating
the secretion of mucus, maintaining the local blood ow and
increasing the resistance of epithelial cells to potential damage by
cytotoxins (Takeuchi, 2010). Our results illustrate that the administration of a non-selective COX inhibitor (indomethacin) completely abolished the gastroprotective action of the FrAq. This result
highlights the relevance of PGs in the antiulcer action of this
fraction and agreement with the previously results of fraction in
increase in the amount of adherent mucus. According to Takeuchi
(2010) one of the mechanisms underlying the action of PGE2 is
exactly the increase in mucus secretion that augments the protective factors on the gastric mucosa.
Based on the results regarding the gastroprotective effect of the
FrAq against gastric injury induced by different ulcerogenic agents,
we conrmed that the action of this fraction was mediated by the
activation of defensive mucosa-protective factors such as increased
mucus production, NO pathways and endogenous PGs. However, it
is desirable for any new antiulcer drug that the preventive effect is
also accompanied by ulcer healing effects.
Antiulcer drugs such as H2-receptor antagonists fail in promoting the healing of gastric ulcers when the regenerated mucosa
presents with poor histological maturity and ulcer relapse is not
prevented by cimetidine (Arakawa et al., 2012).
This study also determined the effect of the FrAq on the healing
of gastric ulcers induced by acetic acid in rats treated for 7 or 14
consecutive days. The acetic acid induced a deep necrotic lesion,
involving the entire mucosal depth and penetrating through the
muscularis mucosae. Ulcer healing is a dynamic process of lling
mucosal defects with proliferating and migrating epithelial cells
and connective tissue, which results in the reconstruction of the

mucosal architecture (Okabe and Amagase, 2005). Chronic treatment with FrAq (25 mg/kg) demonstrated a dramatic reduction in
the ulcerative lesion area by treatment for 7 days (80%), which was
more effective than 14 days of treatment (37%). This result
demonstrated the FrAq (25 mg/kg) accelerates the healing within
7 days of treatment and the healing effect remains after treatment
of 14 days when compared to control group or lansoprazole group.
The injection of acetic acid into gastric mucosa induces the
development of deep gastric ulceration and gastric mucosal damage
directly associated with ECM degradation, in which the zincdependent matrix metalloproteinases (MMPs) play a crucial role.
In several animal studies of gastric ulcer, attention has focused
on the role of MMPs, mainly MMP-2 and MMP-9 (Sen-Li et al.,
2013). Wound formation and the following healing are dynamic
processes of ECM remodeling that are mainly inuenced by MMP-2
and MMP-9 (Gyenge et al., 2013).
According to Yeh et al. (2012) MMP-9 is the protease most
signicantly involved in the degradation of the basement membrane
and is associated with pathological states that include inammation
and cancer. Sen-Li et al. (2013) described enhanced expression of
MMP-9 in the margin of the ulcer in patients with this disease. Their
study suggested that the presence of MMP-9 at the margin of the
ulcer may be indicative of inammation and poor wound healing.
Ulcerogenic agents, such as indomethacin, exhibited 12-fold higher
pro-MMP-9 activity and ethanol exhibited 22-fold higher pro-MMP-9
activity in rat gastric tissues relative to untreated tissues (Mei et al.,
2013). Our results showed MMP-9 activity only after treatments with
the vehicle and lansoprazole (7 days). These results determined that
the presence of MMP-9 activity at the gastric mucosa in groups
treated with the vehicle was related with the absence of healing in
ulcers of the gastric mucosa. Our results also show that the seemingly
healing macroscopic 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 persistence of the inammatory process at the gastric mucosa.
Our results shown that treatment of the acetic acid induced chronic
ulcers with FrAq (25 mg/kg) for 7 and 14 days inhibited the MMP-9
activity. Our results could be strengthened by the study from Yeh
et al. (2012) in which extract from Terminalia catappa leaves exerts an
antimetastic effect by attenuating MMP-9 mediated inammation in
hepatocellular carcinoma. In addition to our results obtained regarding MMP-9, our results also reported high MMP-2 activity in gastric
ulcers of the control group treated with vehicle (7 and 14 days), and
lower MMP-2 activity was observed after the treatment of animals

Please cite this article as: Pinheiro Silva, L., et al., Terminalia catappa L.: A medicinal plant from the Caribbean.... Journal of
Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.11.025i

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Relative Abundance

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35
541.15

30

781.45
1013.13
954.49

25

1132.91

639.27

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312.07

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1397.19
1562.07

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5

1712.29

0
500

1000

1500

2000

m/z

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 1002000 Da.

