722 Vol. 37, No. 5Biol. Pharm. Bull. 37(5) 722–730 (2014)

© 2014 The Pharmaceutical Society of Japan

Regular Article

The Gastroprotective Effects of Eugenia dysenterica (Myrtaceae) Leaf Extract: The Possible Role of Condensed TanninsLigia Carolina da Silva Prado,a Denise Brentan Silva,c Grasielle Lopes de Oliveira-Silva,a Karen Renata Nakamura Hiraki,b Hudson Armando Nunes Canabrava,a and Luiz Borges Bispo-da-Silva*,a

a Laboratory of Pharmacology, Institute of Biomedical Sciences, Federal University of Uberlândia; b Laboratory of Histology, Institute of Biomedical Sciences, Federal University of Uberlândia; ICBIM-UFU, Minas Gerais 38400–902, Brazil: and c Núcleo de Pesquisas em Produtos Naturais e Sintéticos, Faculty of Pharmaceutical Science at Ribeirão Preto, University of São Paulo; NPPNS-USP, São Paulo 14040–903, Brazil.Received June 26, 2013; accepted February 6, 2014

We applied a taxonomic approach to select the Eugenia dysenterica (Myrtaceae) leaf extract, known in Brazil as “cagaita,” and evaluated its gastroprotective effect. The ability of the extract or carbenoxolone to protect the gastric mucosa from ethanol/HCl-induced lesions was evaluated in mice. The contributions of nitric oxide (NO), endogenous sulfhydryl (SH) groups and alterations in HCl production to the extract’s gastroprotective effect were investigated. We also determined the antioxidant activity of the extract and the possible contribution of tannins to the cytoprotective effect. The extract and carbenoxolone protected the gastric mucosa from ethanol/HCl-induced ulcers, and the former also decreased HCl production. The blockage of SH groups but not the inhibition of NO synthesis abolished the gastroprotective action of the extract. Tannins are present in the extract, which was analyzed by matrix assisted laser desorption/ioniza-tion (MALDI); the tannins identified by fragmentation pattern (MS/MS) were condensed type-B, coupled up to eleven flavan-3-ol units and were predominantly procyanidin and prodelphinidin units. Partial removal of tannins from the extract abolished the cytoprotective actions of the extract. The extract exhibits free-radical-scavenging activity in vitro, and the extract/FeCl3 sequence stained gastric surface epithelial cells dark-gray. Therefore, E. dysenterica leaf extract has gastroprotective effects that appear to be linked to the inhibition of HCl production, the antioxidant activity and the endogenous SH-containing compounds. These pleiotropic actions appear to be dependent on the condensed tannins contained in the extract, which bind to mucins in the gastric mucosa forming a protective coating against damaging agents. Our study highlights the biophar-maceutical potential of E. dysenterica.

Key words Eugenia dysenterica; gastroprotection; condensed tannin; Myrtaceae; matrix assisted laser de-sorption/ionization (MALDI)

Numerous plant-derived extracts are gastroprotective and represent a great source of bioactive compounds with the ther-apeutic potential to treat gastric and duodenal ulcers, diseases that are considered to be worldwide heath issues.1) Thus, stud-ies of this subject are important because they provide data that assist governmental institutions in their policies concerning the use of plants in the phytotherapeutic industry, the sustain-able use of the biodiversity and consequently, the application of phytotherapeutics to health promotion programs.

Many plants exhibiting gastroprotective actions that have been studied belong to the Myrtaceae family and include Campomanesia lineatifolia,2) Campomanesia xanthocarpa,3) Eugenia jambolana,4,5) Myrtus communis,6) Plinia edulis,7) Syzygium aromaticum,8,9) and Syzygium cumini.10) Eugenia dy-senterica DC. (Myrtaceae), also known in Brazil as “cagaita” or “cagaiteira,” is a shrubby ornamental and melliferous tree that is widely distributed throughout the second largest Bra-zilian biome, the Cerrado or upland savannah.11) The fruit is edible and has traditionally been used as a cathartic agent; the leaves, in contrast, have been used to treat diarrheic dis-eases.11) Recently published data provide scientific support, at least in part, for these folk uses.12,13)

