Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

1 Contents lists available at ScienceDirect

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4 Journal of Ethnopharmacology
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journal homepage: www.elsevier.com/locate/jep
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Research Paper
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Terminalia catappa L.: A medicinal plant from the Caribbean
13 pharmacopeia with anti-Helicobacter pylori and antiulcer action
14
15 in experimental rodent models
16
17 Laísa Pinheiro Silva a, Célio Damacena de Angelis a, Flavia Bonamin a, Hélio Kushima a,
18 Francisco José Mininel b, Lourdes Campaner dos Santos b, Flavia Karina Delella c,
19
Sergio Luis Felisbino c, Wagner Vilegas d, Lucia Regina Machado da Rocha a,
20
21 Matheus Aparecido dos Santos Ramos f, Tais Maria Bauab f,
e,n,1
22 Q1 Walber Toma , Clelia Akiko Hiruma-Lima a,n,1
23 a
Univ. Estadual Paulista-UNESP – Departamento de Fisiologia, Instituto de Biociências, CEP 18618-970 Botucatu, SP, Brazil
24 b
Univ. Estadual Paulista-UNESP – Departamento de Química Orgânica, Instituto de Química, CEP 14800-900, Araraquara, SP, Brazil
25 c
Univ. Estadual Paulista-UNESP – Departamento de Morfologia, Instituto de Biociências, CEP 18618-970, Botucatu, SP, Brazil
d
26 Univ. Estadual Paulista-UNESP – Campus Experimental do Litoral Paulista, CEP 11330-900 São Vicente, SP, Brazil
e
Universidade Santa Cecília – Pós-Graduação em Sustentabilidade de Ecossistemas Costeiros e Marinhos, Rua Oswaldo Cruz, 266,
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Boqueirão, CEP 11045907 Santos, SP, Brazil
28 f
Univ. Estadual Paulista-UNESP – Departamento de Ciências Biológicas, Faculdade de Ciências Farmacêuticas, 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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33 Article history: Ethnopharmacological relevance: Terminalia catappa L. (Combretaceae) is a medicinal plant listed as a
34 Received 22 August 2014 pharmacopeia vegetable from Caribbean to treat gastritis. The objective of this study was to evaluate the Q7
35 Received in revised form gastroprotective and healing effect of the aqueous fraction (FrAq) obtained from the leaves of Terminalia
36 3 November 2014
catappa and to determine the antiulcer mechanism of action in experimental rodent models and its
Accepted 13 November 2014
37 activity to Helicobacter pylori.
38 Material and methods: In rodents, the FrAq was challenged by different necrotizing agents, such as
Keywords: absolute ethanol and ischemia–reperfusion injury. The antiulcer mechanism of action of FrAq was
39
Terminalia catappa L. assessed and the healing effects of the fraction after seven and 14 days of treatment was evaluated by
40 Combretaceae
matrix metalloproteinase activity (MMP-2 and MMP-9). The toxicological effect of subacute treatment
41 Gastric healing action
with FrAq during 14 days of treatment was also analyzed. The anti-Helicobacter pylori activity was
42 Helicobacter pylori
MMP-9 determined by microdilution. The phytochemical study of the fraction was analyzed by experiments with
43
MMP-2 FIA-ESI-IT-MSn (Direct Flow Analysis-ionization Electrospray Ion Trap Tandem Mass Spectrometry) and
44 high performance liquid chromatography (HPLC) coupled to a photodiode array (PDA).
45 Results: Oral treatment with FrAq (25 mg/kg) significantly decreased the number of ulcerative lesions
46 induced by ethanol and ischemia/reperfusion injury. The action of FrAq was mediated by the activation of
47 defensive mucosa-protective factors, such as increases in mucus production, the nitric oxide (NO)
48 pathway and endogenous prostaglandins. Oral treatment with FrAq for seven and 14 days significantly
49 reduced the lesion area (80% and 37%, respectively) compared to the negative control group. Analyses of
50 MMP-9 and MMP-2 activity from gastric mucosa confirmed the accelerated gastric healing effect of FrAq.
51 This extract also presented considerable activity against Helicobacter pylori. The mass spectrum and
MS/MS of the aqueous fraction indicates the existence of many different phenolic compounds, including
52
punicalagin, punicalin, and gallagic acid, among others.
53
Conclusions: We concluded that FrAq from Terminalia catappa leaves has excellent preventive and
54 curative effects on acute and chronic induced gastric ulcers and showed an important profile against
55 Helicobacter pylori.
56 & 2014 Published by Elsevier Ireland Ltd.
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58 67
59 68
60 69
61 n
Corresponding authors. Tel.: þ 55 14 38800312; fax: þ55 1438153744. 70
62 E-mail addresses: walbertoma@gmail.com (W. Toma), hiruma@ibb.unesp.br (C. Akiko Hiruma-Lima). 71
1
63 Contributed equally to the supervision of this study.
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64 http://dx.doi.org/10.1016/j.jep.2014.11.025 73
65 0378-8741/& 2014 Published by Elsevier Ireland Ltd. 74
66