T_130129165016 #1-1000 RT:0.60-0.96 AV:70 NL:2.71E2

F: ITMS - c ESI Full ms2 1083.00@cid30.00 [295.00-1200.00]
100
95
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Relative Abundance

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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 m/z 300-1200 Da.

with lansoprazole and FrAq. Recent studies have highlighted the

function of MMP-2 in the healing process of rat gastric ulcers induced
by acetic acid (Gyenge et al., 2013), but the function of MMP-2 is not
well understood.
The antiulcerogenic actions demonstrated by FrAq could be
attributed to the phenolic compounds present in this fraction.
Mininel et al. (2014) analyzed mass spectra in a full-scan of the
extract and this fraction and showing similarity to each other,
highlighted by the precursor ions m/z 1083 (punicalagin), m/z 781
(pulicalin), m/z 601 (gallagic acid) and m/z 301 attributed to ellagic
acid. In these studies, the chromatogram from HPLC-PDA of the
aqueous fraction of Terminalia catappa conrmed the majority of the

compounds punicalagin (anomers and ) and punicalin (anomers

and ) are similar to the hydroalcoholic extract studied by Mininel
et al. (2014). Ellagic acid (EA) is one of the naturally occurring
polyphenols found in the FrAq from Terminalia catappa leaves.
Numerous pharmacological studies have suggested that EA provides
mucosal protective action in the stomach against ethanol or ischemiareperfusion injury (Iino et al., 2001; Iino et al., 2002) and several
gastroprotective mechanisms are attributed to this compound. For
example, Chatterjee et al. (2012) showed that EA enhanced prostaglandins when compared with the ulcerated untreated group.
In our nal experimental series, we evaluated the subacute
toxicological parameters from groups that received vehicle,

Please cite this article as: Pinheiro Silva, L., et al., Terminalia catappa L.: A medicinal plant from the Caribbean.... Journal of
Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.11.025i

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10

T. catappa FrAq - CH5

2000000

1000000

2 - e punicalin

0
0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

Retention Time [min]

Fig. 10. Chromatogram from HPLCPDA of the aqueous fraction of the leaves of
Terminalia catappa. Hydro Column ow 1 mL/min, gradient method 560% MeOH,
40 min HPLCPDA (Jascos), 270 nm.

lansoprazole or the FrAq over 14 days. Important toxicity parameters, including changes in body weight, mortality and weight of
the vital organs, were determined as shown in Table 2. Treatment
with FrAq for 14 days with 25 mg/kg did not signicantly alter the
body weight of animals compared to the vehicle control over the
14-days treatment period (data not shown) and the average
weights of the major organs of the FrAq-treated subjects were
comparable to those of the control group. Table 2 also shows that
FrAq did not affect several biochemical parameters. Treatment did
not signicantly alter the body weight, the weight of vital organs
and biochemical parameters of serum. However, Mininel et al.
(2014) evaluated the mutagenicity of the hydroalcoholic extract
from the leaves of Terminalia catappa with the Ames test and
illustrated that this extract exhibited mutagenic activity in vitro at
high concentrations, leading them to recommend caution when
using this compound for medicinal purposes.
This study also evaluated the activity of aqueous fraction (FrAq)
against Helicobacter pylori and determination of minimal inhibitory
concentration (MIC) of 125.0 mg/mL considered an important antimicrobial activity. The literature do not show a consensus in relation
to MIC values obtained for natural products. Aligannis et al. (2001)
consider MICs with values equal to or less than 500 mg/mL as potent
inhibitors; MICs between 600 and 1500 mg/mL and MIC moderate
inhibitors as above 1600 mg/mL as weak inhibitors. Webster et al.
(2008) established as another satisfactory MIC value equal to or less
than 1000 mg/mL. In this sense, the result reported in this investigation demonstrated the inhibitory potential of Terminalia catappa
against this important bacteria responsible to gastric ulcer.

5. Conclusions
We determined that the FrAq from Terminalia catappa leaves
has an important anti-Helicobacter pylori activity, excellent preventive and curative effects on acute and chronically induced
gastric ulcers. The mechanisms involved in the gastroprotection
are related to the NO pathway, an increase in the mucus and the
endogenous prostaglandins, and this fraction was able to heal
ulcers through the inhibition of MMP-9 and MMP-2 activities.

Uncited references
Li et al. (2003), Seeram et al. (2005).

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This study was supported by the Biota-FAPESP project (Fundao Q4 69
70
de Amparo Pesquisa do Estado de So Paulo), CNPq (Conselho
Nacional de Desenvolvimento Cientco e Tecnolgico) and CAPES Q5 71
72
(Coordenao de Aperfeioamento Pessoal de Nvel Superior).
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1 - e punicalagin

Intensity [V]

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Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.11.025i

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Please cite this article as: Pinheiro Silva, L., et al., Terminalia catappa L.: A medicinal plant from the Caribbean.... Journal of
Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.11.025i