In considering the selection of a plant for pharmacological studies many approaches can be used. The taxonomic ap-

proach to plant collection relies on the premise that related taxa have inherited the genetic ability to produce similar pharmacologically active secondary metabolites.14,15) There-fore, considering that many species of the Myrtaceae family including those from the Eugenia genus have been reported to possess gastroprotective activities, we applied the taxonomic approach to select E. dysenterica and study its cytoprotective actions on the stomach. In a pilot experiment published only as part of an academic report, our co-author (Canabrava) and his colleagues observed that E. dysenterica leaf extract indeed protects the gastric mucosa of rats from damage caused by the administration of indomethacin, a cyclooxygenase inhibitor.16) To study this subject further and to pharmacologically char-acterize the E. dysenterica leaf extract, we investigated the mechanisms involved in its gastroprotective effect on ethanol/HCl-induced ulcers in mice; moreover, a group of secondary compounds that are putatively responsible for this effect were analyzed and identified by matrix assisted laser desorption/ionization (MALDI)-MS and MS/MS.

MATERIALS AND METHODS

Animals The experiments were performed using male Swiss mice (35–45 g). The animals were housed in a room at 21°C with 12 h light/dark cycles and were provided free access to tap water and standard chow. All the protocols used were

* To whom correspondence should be addressed. e-mail: bispodsilva@yahoo.com.br; bispodasilva@icbim.ufu.br

The authors declare no conflict of interest.

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reviewed and approved by the Ethics Committee on Animal Use of the Federal University of Uberlândia (CEUA/UFU; process n° 022/11, addendum 175/11).

Plant Material Eugenia dysenterica DC. (Myrta-ceae) leaves were collected at the Santa Rita farm located in Lassance city, state of Minas Gerais, Brazil (17°59′14.23″S; 44°44′38.00″W) in September, 2011. The species was identi-fied by Dr. Adriana Arantes, and a voucher specimen was de-posited at the Herbarium of the Federal University of Uberlân-dia (Uberlândia, MG, Brazil) with the number HUFU-45956.

Preparation of the Leaf Extract The leaves were washed (immersed for 10 min in 70% ethanol, 5 min in 0.2% hypochlo-rite and 10 min in running water; this procedure was applied to disinfect the surface of the leaves) and dried in an oven at 40°C for 48 h. The powdered dried leaves were extracted with distilled water (20%, w/v) for 48 h at room temperature. The extract was lyophilized and then stored at −20°C and protect-ed from light until use (yield: 0.8%). The surface disinfection procedure neither altered the biological effect of the extract on the gastric tissue, nor changed the structure of its condensed tannins when compared to extract obtained with leaves that were washed only with water (data not shown).

Qualitative Determination of the Tannins in the Extract The presence of tannins was screened by reacting the extract samples with gelatin or FeCl3 according to the method de-scribed by Costa (2000), with some modifications as follows: (I) 1 drop of HCl (10%) was added to 1 mL of the extract solution (1 mg/mL), and gelatin solution (2.5% in 10% NaCl) was added dropwise; the formation of a precipitate indicated a positive reaction. (II) FeCl3 (1%) was added dropwise to 5 mL of the extract solution (1.0 mg/mL); the solution was monitored for a brownish green or a blue-black color and/or precipitation.

Evaluation of the Gastroprotective Effect of the Extract The acidified ethanol-induced gastric lesion model17) was used to evaluate the gastroprotective effect of the E. dysenterica leaf extract. Mice were divided into 6 groups (n=6–10) and fasted for 24 h prior to the oral administration (total volume: 0.8 mL) of saline (0.9% NaCl), carbenoxolone (250 mg/kg)9) or E. dysenterica leaf extract (100, 300, 550, 1000 mg/kg); 50 min after the treatments, all the animals orally received 0.26 mL of 0.3 M HCl/60% ethanol. The animals were sacrificed by cervical dislocation under anesthesia (sodium thiopental, 60 mg/kg, intraperitoneally (i.p.)) 1 h after the administration of the HCl/ethanol solution. The stomachs were removed, opened along the greater curvature, fixed between 2 glass plates and scanned. ImageJ software (http://rsb.info.nih.gov/ij/) was used to analyze the stomach images, and the results were expressed as the ulcerative index (U.I.). To calculate the U.I., the lesions were scored according to the severity of the gastric mucosal injury as follows: hemorrhagic lesions (3), high hyperemic area (2) and moderate or soft hyperemic area (1). Thus, the U.I. was determined as previously described,18) with some modifications: U.I.=3×hemorrhagic lesion area (mm2)+2×high hyperemic area (mm2)+1×moderate/soft hy-peremic area (mm2).