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

1 1. Introduction 2.2. Chemicals and reagents 67

2 68
3 Terminalia catappa is a species of the family Combretaceae and HPLC–grade methanol (MeOH) and acetonitrile were purchased 69
4 popularly known in Brazil as “amendoeira”, “amendoeira-da-praia”, from J.T. Baker (Baker-Mallinckrodt, Phillipsburg, NJ, USA). HPLC– 70
5 “amendoeira-da-Índia”, “cuca”, “guarda-sol”, “castanheira da Índia”, grade water (18 MΩ cm) was obtained using a direct Milli-Q pur- 71
6 “castanhola” and “chapéu-de-Sol”. This plant is widely distributed ification system (Millipore Co., Bedford, MA, USA). Sep-Pak RP18 72
7 in countries with tropical and subtropical climates, especially in cartridges (500 mg/mL) for solid-phase extraction (SPE) were pur- 73
8 coastal regions due to the plant's ability to easily adapt to salinity chased from Phenomenex Co. (Torrance, CA, USA). 74
9 and winds (Thomson and Evans, 2006). In Asian countries, the 75
10 leaves of this species are commonly used for the treatment of 2.3. Apparatus 76
11 dermatitis, hepatitis, diarrhea and pyresis (Chen et al., 2000). This 77
12 plant was also listed in Pharmacopeia vegetables of the Caribbean, The mass spectrometry experiments were performed on LCQ 78
13 where the leaves of this plant are used in a decoction for gastritis Fleet equipment (Thermo Scientifics) equipped with the dispersal of 79
14 and urinary infection (Germosén-Robineau, 2014). The literature the directly introduced sample via flow injection analysis (FIA). The 80
15 also shows that the polar extract from different parts (leaves, fruits studied matrix was analyzed by electrospray ionization (ESI), and 81
16 and bark) of Terminalia catappa have shown the following biological multiple stages of fragmentation (MS2, MS3, MSn) were performed at 82
17 activities: antimicrobial, antifungal (Fyhrquist et al., 2002), antiox- an ion trap (IT) interface. The negative mode was selected for the 83
18 idant (Masuda et al., 1999; Chen and Li, 2005; Pandya et al., 2013), generation and analysis of the mass spectra for the first order (MS) 84
19 antimetastatic (Yeh et al., 2012, 2014), anti-inflammatory (Fan et al., and for the remaining multi-stage experiments under the following 85
20 2004; Lin et al., 1999), hepatoprotective (Lin et al., 1997; Chen et al., conditions: capillary voltage,  25 V; voltage spray,  5 kV; capillary 86
21 2000; Tang et al., 2006; Chen and Li, 2005), mutagenic (Mininel temperature, 275 1C. A carrier gas (N2) with a flow of 8 arbitrary 87
22 et al., 2014), aphrodisiac (Ratnasooriya and Dharmasiri, 2000) and units (A.U.) was used, and the collision gas was helium (He). The 88
23 antidiabetic (Nagappa et al., 2003). Nunes et al. (2012) described the track acquisition was 100–2000 m/z. Xcalibur version 1.3 software 89
24 gastroprotective effect of the ethanolic extract obtained from bark (Thermo Finigans) was used to acquire and process the data. 90
25 of this species and Kumar et al. (2014) previously reported the For the FIA-ESI-IT-MSn assay, 10 mg of the aqueous fraction was 91
26 antisecretory effect of the ethanolic extract from leaves, although dissolved in 1 mL of MeOH:H2O (1:1, v/v) after using an ultrasonic 92
27 the mechanism responsible for this preventive effect remains bath for 5 min. The samples were then filtered through a 0.22 mm 93
28 unknown, thus the use of this specie against gastric bacteria is PTFE filter, and aliquots of 20 mL were directly injected into the 94
29 relevant. Infection caused to Helicobacter pylori is considered the FIA-ESI-IT-MSn system. 95
30 most prevalent cause of gastric diseases such as ulcers, dyspepsia For the HPLC–PDA a clean-up step was performed to remove any 96
31 and stomach cancer (Camargo et al., 2014). The drug therapy availa- contaminants; the solution was purified by solid phase extraction 97
32 ble for the treatment of diseases caused by this microorganism has (SPE) using Phenomenex Strata C18 cartridges (500 mg of stationary 98
33 limitation such as the high rate of resistance to conventional drugs phase) that were previously activated with 5 mL of MeOH and 99
34 and inappropriate patient treatment, as presented to numerous side equilibrated with 5 mL of MeOH:H2O (1:1, v/v). The dried aqueous 100
35 effects (Megraud et al., 2013). In this sense, the use of natural fraction was diluted to 10 mg/mL in HPLC solvent. A 20 mL aliquot 101
36 products as an alternative to control this bacterium has shown to be was injected directly into the HPLC–PDA with detection at 270 nm. 102
37 satisfactory, what drives the improvement of investigations of medi- The identification of the different compounds in the chromato- 103
38 cinal plants as adjuvant or new antimicrobial drugs (Parreira et al., graphic profile of the aqueous fraction was done by comparing their 104
39 2014; Takeuchi et al., 2014). retention times (tr) and the UV spectra with those isolates pre- 105
40 The aims of this present work were to characterize the anti- viously described in the literature (Mininel et al., 2014). 106
41 Helicobacter pylori activity and antiulcer effect of the aqueous 107
42 fraction obtained from leaves of Terminalia catappa and determine 108
43 the mechanism of action for this medicinal species. 2.4. Animals 109
44 110
45 Male Wistar rats weighing 180–250 g were obtained from breed- 111
46 ing at the Biotério Central at the Universidade Estadual Júlio de 112
47 2. Material and methods Mesquita Filho (UNESP), Botucatu-SP. Animals were fed with a 113
48 certified Nuvilab CR-diet, with free access to tap water and were 114
49 2.1. Preparation of the aqueous fraction housed on a 12 h light/dark cycle at 6071% humidity and a 115
50 temperature of 2272 1C. The UNESP Institutional Animal Care and 116
51 The leaves of Terminalia catappa were collected in January Use Committee, following the recommendations of the Canadian 117
52 (2010) by Msc. Laísa Pinheiro Silva in Santos/SP, Brazil. The speci- Council on Animal Care (Olfert et al., 1993), approved all of the 118
53 men was identified by Msc. Paulo Salles Penteado Sampaio, and employed protocols under number 18/05. 119
54 the voucher specimen was deposited to the Herbarium at the 120
55 Universidade Santa Cecília, Santos, SP, Brazil, under the register 2.5. Evaluation of the gastroprotection activity of the FrAq 121
56 M. Tomaz 01, for future reference. The leaves from Terminalia 122
57 catappa (378.54 g) were dried for six days at 50 1C, powdered 2.5.1. Gastric ulcer induced by absolute ethanol 123
58 (3 mm) and subjected to percolation with absolute ethanol (2 L) The rats were divided into five treatment groups (n¼ 7–14) 124
59 for 2 h with a flux of 2.0 mL/min/kg. The hydroalcoholic extract and fasted for 12 h prior to receiving an oral dose of the vehicle 125
60 submitted to the rotary evaporator (45 1C) resulted in 33.1 g of (saline 10 mL/kg), carbenoxolone (100 mg/kg) and FrAq (12.5, 25 126
61 product (a yield of 8.75%). This extract was partitioned into three and 100 mg/kg). After 60 min, all groups were orally treated with 127
62 fractions: a hexane fraction – FrHex (7.09 g, 24.96%), an ethyl 1 mL of absolute ethanol for the gastric ulcer induction. One hour 128
63 acetate fraction – FrEtOAc (12.22 g, 43.02%) and an aqueous later, animals were killed and their stomachs excised. The incision 129
64 fraction – FrAq (8.43 g, 26.16%). As the commonly used formula- into each stomach was performed along the greater curvature and 130
65 tion of this medicinal plant is a decoction, we chose to utilize the examined for linear hemorrhagic lesions in the glandular region 131
66 FrAq in the experimental pharmacological protocols. (Morimoto et al., 1991). Then, the stomachs were photographed and 132

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 ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 3

1 the extent of the lesions was measured by ulcerative lesion area 2.7. Effect of FrAq healing acetic acid-induced gastric lesions 67
2 (U.L.A.) in mm2 by the program AVSoft BioView Spectras. 68
3 The experiment was performed according to the method descri- 69
4 bed by Okabe et al. (1971). Six groups (n¼5–6) of male Wistar rats 70
2.5.2. Gastric ulcer induced by ischemia–reperfusion
5 were fasted for 12 h before this experiment. Under anesthesia, a 71
Q3 Ischemia–reperfusion damage was produced in rats by a
6 laparotomy was done in all animals through a midline epigastric 72
method proposed by Ueda et al. (1989). Rats (n ¼7–9) were orally
7 incision. A plastic 4.2 mm internal diameter tube was firmly applied 73
administered saline (10 mL/kg), lansoprazole (30 mg/kg) and FrAq
8 to the serosal surface of the stomach wall, and 70 ml of an 80% 74
(the lower effective dose of 25 mg/kg). Thirty minutes later, the
9 solution of acetic acid was applied for 20 s on the serosal surface of 75
animals were anaesthetized by intramuscular injection of keta-
10 the stomach and then completely removed. The stomach was bathed 76
mine (50 mg/kg) and xylazine (10 mg/kg). The left side of the
11 with saline (20 1C) to avoid adherence to the external surface of the 77
abdomen was shaved, and an incision was made. Briefly, the celiac
12 ulcerated region and then the abdomen was then closed and all 78
artery was dissected, freed of excess fat and clamped for 30 min
13 the animals were fed normally. This process resulted in a chronic 79
(ischemia phase) using a micro-bulldog clamp. Re-oxygenation
14 ulceration of the mucosa and submucosa, with an approximate ulcer 80
was allowed by removing the clamp for 60 min (reperfusion
15 area of 13.8 mm2. FrAq (25 mg/kg) from Terminalia catappa, lanso- 81
phase). At the end of this period, the animals were killed and
16 prazole (30 mg/kg) or saline (10 mL/kg) were administered for the 82
the stomachs were excised and opened along the great curvature
17 determination of the healing effects by the subacute treatment 83
for the detection of the U.L.A.
18 during 7 and 14 days. All treatments were done orally once a day 84
19 beginning one day after surgery. One day after the last drug 85
20 2.6. Determination of mechanisms of action from FrAq administration, the rats were killed and the stomachs were removed. 86
21 The gastric lesions were evaluated by examining the inner gastric 87
22 2.6.1. Gastric secretion in lesions induced by pylorus ligature surface with a dissecting magnifying glass. 88
23 The method of Shay et al. (1945) was used with modification. Rats 89
24 (n¼ 6–8) were fasted for 12 h and, immediately after pylorus ligature, 90
2.7.1. Extraction of total protein and zymography
25 saline (10 mL/kg), cimetidine (100 mg/kg) or FrAq (25 mg/kg) was 91
Tissue with gastric ulcer from each experimental group described
26 administered intraduodenally or orally. The rats were killed 4 h later, 92
previously was used to extract total protein. The extraction was
27 their abdomens were opened and the stomachs removed. The gastric 93
carried out following the ratio of 30 mg of tissue: 0.1 mL of 50 mM
28 content was collected to determine the total amount of gastric-juice 94
Tris–HCl solution, pH 7.5, containing 0.25% Triton X-100, 10 mM
29 acid (mL). Distilled water was added, and the resultant solution was 95
CaCl2 and protease inhibitor cocktail (P-8849 – Sigma-Aldrich,
30 centrifuged at 3000g for 10 min. The hydrogen ion concentrations 96
St Louis, MO, USA) by crushing with the Polytron type homogenizer.
31 (mEq/mL/4 h) were recorded in the gastric secretion by adjusting the 97
The homogenate was centrifuged at 4000g for 20 min at 4 1C. The
32 supernatant volume by titration to pH 7.0 with 0.01 N NaOH. 98
supernatant was collected and protein content was quantified by the
33 99
Bradford (1976).
34 100
2.6.2. Determination of the gastric mucus content Samples of extracted proteins (28 μg) of the gastric ulcer from
35 101
This assay was done as described by Rafatullah et al. (1990) different experimental groups treated during 7 and 14 days were
36 102
with modification. After a 12 h fast, rats (n ¼ 6) received saline subjected to electrophoresis under non-reducing conditions on 8%
37 103
(10 mL/kg), carbenoxolone (100 mg/kg) and FrAq from Terminalia polyacrylamide gel copolymerized with 0.1% purified gelatin
38 104
catappa (25 mg/kg) orally. The pylorus was ligated thirty minutes (Sigma-Aldrich Co. LLC. St. Louis, MO, U.S.A.). After electrophoresis,
39 105
after treatment. The animals were killed 4 h after pylorus ligation the gels were subjected to two washes of 15 min in a solution of
40 106
and the glandular portion of the stomachs was removed and 2.5% Triton X-100 to remove SDS, and two washes of 5 min in
41 107
weighed. Each segment was immediately immersed in 10 mL of 50 mM pH 8.0 Tris–HCl buffer. Subsequently, gels were incubated
42 108
0.1% Alcian blue solution (0.16 M sucrose/0.05 M sodium acetate, in 50 mM Tris–HCl buffer, pH 8.0, containing 5 mM of CaCl2 for
43 109
pH 5.8) for 2 h, followed by two successive rinses with 10 mL of 22 h at 37 1C. Finally, the gels were stained with Coomassie
44 110
0.25 M sucrose (the first for 15 min and the second for 45 min) to Brilliant Blue R-250 (Sigma-Aldrich Co. LLC. St. Louis, MO, U.S.A.).
45 111
remove the excess dye. Each stomach was then transferred to The relative molecular weight of the bands was determined
46 112
0.5 M magnesium chloride solution for 2 h. Four milliliters of the according to the molecular weight standard (Precision Plus Pro-
47 113
dye solution was then vigorously shaken with an equal volume of tein™, BIO-RAD) used in electrophoresis. The bands obtained
48 114
ether, the resulting emulsion was centrifuged at 2000g, and the through zymography were scanned and analyzed by densitometry.
49 115
absorbance of the aqueous layer was measured at 580 nm. The The bands representative of the gelatinase activity of MMP-2 and
50 116
amount of blue dye extracted per gram of wet glandular tissue -9 were analyzed by obtaining the integrated optical density (IOD)
51 117
was then calculated from a standard curve of dye prepared in a of the bands using Image J software. Due to limited amount of
52 118
sucrose–acetate solution. tissue for this analysis, the tissue from 5 different animals from
53 119
each experimental group was pooled together for extraction. The
54 120
zymography with pooled samples was repeated three time. Values
55 2.6.3. Determination of the role of nitric oxide (NO), prostaglandin 121
were plotted in a histogram showing the ratio of the IOD of the
56 (PGE) and sulfhydryl compounds (SH) in gastric protection 122
treated groups to the control group IOD.
57 Male rats (n ¼5) were divided into nine groups and pretrea- 123
58 ted with either saline, L-NAME (N-nitro-L-arginine methyl ester, 124
59 70 mg/kg) – an inhibitor of NO synthesis, INDO (indomethacin, 2.7.2. Toxicological evaluation 125
60 30 mg/kg) – an inhibitor of PGE or NEM (N-ethylmaleimide, Some toxicological parameters were also obtained from three 126
61 10 mg/kg) – a blocker of SH compounds (Arrieta et al., 2003). groups of animals subjected to the healing gastric ulcer model and 127
62 Thirty minutes after the pretreatment, the animals were adminis- treated orally during 14 consecutive days once a day with: FrAq 128
63 tered (p.o.) saline (10 mL/kg), carbenoxolone (100 mg/kg) and FrAq (25 mg/kg) from Terminalia catappa leaves, lansoprazole (30 mg/kg) 129
64 (25 mg/kg). After 60 min, all groups received 1 mL absolute and saline (10 mL/kg). Body weight was recorded daily throughout 130
65 ethanol to induce gastric ulcers. One hour after receiving ethanol the experimental period, and the macroscopic analyses and weight of 131
66 the rats were killed for the determination of gastric lesions. vital organs (liver, kidneys, heart, spleen and lungs) were compared 132