Determination of the Gastroprotective Effect of the Extract with a Reduced Tannin Content Tannins were partially removed from the extract as previously described,19) with modifications as follows: 1.5 mL of a gelatin solution (2.5%) in 0.2 mol/L acetate buffer, pH 5.0 with 0.17 mol/L NaCl was added to 1.5 mL of the extract (80 mg/mL) and

mixed carefully. After 15 min, the samples were centrifuged at 3000 rpm for 15 min. The supernatant was removed and used to treat mice (GD-ED group) that had been fasted for 24 h (1.0 mL/40 g, i.e., 1000 mg/kg) 30 min before the administra-tion of the ethanol/HCl solution. A control extract (ED group) was prepared by adding 1.5 mL of the acetate buffer without gelatin to the same volume of the extract (80 mg/mL). To verify the effectiveness of this method in removing polyphe-nolic compounds from the extract, 2.980 µL of FeCl3 (0.01 M in 0.01 M HCl) was added to 20 µL of the supernatant from the gelatin-treated, control extract or distilled water (blank) to determine its absorbance at 510 nm 45 min later. The data from both groups were expressed as the absorbance units/mg of the extract.

Determination of the Ability of the Extract to Bind to the Gastric Mucosa and Stain with Ferric Chloride This experiment was conducted as previously described20) with some modifications. Briefly, animals without any treatment were sacrificed by cervical dislocation under anesthesia (sodium thiopental, 60 mg/kg, i.p.); the stomachs were then removed, fixed in phosphate-buffered 4% formalin, embed-ded in paraffin and further sectioned in 5 µm slices. Depar-affinized and hydrated sections were then treated with a 5% solution of E. dysenterica leaf extract for 1 h, rapidly rinsed in water (3 times), treated with 2% FeCl3 for 15 min, washed in water, alcohol and xylene and mounted in gum damar. No counterstain was used, and the slices were observed under a light microscope.

Effect of the Extract on Gastric Secretion The assay was performed according to the method of Shay et al.,21) with some modifications as follows: the mice were fasted for 36 h with free access to water; immediately after laparotomy and pylorus ligature under anesthesia (10 mg/kg xylazine and 100 mg/kg ketamine, i.p.), E. dysenterica leaf extract (1000 mg/kg), cimetidine hydrochloride (100 mg/kg)9) or saline were administered intraduodenally, and the abdomen was sutured. The animals were sacrificed 4 h later by cervical dis-location while they were under thiopental sodium anesthesia (60 mg/kg, i.p.); the abdomen was then reopened, and another ligature was placed at the esophageal end near the diaphragm. The stomachs were removed, the gastric content was collected and weighed (in mg), and the gastric content volume was ad-justed to 5 mL by the addition of distilled water. The solution was centrifuged at 3000 rpm for 10 min. The pH was deter-mined using a pH meter, and the total acid in the supernatant was determined by titrating to pH 7.0 using a 0.01 mol/L NaOH solution and phenolphthalein as an indicator. The free and total acidity values were expressed as pH values and µeqH+/g of the gastric content, respectively.

Evaluation of the Participation of Nitric Oxide (NO) and Endogenous SH-Groups in the Gastroprotective Action of the Extract These experiments were conducted as previous-ly described,22) with some modifications: mice were fasted for 24 h and divided into groups (n=9–10) that were treated with NEM (N-ethylmaleimide, 10 mg/kg, i.p.), (NG-nitro-L-arginine methyl ester (L-NAME), 70 mg/kg, i.p.), or saline (0.5 mL, i.p.).9) Thirty minutes later, animals of all groups received oral doses of the vehicle (saline), carbenoxolone (250 mg/kg) or E. dysenterica leaf extract (1000 mg/kg). After 50 min, gastric mucosa lesions were induced. The animals were sacrificed 1 h after the administration of the HCl/ethanol solution, the

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stomachs were removed, and the gastric mucosa lesions were measured as described above.