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

1 between either the FrAq or lansoprazole and the vehicle-treated 400 67

2 groups to evaluate subacute toxicity. On the day after the last drug 68
3 administration, the rats from each treatment group were killed, and 69
n.s.
4 their blood was collected. The blood samples were then centrifuged 70
300
5 (3000g for 10 min), and the serum obtained was frozen at  20 1C 71

ULA (mm )
2
6 until biochemical analysis. Serum biochemical parameters, including 72
7 glucose, urea, creatinine, γ-glutamyl transpeptidase (γ-GT), aspartate *** ** 73
8 aminotransferase (AST) and alanine aminotransferase (ALT), were 200 74
9 measured using an automated biochemical analyzer (SBA-200, 75
10 Companhia Equipadora de Laboratórios Modernos, São Paulo, Brazil). 76
11 100 77
12 2.8. Minimum inhibitory concentration (MIC) 78
13 *** 79
14 We used Helicobacter pylori ATCC 43504 using a microdilution 0 80
15 technique following by CLSI (2006) with modifications to determine Vehicle Carbenoxolone 12.5 25 100 81
16 the minimal inhibitory concentration (MIC) values. Helicobacter pylori 100 mg/kg 82
FrAq (mg/kg)
17 was inoculated on Mueller-Hinton agar plates containing 5% sheep 83
18 blood and incubated at 36 1C for 72 h, in 10% CO2 atmosphere. Fig. 1. Effect of pretreatment with the aqueous fraction (FrAq) obtained from the 84
19 Inoculates were prepared in the same medium at a density adjusted leaves of Terminalia catappa on ethanol-induced gastric ulcers in rats. The animals 85
orally received saline solution (vehicle), carbenoxolone or FrAq. The results are
20 to a 2.0 McFarland turbidity standard. The working suspension for 86
expressed as the mean 7 S.E.M. (n ¼7–14), and statistical significance was deter-
21 microorganism was diluted 1:10 and a 100 mL volume was added to mined by one-way analysis of variance (ANOVA) followed by Dunnett's test 87
22 each well of microplates. A 100 mL volume of Mueller-Hinton broth (nn p o0.01, nnn p o0.001 and n.s. – no significant differences). 88
23 supplemented with 10% fetal bovine serum was added each well of 89
24 microplates. The concentrations for each substance, prepared in 2% 90
25 DMSO, ranging from 0.5 to 1000 mg/mL were obtained when a 100 mL 80 91
26 volume of the fraction was transferred to the first well of each row, 92
27 and serial 2-fold dilutions were performed. Amoxicillin was used as 93
28 reference antimicrobial compound and the MICs were recorded after 60 94
29 incubation of the microplates at 36 1C for 72 h in a 10% CO2 atmo- 95
ULA (mm²)

30 sphere. The MICs were recorded as the lowest concentration at 96

31 which no growth was observed. This record was facilitated by 97
40
32 addition of 20 mL of resazurin solution (100 mg/mL) as revelator to 98
33 each of well and incubation until 2 h. A pink color indicated bacterial 99
34 growth and a blue color indicated a non-bacterial growth. 100
20
35 101
36 2.9. Statistical analysis 102
37 103
38 0 104
The results were expressed as the mean 7standard error of the Vehicle Lansoprazole FrAq
39 mean (S.E.M.) of the parameters obtained. Statistical comparisons 105
30 mg/kg 25 mg/kg
40 were done by one-way analysis of variance (ANOVA) followed by 106
41 Dunnett's or Tukey's post hoc test, with the level of significance set Fig. 2. Effect of pretreatment with the aqueous fraction (FrAq) obtained from the 107
42 leaves of Terminalia catappa on ischemia–reperfusion induced gastric ulcers in rats. 108
at np o0.05; nnp o0.01 and nnnp o0.001. The animals orally received saline solution (vehicle), lansoprazole or FrAq. The
43 results are expressed as the mean 7 S.E.M. (n ¼7–9), and statistical significance was
109
44 determined by one-way analysis of variance (ANOVA) followed by Dunnett's test, 110
45 3. Results with the level of significance set at nnp o 0.01 and nnnp o0.001. 111
46 112
47 In the present study, we evaluated the protective effect of FrAq well as lansoprazole (30 mg/kg) significantly decreased the extent of 113
48 obtained from the leaves of Terminalia catappa against the gastric ulcerative lesions by 33% and 71%, respectively, when compared to 114
49 mucosa damage induced by absolute ethanol. To establish a dose– stomachs from the vehicle-treated control group (po0.01). 115
50 response profile for the antiulcer activity of FrAq, we used varying After confirming the gastroprotective effect of FrAq against 116
51 doses of FrAq (12.5, 25 and 100 mg/kg body weight) to identify the lesions induced by ethanol and I/R, the next step was to evaluate 117
52 lowest dose that could elicit an optimal gastroprotective effect the antiulcer mechanisms of action for this fraction. We studied the 118
53 (Fig. 1). The oral administration of ethanol rapidly penetrated the effect of FrAq on gastric juice parameters to evaluate their possible 119
54 gastric mucosa and caused membrane damage, erosion, and hemor- anti-secretory action by the pylorus ligation procedure (Table 1). We 120
55 rhagic ulcerations. The pre-treatment of rats with FrAq significantly observed that the administration of FrAq by an intraduodenal or 121
56 inhibited the formation of gastric lesions by 41% (25 mg/kg) and 36% oral route was not able to change the H þ concentration of gastric 122
57 (100 mg/kg) compared to vehicle-treated control group (po0.001). juice when compared to vehicle-treated animals (p40.05). This 123
58 A lower dose of FrAq (12.5 mg/kg) evaluated in this study did not result excluded the possibility of the antisecretory effect of FrAq. 124
59 show a gastroprotective effect against ethanol (p40.05). We also However, this assay also revealed that the oral treatment with FrAq 125
60 observed no significant differences in the gastroprotective effect of was able to significantly increase gastric volume (42%). This effect 126
61 FrAq at doses of 25 or 100 mg/kg (p40.05). Thus, the subsequent could represent that FrAq was able to induce an increase in the 127
62 experiments with FrAq were carried out the lower dose of 25 mg/kg amount of adherent gastric mucus. Next, we evaluated the effects of 128
63 administered orally. the oral administration of FrAq on this gastric mucus in pyloric 129
64 This study evaluated the ability of FrAq to protect the gastric ligature rats (Fig. 3). We observed that oral treatment with FrAq 130
65 mucosa of rats from oxidative damage induced during an I/R proce- caused an increase (43%) in the amount of adherent gastric mucus 131
66 dure (Fig. 2). We observed that oral treatment with FrAq (25 mg/kg) as compared to the vehicle-treated group (po0.05), confirming our 132