Determination of the in Vitro Antioxidant Activity of the Extract The free-radical-scavenging activity of the E. dysenterica leaf extract was measured in vitro using the 1,1-diphenyl-2-picryl-hydrazyl (DPPH·; Sigma-Aldrich Chemi-cal Co., St. Louis, MO, U.S.A.) free radical method.23) Briefly, 0.5 mL of water or extract dissolved in water was added to 1.5 mL of an ethanolic DPPH· solution to reach the following final concentrations in the reaction tube: 0.06 mM DPPH· and 0.1, 0.2, 0.3, 1.0, 2.0, 3.0, 10.0 or 30.0 µg/mL extract. After a 30 min incubation at room temperature in a low luminosity environment, the absorbance of each sample was determined at 515 nm. The difference between the absorbance of the solu-tions containing only DPPH· and that of solutions containing DPPH· plus extract was determined. Tests were performed in duplicate using solutions without reagents as blanks and ascorbic acid as a positive control. The sample concentration providing 50% inhibition (IC50) was calculated by nonlinear regression analysis (hyperbolic equation) of the inhibition percentage plotted against the sample concentration using the GraphPad Prism® software.

Liquid Chromatography-Tandem Mass Spectrometry The LC analyses were performed with an UPLC-DAD-ESI TQ Acquity (Waters®, Milford, MA, U.S.A.), using a C18 Shim-pack XR-ODS (2.2 µm; 2.0 mm×50 mm, Shimadzu) column. The mobile phase was acetonitrile (solvent B) and water containing 0.1% formic acid, and the flow rate was 0.3 mL·min−1. The column temperature was 30°C and the injection volume was 5 µL. The elution profile was the fol-lowing: 5% B (0 to 0.9 min), 5 to 20% B (0.9 to 5.1 min), 20 to 100% B (5.1 to 8.5 min), 100% B (8.5 to 9.2 min). The MS conditions were the following: cone energy of 25 kV, colli-sion energy of 25 eV, capillary energy of 2.5 kV. Nitrogen was used as the nebulizing and drying gas (650 L h−1, 350°C) and argon was used as the collision gas. TIC chromatograms were recorded between m/z 50 and 1000 in both negative and posi-tive modes.

MALDI-MS and MALDI-MS/MS Analyses The MS and MS/MS analyses were performed using the UltrafleX-treme MALDI-TOF/TOF equipment (Bruker Daltonics, Bremen, Germany). A mixture of peptides (standard II of Bruker®) was used for the external and internal calibrations. The ions were accelerated at 20 kV. The experimental condi-tions were the following: pulsed ion extraction of 120 ns, laser frequency of 1000 Hz, reflectron mode, positive ion mode; 800 shots were averaged to record a mass spectrum. For MS/MS analyses, the selected ions were accelerated to 19 kV in the LIFT cell for MS/MS analyses. 2,5-Dihydroxybenzoic acid (DHB) was used as the matrix (20 mg/mL with 30% acetoni-trile and 70% H2O with 0.1% trifluoracetic acid). The aqueous extract was solubilized with ACN : H2O (3 : 7) and DHB con-taining 0.1 M NaCl (1 : 1 : 0.1) was added. These mixtures (1 µL) were spotted onto a ground stainless steel MALDI target. The compounds were identified by their MS data, fragmentation pattern and accurate mass measures.

Drugs Carbenoxolone, N-ethylmaleimide and NG-nitro-L-arginine methyl were purchased from Sigma-Aldrich Chemi-cal Co., cimetidine was purchased from Fluka Analytical (Buchs, Switzerland), xylazine and ketamina were purchased from Syntec do Brasil LTDA (Cotia, SP, Brazil), thiopental

was purchased from Cristália Produtos Químicos e Farmacêu-ticos LTDA (Itapira, SP, Brazil).

Statistical Analysis The data were expressed as the means± S.E.M. and were compared by ANOVA followed by Dunnett’s test or by Student’s t-test, as appropriate. The data were considered to be statistically significant when p<0.05.