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 ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 5

1 Table 1 14 days (Fig. 5b) demonstrated that this fraction was able to 67
2 Effects of the aqueous fraction (FrAq) from the leaves of Terminalia catappa (25 mg/kg) accelerate the healing of chronic gastric ulcers in rats. At the dose 68
administered through the intraduodenal (i.d.) or oral (p.o.) route on the biochemical
3 of 25 mg/kg FrAq, 7 and 14 days of treatment significantly decreased 69
parameters of gastric juice obtained from pylorus-ligature rats (n¼6–8).
4 the area of lesion in 80% and 37%, respectively when compared to the 70
5 Treatment Route Dose (mg/kg) Gastric juice (mL) [H þ ] (μEq/mL/4 h) control group treated with vehicle (po0.01). In the group treated 71
6 with lansoprazole at 30 mg/kg, the healing effect was also observed 72
7 Vehicle i.d. – 7.02 7 2.25 6.99 7 1.84 with the decrease of gastric lesion at 83% (7 days) and 64% (14 days). 73
Cimetidine 100 4.29 7 1.19nn 2.777 1.30nn
8 FrAq 25 8.02 7 2.18 7.34 7 0.65
To determine in better detail the healing process of FrAq, we also 74
9 analyzed the stomach by zymograph. Fig. 6 presents the activities of 75
Vehicle p.o. – 3.747 1.01 5.25 7 1.51
10 MMP-2 and MMP-9 on the gastric mucosa after treatment with 76
Cimetidine 100 2.63 7 0.67 1.94 7 0.87nn
11 FrAq 25 5.30 7 1.67 n 6.337 0.43 vehicle, lansoprazole and FrAq for 7 or 14 days. Our results show that 77
12 MMP-9 activity was present only after treatment during 7 days in 78
13 Data represents the means7 S.E.M. The asterisks denote the significance levels, stomachs from the vehicle and lansoprazole-group. We also observed 79
14 when compared with the control group. that this MMP-9 activity was absence for all treatments during 14 80
n
15 po 0.05. days. In contrast, MMP-2 activity was present in all treated groups 81
nn
p o0.01 by one-way ANOVA followed by Dunnett's test.
16 including the sham group (without gastric lesion). The treatment of 82
17 animals that exhibited major MMP-2 activity after 7 and 14 days was 83
18 compared to the animals treated with vehicle. Lower activity of 84
19 n.s. MMP-2 was observed after treatment of the animals with lansopra- 85
20 zole and FrAq for 7 days (Fig. 7a,  40 and 26%, respectively, in rela- 86
21 3000 tion to the vehicle) and 14 days (Fig. 7b,  54 and 73%, respectively, 87
22 in relation to the vehicle). 88
23 Other important data obtained from this test based on the 89
(mg/g wet tissue)

24 treatment of animals with FrAq for 7 and 14 consecutive days was 90

2000
Alcian blue

25 the absence of death in these groups and also absence body 91

26 weight changes (data not shown), and some organ weights and 92
27 biochemical parameters of serum (Table 2). These results, there- 93
28 fore, ensure that based on the parameters used in this study, the 94
29
1000 FrAq at doses of 25 mg/kg does not cause significant toxic effects 95
30 after 7 or 14 consecutive days of treatment. 96
31 This study also evaluated the activity of FrAq against Helico- 97
32 bacter pylori and presented minimal inhibitory concentration 98
0
33 Vehicle Carbenoxolone FrAq (MIC) of 125.0 mg/mL considered a accentuated activity and the 99
34 100 mg/kg 25 mg/kg positive control (amoxicillin) presented MIC of 15.0 mg/mL. 100
35 The analysis of mass spectra of FrAq (Fig. 9) showed the same 101
Fig. 3. Effects of the aqueous fraction (FrAq) obtained from the leaves of Terminalia
36 compounds identified in the hydroalcoholic extract studied by 102
catappa (25 mg/kg) on the production of adherent gastric mucus (measured as the
37 amount of bound Alcian blue) in pylorus-ligated rats relative to the control values
Mininel et al., (2014) as shown in Table 3. The spectra (Fig. 8) show 103
38 (n¼ 6). The results are the mean7 S.E.M. Statistical significance was determined by the ions with m/z 1083 (punicalagin); m/z 601 attributed to 104
39 ANOVA followed by Dunnett's t test (np o0.05; nnn p o0.01 and n.s. – no significant gallagic acid and at m/z 301 attributed to ellagic acid, illustrating 105
40 differences). the similarity with the hydroalcoholic extract (Mininel et al., 106
41 2014). The fragmentation of the second order (MS2) ion at m/z 107
42 hypothesis. As expected, carbenoxolone (used as positive control) 1083 (punicalagin) leads to ions at m/z 781 (punicalin) and the ion 108
43 enhanced the mucus production and there was no statistical with m/z 601 (gallagic acid) as shown in Fig. 9. The chromato- 109
44 difference between the groups treated with carbenoxolone and graphic profile of the water fraction is also shown in Fig. 10. In the 110
45 FrAq (p40.05). chromatogram, two peaks are highlighted, eluting at tr 17.2 min 111
46 In addition to mucus as defense mechanism elicited by FrAq, and tr 23.3 min, indicating the presence of the major compound 112
47 other factors involved with the strengthening of gastric mucosa were punicalagin (α and β anomers). Two peaks with tr 28.0 min and tr. 113
48 also investigated, such as NO (nitric oxide), sulfhydryl (SH) com- 30.1 min (region 2) were observed, corresponding to the com- 114
49 pounds and PGs, which are factors that maintain mucosal integrity. pound punicalin (α and β anomers), derived from punicalagin. 115
50 Our results (Fig. 4a) shown that the pretreatment of animals with 116
51 NEM (sulfhydryl inhibitor) did not change the gastroprotective effect 117
52 of FrAq (p40.05) compared to the vehicle-pretreated FrAq group. 4. Discussion 118
53 Therefore, this result excluded the involvement of sulfhydryl groups 119
54 in the gastroprotective effect of FrAq. However, the group of animals The development of gastric ulcers is a complex and multi- 120
55 pretreated with L-NAME (a NO-synthase inhibitor) and treated with factorial process including bacterial infections, the increase of acid 121
56 FrAq display a significant increase in gastric lesions (Fig. 4b). A similar secretion, generation of reactive oxygen species (ROS), inhibition 122
57 picture emerged when the effect of PGs was studied. Fig. 4c shows of the endogenous PGs, and the degradation of the extracellular 123
58 that administration of indomethacin markedly increased the size of matrix (ECM) (Laine et al., 2008; Mei et al., 2013). Research during 124
59 the ulcerative lesion and completely abolished the previously the last decade has offered new insights in the preventative 125
60 observed protective effect of FrAq. Our results illustrated that beside therapy and the healing of gastric ulcers and the synergistic 126
61 the increase in gastric mucus barrier induced by FrAq, the gastro- efficiency of a multi-target approach based on individual mechan- 127
62 protective effect of this fraction also involved the action of NO and isms of action could be the new perspective for treatment of this 128
63 endogenous PGs levels. After determining the factors involved with disease (Wagner, 2011; de Carvalho et al., 2014). 129
64 FrAq action, the possible healing effect of FrAq was evaluated with Terminalia catappa, is a medicinal plant widely distributed in 130
65 the acetic acid method (the most similar to human gastric ulcer). Oral tropical and subtropical regions and it has been previously reported 131
66 treatment with FrAq during 7 consecutive days (Fig. 5a) and also that this species exhibits a variety of biological effects. However, the 132

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1 67
2 68
3 69
4 70
5 71
6 72
7 73
8 74
9 75
10 76
11 77
12 78
13 79
14 80
15 81
16 82
17 83
18 84
19 85
20 86
21 87
22 88
23 89
24 90
25 91
26 92
27 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 93
28 (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 94
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
29 95
(25 mg/kg). The results are reported as the mean7 S.E.M. Statistical significance was determined by ANOVA followed by Dunnett's test. np o0.05; nnp o 0.01; nnnp o 0.001
30 compared to the corresponding vehicle group. # o 0.05; ##p o 0.01 compared to the corresponding NEM þvehicle, L-Nameþ vehicle or INDO þvehicle. N.S. – no significant
96
31 differences. 97
32 98
33 99
34 20 10 100
35 101
36 102
37 8 103
15
38 104
ULA (mm²)
ULA (mm²)

39 6 105
40
10 ** 106
41 107
42
4 108
***
43 109
5
44 *** *** 2 110
45 111
46 112
0 0
47 113
Vehicle Lansoprazole FrAq Vehicle Lansoprazole FrAq
48 114
30 mg/kg 25 mg/kg 30 mg/kg 25 mg/kg
49 115
50 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 116
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
51 117
as the mean7 S.E.M. Statistical significance was determined by ANOVA followed by Dunnett's test. nnp o 0.01;nnnpo 0.001 compared to the corresponding vehicle group.
52 118
53 119
54 underlying mechanisms of the gastroprotective and healing effects extract of bark from this species against gastric ulcers induced by 120
55 of the aqueous fraction (FrAq) obtained from the leaves have yet to ethanol. Aside from using a different part of plant (the bark instead 121
56 be evaluated. leaves), this study has demonstrated gastroprotection at a dose 10 122
57 In the present study, FrAq showed a marked gastroprotective times higher than our study (250 mg/kg vs. 25 mg/kg). Our studies 123
58 effect, evidenced by the dramatic inhibition of the gastric damage also highlighted the use of the renewable part of this plant (leaves) 124
59 produced by ethanol. Ethanol induced erosion, ulcerative lesions, that would enable the management of this plant for the future 125
60 and petechial bleeding in the mucosa of the stomach in humans production of a phytotherapeutic against gastric ulcer. 126
61 and an ethanol-induced gastric ulcer model is commonly used to Gastric ulcer induced by ethanol is commonly associated with 127
62 study both the pathogenesis and new therapeutics for the treat- reduced mucosal blood flow and ischemia that causes deleterious 128
63 ment of gastric ulcer disease (Oyagi et al., 2010). effects on the gastric mucosa, mainly in the aging gastric mucosa 129
64 Our results have shown the effective gastroprotective effect of the (Tarnawski et al., 2014). Ischemia weakens the gastric mucosal barrier 130
65 aqueous fraction obtained from the leaves of Terminalia catappa. and increases the acid back-diffusion, predisposing the gastric mucosa 131
66 A previous study by Nunes et al. (2012) evaluated the ethanolic to damage (Rao and Vijayakumar, 2007). The restoration of blood flow 132

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 L. Pinheiro Silva et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 7

1 and punicalin, both polyphenols presents in Terminalia catappa, were 67

2 able to attenuate oxidative stress and hypoxia-induced apoptosis. 68
3 These results therefore confirm the gastroprotective effect of 69
4 this fraction and encourage the continuation of the studies of the 70
5 effects of FrAq treatment and the characterization of its mechan- 71
6 isms of action. 72
7 Studies already performed with the hydroalcoholic extract of 73
8 Fig. 6. Representative zymography results of the gastric ulcers from the different Terminalia catappa and other species of this genus such as 74
9 groups illustrating MMP-2 and MMP-9 activity. Treatment for 7 days – vehicle (line 1), Terminalia chebula and Terminalia fagifolia have demonstrated the 75
lansoprazole (line 2), aqueous fraction (FrAq) obtained from leaves of Terminalia
10 catappa at dose of 25 mg/kg (line 3) and sham (line 4). Treatment for 14 days – vehicle
antisecretory effect of these species (Kumar et al., 2014; Mishra 76
11 (line 5), lansoprazole (line 6), aqueous fraction (FrAq) obtained from leaves of et al., 2013; Nunes et al., 2014). Unlike these studies, our results 77
12 Terminalia catappa at dose of 25 mg/kg (line 7) and sham (line 8). Gelatinolytic bands have shown that the oral and intraduodenal treatment with FrAq 78
13 of 92, 72, 64 and 57 kDa were observed, which correspond to active-MMP-9, (25 mg/kg) does not promote the antiulcerogenic effect in an 79
pro-enzyme, intermediate and active-MMP-2, respectively.
14 antisecretory manner. However, our result also illustrated that 80
15 oral treatment with FrAq was able to significantly increase the 81
16 gastric volume in relation to the control group treated with vehicle 82
17 (5.30 mL and 3.74 mL, respectively). Our hypothesis was that the 83
18 FrAq elicits the increase of gastric juice because of the amount of 84
19 adherent gastric mucus. 85
20 The success of the pharmacological treatment for gastric ulcers 86
21 relies on the augmentation of the protective factors of the gastric 87
22 mucosa such as the mucus barrier (Al-Jiboury and Kaunitz, 2012; 88
23 Brzozowska et al., 2013). The adherent gastric mucus is the first line 89
24 of defense against acid and serves as a barrier against self-digestion; 90
25 the functional integrity of the gastric mucosa depends on this 91
26 barrier (Wallace, 2008). Our study reveals a significant increase in 92
27 the amount of adherent mucus in the animals treated with FrAq, 93
28 thus justifying the increase in gastric volume by treatment with this 94
29 fraction. This study involving the FrAq is highly significant in the 95
30 ongoing search for an antiulcerogenic therapy, as the prolonged use 96
31 of antisecretory drugs such as proton-pump inhibitors and H2 97
32 blockers can provoke serious side effects (Ozdil et al., 2013; Eom 98
33 et al., 2011). 99
34 In addition to the increased production of the mucus barrier, 100
35 several studies have demonstrated a complex defense system invol- 101
36 ving the gastric mucosa, which include the regulation of gastric 102
37 mucosal blood flow, the formation of sulfhydryl compounds, NO and 103
38 endogenous PGs (Brzozowski et al., 2000; Pawlik et al., 2001). 104
39 NO is considered to be one of the most important defensive 105
40 endogenous agents in the gastric mucosa and plays a physiological 106
41 role in the homeostasis of the gastrointestinal tract (Brzozowski 107
42 et al., 2006). Together with the endogenous prostaglandins, NO also 108
43 preserves the gastric mucosa integrity and the inhibition of NO 109
44 release can result in disturbances in the gastrointestinal motility, 110
45 blood flow, and acid secretion (Brzozowski et al., 2006; Khalifa 111
46 et al., 2002; Wallace and Ma, 2001). In addition to the increase in 112
47 gastric mucus, our results show that the restoration of the endo- 113
48 genous NO represents an additional mechanism responsible for the 114
49 gastroprotective effects of FrAq, as a NO-synthase inhibitor elicits a 115
Fig. 7. Effect of treatment with the vehicle, lansoprazole (30 mg/kg) or the aqueous
50 fraction (FrAq) obtained from Terminalia catappa (FrAq) on the gelatinolytic activity
significant increase in gastric lesion in stomachs previously treated 116
51 of MMP-9 (black column) and MMP-2 (white column) in the gastric mucosa after 7 with the fraction. The gastroprotective effect of the FrAq through 117
52 (panel a) and 14 days (panel b) of treatment. Values show the ratio of the IOD from endogenous NO agrees with the protective effect of this fraction 118
53 the treatment groups to the control group (vehicle) IOD value. against I/R in rats by inhibiting the excessive formation of ROS. 119
54 Our study is also in concordance with those performed by 120
55 (reperfusion) after a period of ischemia initiates a cascade of changes, Pandya et al. (2013) in which they evaluated the status of the 121
56 including the release of local ROS, inflammatory responses, tissue ethanolic extract of Terminalia catappa (at dose of 200 mg/kg) and 122
57 damage by fragmenting cellular DNA and an increase in the adhesion illustrated an augmentation of the antioxidant defense system 123
58 of neutrophils to endothelial cells, a phenomenon known as ische- against Ehrlich carcinoma. Kinoshita et al. (2007) also described 124
59 mia–reperfusion (I/R) (Smith et al., 1996; De Foneska and Kaunitz the antioxidant and hepatoprotective actions from the aqueous 125
60 2010). extract of the leaves of Terminalia catappa. As well as NO, the 126
61 The current study shows that oral treatment with FrAq (25 mg/kg) increase in gastric SH content is important for the limiting the 127
62 significantly decreased the extent of ulcerative lesions in the gastric production of oxygen-derived free radicals (Genestra, 2007). 128
63 tissue submitted to I/R injury compared to stomachs from the vehicle- Our study showed that the pre-treatment of animals with NEM 129
64 treated control group. Based on the physiopathology of the I/R (a sulfhydryl inhibitor) did not change the gastroprotective effect 130
65 experimental model, our results are in concordance with Chen et al. of the FrAq (p 40.05). Therefore, our results exclude the involve- 131
66 (2012) and Wang et al. (2013) in which they observed that punicalagin ment of SH content in the gastroprotective effect of FrAq. 132