RESULTS

E. dysenterica leaf extract decreased the U.I. (in mm2) in a dose-dependent manner and was as effective as the reference drug carbenoxolone (Fig. 1). The addition of gelatin and FeCl3 to the E. dysenterica leaf extract resulted in the formation of a precipitate and the development of a blue-black color, respectively. Moreover, the addition of gelatin to the extract decreased the extract’s absorbance at 510 nm after the addition of FeCl3 (Fig. 2A) and greatly decreased its gastroprotective effect; indeed, there was no significant difference between the U.I. observed in saline (control) and GT-ED treated groups (Fig. 2B). Moreover, the E. dysenterica extract/FeCl3 sequence stained the surface epithelial cells dark-gray (Fig. 3); neither E. dysenterica extract nor FeCl3 alone stained the epithelial surface (not shown). The extract reduced DPPH· to a yellow product in a concentration-dependent manner, with approximately half the potency of the standard antioxidant agent, ascorbic acid (IC50: 3.97± 0.05 µg/mL vs. 2.09± 0.01 µg/mL; extract vs. ascorbic acid). The intraduodenal administra-tion of E. dysenterica leaf extract or cimetidine decreased the total and free acidity of the gastric juice of mice subjected to the pyloric-ligation procedure (Table 1). However, neither the extract nor cimetidine significantly altered the mass of the gastric content (Table 1). The U.I. observed after E. dysen-terica leaf extract administration did not differ between the control (saline-treated) animals and the NEM-treated mice (Fig. 4A). L-NAME administration decreased the ability of carbenoxolone but not the extract to protect the gastric mucosa from ethanol/HCl-induced lesions (Fig. 4B).

The aqueous extract of E. dysenterica was analyzed by LC-DAD-MS and LC-DAD-MS/MS. The compounds were identified by analysis of the MS, MS/MS, and UV data, in comparison with previously published fragmentation patterns

Fig. 1. Evaluation of the Gastroprotective Effect of the Aqueous Euge-nia dysenterica Leaf Extract

The effects of E. dysenterica leaf extract (100–1000 mg/kg, p.o.) and carbenoxo-lone (CBX, 250 mg/kg, p.o.) on gastric lesions induced by the administration (p.o.) of acidified hydroalcoholic solution were analyzed in mice (n=6–10). The results represent mean±S.E.M. of the ulcer index (U.I.) in mm2. * p<0.05 and ** p<0.01 versus control (SAL).

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and standard injections. The substances procyanidin B-1 (5), catechin (4) and a dimeric procyanidin gallate were identified (Table 2, Fig. 5). The aqueous extract of E. dysenterica was also analyzed by MALDI. Figure 6 shows the MALDI-TOF spectrum of the polymeric tannin mixture in the extract. MS/MS data were analyzed to determine the presence of oligo-meric units of flavan-3-ol or galloyl. The identified tannins were condensed type-B, coupled up to eleven flavan-3-ol units. Moreover, E. dysenterica condensed tannins were found to consist predominantly of procyanidin units and were also combined with prodelphinidin units in the series 2 and 3 (Table 3, Fig. 5).

DISCUSSION

Our data demonstrated that E. dysenterica leaf extract dose-dependently protects the mouse gastric mucosa from damage induced by administration of ethanol/HCl. This observation confirms preliminary data from our laboratory (using cycloox-ygenase inhibitor to induce gastric lesion in rat)16) and shows that the gastroprotective effect of E. dysenterica leaf extract is not species- or model-specific, which may reflect its ability to

Fig. 2. Determination of the Gastroprotective Effect of the Aqueous Eugenia dysenterica Leaf Extract with a Reduced Tannin Content

Absorbance of Eugenia dysenterica leaf extract (ED) and gelatin-treated E. dysenterica leaf extract (GT-ED) at 510 nm after the addition of FeCl3 (A). Effects of saline (Sal, p.o.), ED (1000 mg/kg, p.o.) and GT-ED (1000 mg/kg, p.o.) on gastric lesions induced by the administration (p.o.) of acidified hydroalcoholic solution in mice (n=6–7) (B). The results represent mean±S.E.M. of the absorbance or of the ulcer index (U.I.) in mm2. * p<0.05 versus control.

Fig. 3. Determination of the Ability of the Aqueous Eugenia dysenterica Leaf Extract to Bind to the Gastric Mucosa and Stain with Ferric ChlorideMice gastric mucosa section (glandular portion) sequence treated with E. dysenterica and FeCl3. No counterstain. Note the dark-gray staining of the gastric mucosal

surface after the addition of FeCl3 (arrows).