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1 Table 2 67
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 68
selected toxicological parameters (n¼ 5–6).
3 69
4 Treatment (p.o.) 70
5 71
6 Vehicle Lansoprazole FrAq 72
7 73
Organ weights (g)
8 Heart 3.85 7 0.48 3.88 7 0.32 3.677 0.13
74
9 Lung 4.497 0.64 5.34 7 1.43 4.20 7 0.40 75
10 Kidney 5.147 0.63 4.917 0.18 4.917 0.07 76
11 Liver 10.107 1.23 9.99 7 0.28 9.707 0.17 77
Spleen 2.69 7 0.28 2.83 7 0.24 2.677 0.27
12 78
13 Biochemical parameter 79
14 Glucose 136.90 7 7.00 136.40 7 5.80 151.007 8.50 80
Creatinine 0.56 7 0.02 0.56 7 0.03 0.53 7 0.04
15 Urea 38.107 1.12 39.40 7 5.35 39.60 7 4.23
81
16 γ-GT 0.96 7 0.17 2.02 7 0.55 1.247 0.25 82
17 AST 157.117 13.4 181.20 7 17.51 190.60 7 14.50 83
18 ALT 44.337 2.51 48.007 5.04 43.17 3.11 84
19 85
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,
20 AST – aspartate aminotransferase, γ–GT – gamma-glutamyl transpeptidase) and mg/dL (urea and creatinine). 86
21 87
22 88
23 Table 3 mucosal architecture (Okabe and Amagase, 2005). Chronic treat- 89
24 Identification of the substances obtained in the aqueous fraction (FrAq) from the ment with FrAq (25 mg/kg) demonstrated a dramatic reduction in 90
leaves of Terminalia catappa by FIA-ESI-IT-MSn.
25 the ulcerative lesion area by treatment for 7 days (80%), which was 91
26 [M–H]  MSn ions Identification more effective than 14 days of treatment (37%). This result 92
27 demonstrated the FrAq (25 mg/kg) accelerates the healing within 93

28 1083 781 [M-152-152-H] Punicalagin (1) 7 days of treatment and the healing effect remains after treatment 94
29 601 [M-152-152-180-H]  of 14 days when compared to control group or lansoprazole group. 95
781 601 [M-180-H]  Punicalin (2)
30 601 409 [M-191-H]  Gallagic acid (3)
The injection of acetic acid into gastric mucosa induces the 96
31 301 257 [M-44-H]  Ellagic acid (4) development of deep gastric ulceration and gastric mucosal damage 97
32 229 [M-44-28-H]  directly associated with ECM degradation, in which the zinc- 98
33 dependent matrix metalloproteinases (MMPs) play a crucial role. 99
Adapted from Mininel et al. (2014).
34 In several animal studies of gastric ulcer, attention has focused 100
35 on the role of MMPs, mainly MMP-2 and MMP-9 (Sen-Li et al., 101
36 Among the humoral factors in the gastric mucosa, endogenous 2013). Wound formation and the following healing are dynamic 102
37 PGs play an important role in the protective effect by stimulating processes of ECM remodeling that are mainly influenced by MMP-2 103
38 the secretion of mucus, maintaining the local blood flow and and MMP-9 (Gyenge et al., 2013). 104
39 increasing the resistance of epithelial cells to potential damage by According to Yeh et al. (2012) MMP-9 is the protease most 105
40 cytotoxins (Takeuchi, 2010). Our results illustrate that the admin- significantly involved in the degradation of the basement membrane 106
41 istration of a non-selective COX inhibitor (indomethacin) comple- and is associated with pathological states that include inflammation 107
42 tely abolished the gastroprotective action of the FrAq. This result and cancer. Sen-Li et al. (2013) described enhanced expression of 108
43 highlights the relevance of PGs in the antiulcer action of this MMP-9 in the margin of the ulcer in patients with this disease. Their 109
44 fraction and agreement with the previously results of fraction in study suggested that the presence of MMP-9 at the margin of the 110
45 increase in the amount of adherent mucus. According to Takeuchi ulcer may be indicative of inflammation and poor wound healing. 111
46 (2010) one of the mechanisms underlying the action of PGE2 is Ulcerogenic agents, such as indomethacin, exhibited 12-fold higher 112
47 exactly the increase in mucus secretion that augments the pro- pro-MMP-9 activity and ethanol exhibited 22-fold higher pro-MMP-9 113
48 tective factors on the gastric mucosa. activity in rat gastric tissues relative to untreated tissues (Mei et al., 114
49 Based on the results regarding the gastroprotective effect of the 2013). Our results showed MMP-9 activity only after treatments with 115
50 FrAq against gastric injury induced by different ulcerogenic agents, the vehicle and lansoprazole (7 days). These results determined that 116
51 we confirmed that the action of this fraction was mediated by the the presence of MMP-9 activity at the gastric mucosa in groups 117
52 activation of defensive mucosa-protective factors such as increased treated with the vehicle was related with the absence of healing in 118
53 mucus production, NO pathways and endogenous PGs. However, it ulcers of the gastric mucosa. Our results also show that the seemingly 119
54 is desirable for any new antiulcer drug that the preventive effect is healing macroscopic ulcer observed by treating animals with lanso- 120
55 also accompanied by ulcer healing effects. prazole (7 days) may not represent a good wound healing because the 121
56 Antiulcer drugs such as H2-receptor antagonists fail in promot- presence of MMP-9 activity in gastric damage further demonstrates 122
57 ing the healing of gastric ulcers when the regenerated mucosa the persistence of the inflammatory process at the gastric mucosa. 123
58 presents with poor histological maturity and ulcer relapse is not Our results shown that treatment of the acetic acid induced chronic 124
59 prevented by cimetidine (Arakawa et al., 2012). ulcers with FrAq (25 mg/kg) for 7 and 14 days inhibited the MMP-9 125
60 This study also determined the effect of the FrAq on the healing activity. Our results could be strengthened by the study from Yeh 126
61 of gastric ulcers induced by acetic acid in rats treated for 7 or 14 et al. (2012) in which extract from Terminalia catappa leaves exerts an 127
62 consecutive days. The acetic acid induced a deep necrotic lesion, antimetastic effect by attenuating MMP-9 mediated inflammation in 128
63 involving the entire mucosal depth and penetrating through the hepatocellular carcinoma. In addition to our results obtained regard- 129
64 muscularis mucosae. Ulcer healing is a dynamic process of filling ing MMP-9, our results also reported high MMP-2 activity in gastric 130
65 mucosal defects with proliferating and migrating epithelial cells ulcers of the control group treated with vehicle (7 and 14 days), and 131
66 and connective tissue, which results in the reconstruction of the lower MMP-2 activity was observed after the treatment of animals 132

Please cite this article as: Pinheiro Silva, L., et al., Terminalia catappa L.: A medicinal plant from the Caribbean.... Journal of
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1 67
2 1083.39 68
100
3 69
4 95 70
5 90 71
6 85 72
7 80 73
8 75 74
9 70 75
10 76
Relative Abundance

65
11 60 77
12 55 78
13 50 79
14 45
80
15 81
40
16 82
35
17 781.45 83
541.15
30
18 1013.13 84
25
19 954.49 85
20 639.27 1132.91
20 86
15 312.07 1349.87
21 87
10 1397.19
22 1562.07 88
1712.29
23 5 89
24 0 90
500 1000 1500 2000
25 91
26 m/z 92
27 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 100–2000 Da. 93
28 94
29 T_130129165016 #1-1000 RT:0.60-0.96 AV:70 NL:2.71E2 95
30 F: ITMS - c ESI Full ms2 1083.00@cid30.00 [295.00-1200.00] 96
31 100 97
32 95 98
33 90 99
85
34 100
80
35 101
75
36 102
70
37 103
Relative Abundance

65
38 60 104
39 55 105
40 50 106
41 45 107
42 40 108
35
43 109
30
44 110
25
45 20
111
46 15 112
47 10 113
48 5 114
49 0 115
300 400 500 600 700 800 900 1000 1100 1200
50 116
51 m/z 117
52 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 118
53 catappa. Range of ions with m/z 300-1200 Da. 119
54 120
55 with lansoprazole and FrAq. Recent studies have highlighted the compounds punicalagin (anomers α and β) and punicalin (anomers α 121
56 function of MMP-2 in the healing process of rat gastric ulcers induced and β) are similar to the hydroalcoholic extract studied by Mininel 122
57 by acetic acid (Gyenge et al., 2013), but the function of MMP-2 is not et al. (2014). Ellagic acid (EA) is one of the naturally occurring 123
58 well understood. polyphenols found in the FrAq from Terminalia catappa leaves. 124
59 The antiulcerogenic actions demonstrated by FrAq could be Numerous pharmacological studies have suggested that EA provides 125
60 attributed to the phenolic compounds present in this fraction. mucosal protective action in the stomach against ethanol or ische- 126
61 Mininel et al. (2014) analyzed mass spectra in a full-scan of the mia–reperfusion injury (Iino et al., 2001; Iino et al., 2002) and several 127
62 extract and this fraction and showing similarity to each other, gastroprotective mechanisms are attributed to this compound. For 128
63 highlighted by the precursor ions m/z 1083 (punicalagin), m/z 781 example, Chatterjee et al. (2012) showed that EA enhanced prosta- 129
64 (pulicalin), m/z 601 (gallagic acid) and m/z 301 attributed to ellagic glandins when compared with the ulcerated untreated group. 130
65 acid. In these studies, the chromatogram from HPLC-PDA of the In our final experimental series, we evaluated the subacute 131
66 aqueous fraction of Terminalia catappa confirmed the majority of the toxicological parameters from groups that received vehicle, 132

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1 1 - α e β punicalagin Acknowledgments 67
2 T. catappa FrAq - CH5 68
3 This study was supported by the Biota-FAPESP project (Fundação Q4 69
4 de Amparo à Pesquisa do Estado de São Paulo), CNPq (Conselho 70
5 Nacional de Desenvolvimento Científico e Tecnológico) and CAPES Q5 71
2000000
6 (Coordenação de Aperfeiçoamento Pessoal de Nível Superior). 72
Intensity [μV]

7 73
8 74
9 References 75
10 1000000 76
11 2 - α e β punicalin Aligannis, N., Kalpotzakis, E., Mitaku, S., Chinou, I.B., 2001. Composition and 77
12 antimicrobial activity of the essential oils of two Origanum species. Journal of 78
Agricultural and Food Chemistry 49, 4168–4170.
13 79
Al-Jiboury, H., Kaunitz, J.D., 2012. Gastroduodenal mucosal defense. Current
14 Opinion of Gastroenterology 28, 594–601. 80
0
15 Arakawa, T., Watanabe, T., Tanigawa, T., Tominaga, K., Fujiwara, Y., Morimoto, K., 81
16 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 2012. Quality of ulcer healing in gastrointestinal tract: its pathophysiology and 82
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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