Table 1. Effects the Eugenia dysenterica Leaf Extract (ED, 1000 mg/kg) or Cimetidine (100 mg/kg) Administered Intraduodenally on Biochemical Parameters of Gastric Contents

Parameters/Treatments Control Cimetidine ED

Gastric contents (g) 0.47±0.07 0.27±0.04 0.35±0.10pH 3.41±0.20 5.77±0.54** 5.36±0.54**Free acidity (µeq H+/g) 6.42±5.69 0.86±0.51* 2.21±1.55*Total acidity (µeq H+/g) 62.60±7.60 36.00±7.00* 34.60±7.60*

Values represent the mean±S.E.M.; * p<0.05, ** p<0.01 versus control. n=5–8 mice.

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influence various defense mechanisms in the gastric mucosa. The inhibitory effect of the extract on gastric lesions was less potent than that of the standard treatment (carbenoxolone, 250 mg/kg), however, the highest dose of the extract used was as effective as carbenoxolone, which is evidence of the power-ful cytoprotective action of E. dysenterica leaf extract.

Polyphenolic compounds, including flavonoids and tannins, are among the secondary metabolites linked to the gastropro-

tective actions of plants from the Myrtaceae family.2,3) Our preliminary phytochemical analyses suggested the presence of polyphenolic compounds in E. dysenterica leaf extract be-cause it became blue-black in the presence of FeCl3, a general characteristic reaction for this class of chemicals.24) Moreover, the extract effectively precipitated gelatin, a hallmark of tan-nic compounds,24) strongly suggesting the presence of tannins among the polyphenolic compounds from the E. dysenterica leaf extract. Based on these observations, we hypothesized that the tannins in the E. dysenterica extract could account for the extract’s cytoprotective activity. To test this hypothesis, we treated animals with a partially purified extract from which the tannin content had been greatly decreased by gelatin pre-cipitation (the effectiveness of our methods in removing poly-phenolic compounds was spectrophotometrically confirmed; in fact, the absorbance at 510 nm of gelatin-treated samples was decreased by almost 1/4 from that of the non-treated samples after FeCl3 administration; Fig. 2A). After this precipitation, the extract lost its gastroprotective activity, suggesting that the tannins are indeed responsible for the E. dysenterica leaf extract-mediated cytoprotection of the mouse gastric mucosa.

In addition to precipitate soluble proteins, tannins also bind to mucins in various tissues, including those on gas-tric surface epithelial cells.20) Thus, our observation that E. dysenterica leaf extract can bind epithelial mucins and that its presence can be detected as a dark-gray complex after ferric chloride treatment suggest that the tannin–mucin com-plex formed after E. dysenterica treatment could represent a protective coating that prevents gastric tissue damage caused by proteolytic enzymes, H+ and by ethanol, which solubilizes the mucus barrier. It is important to mention that this protec-tive coating mechanism has also been proposed to explain the cytoprotective actions of others tannin-rich extracts.2,25)

Therefore, considering that tannins appear to be extremely important for the gastroprotective effect of the extract, LC-DAD-MS/MS analyses were performed in an attempt to identify the tannins in the aqueous extract of E. dysenterica. It was possible to identify the compounds procyanidin B-1, catechin and procyanidin O-gallate; many additional tannins were identified by MALDI analyses. The molecular formulae were confirmed by the accurate mass data (with an internal calibrant), and the tannins were determined by the MS/MS data, which was used to define the series.

The tannins identified in E. dysenterica were condensed with B-type linkages. The polymerization of flavan-3-ol units, as procyanidin, prodelphinidin, prorobinetidin and others,

Fig. 4. Evaluation of the Participation of Nitric Oxide (NO) and Endo-genous SH-Groups in the Gastroprotective Action of the Eugenia dysen-terica Leaf Extract

Effects of E. dysenterica leaf extract (1000 mg/kg, p.o.) or carbenoxolone (CBX, 250 mg/kg, p.o.) on gastric lesions induced by the administration (p.o.) of acidified hydroalcoholic solution in mice (n=9–10) pre-treated or not with NEM (10 mg/kg, i.p.) (A) or L-NAME (70 mg/kg, i.p.) (B). The results represent the mean±S.E.M. of the ulcer index (U.I.) in mm2.* p<0.05 and ** p<0.01 versus respective control (pre-treated with saline, Sal).

Table 2. Identification of Chromatographic Peaks from the Aqueous Extract of E. dysenterica and UV, MS and MS/MS Data

Peak tR (min) Compound UV

(nm)Negative (m/z) Positive (m/z)

MS MS/MS MS MS/MS

1 3.01 Procyanidin B-1a) 278 577 [M−H]− 577 (20 eV)→451, 425, 407, 339, 299, 289, 245, 161, 125

579 [M+H]+ 579 (20 eV)→427, 409, 301, 289, 275, 247, 205, 163, 139, 127

2 3.41 Catechina) 278 289 [M−H]− 579 [2M−H]−

289 (20 eV)→248, 227, 217, 203, 188, 164, 151, 125, 123

291 [M+H]+ 291 (20 eV)→165, 147, 139, 123, 119, 111

3 4.73 Dimeric procyanidin gallate

277 729 [M−H]− — 731 [M+H]+ 731 (20 eV)→471, 440, 427, 410, 317, 301, 290, 247, 180, 163, 140, 127

tR: Retention time. a) Confirmed by injection of standards.

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yields condensed tannins, and the flavan-3-ols units are linked though C4–C6 or C4–C8 bonds between the units26) (Fig. 5). The MS data showed that the series were composed primar-ily of procyanidin units (290 µ) (Table 3, Figs. 5, 6), and the oligomers consisted of 11 units. The prodelphinidin units and galloyl groups were also present in the identified tannins. For the identification of tannins, the MS/MS data were fundamen-tal, starting with the confirmation number of flavan-3-ol units and the molecular weight of these units. The next step is the confirmation of the identity of the units from the fragments produced by Retro Diels Alder (RDA) fragmentation in ring B or the substituents, such as galloyl group. For this reason, the condensed tannin structures can be proposed with more reliable manner. The MS/MS spectra confirmed, for example, that the ions m/z 889, 1177, 1465, 1753, 2041, 2329, 2617, 2905 and 3193 compose this series, such as the MS/MS spectrum of m/z 2329 that revealed the fragment ions m/z 2041, 1753, 1465, 1177 and 889, the same ions observed in the MS spectrum of the extract (see supplemental information). Therefore, the units added from ion m/z 889 have the molecular weight of 290 Da and suggesting the presence of procyanidin unit. The frag-mentation of ion m/z 889 yielded the fragment ions m/z 737, 719, 601, 567, 449 and 313 (see supplemental information). The m/z 737 fragment ion represents the loss of 152 µ and confirms the presence of the ring B substituents of the procy-anidin unit. The fragment ions of m/z 601 and 313 confirmed the presence of the coupling of successive procyanidin units. The other important polymers in this fraction were formed by one galloyl addition and successive procyanidin units up to 11 units (series 4). The MS/MS analysis of the m/z 753 [M+ Na]+ produced the ion m/z 463, which confirms the presence of gal-loyl in the starter unit. We used MS/MS to propose the tannin structures, and not only MS. The identification of tannins is more reliable from MS/MS analyses, but there are few studies reporting the use of MS/MS data to identify tannins. There-

fore, it was possible to determine the sequential monomer units and chemical constitution of individual polymers only by MS/MS analyses. Hydrolysable tannins have been identified in the Eugenia genus,27,28) but only condensed tannins were detected in the extract analyzed in our study. Previous phyto-chemical investigation of E. dysenterica revealed compounds such as a peptide,12) monoterpenes and sesquiterpenes,29) vitamins and carotenoids.30) The catechin derivatives were suggested to be present in E. dysenterica extract, but this re-port was based only on UV data.31) Our studies provide more complete evidence of the chemical nature of E. dysenterica because the compounds were identified with certainty using UV, MS and MS/MS data.

It has been reported that catechins and procyanidins did not show antiulcer activity against HCl/etanol-induced ulcer,25) moreover, these compounds have low or no capability to precipitate protein.32,33) These observations together with the fact that gelatin-treated E. dysenterica leaf extracts lost their gastroprotective effect, strongly suggest that catechins and procyanidin B-1 do not significantly contribute to this effect. However, it has been suggested that the gastroprotec-tive actions of tannic compounds are related to molecule size. Indeed, tannins yielded from procyanidin units as tetramers, pentamers and hexamers exhibit gastroprotective effects whose magnitude is proportional to the size of the oligomeric chain.25) Interestingly, the interaction of tannins with proteins also appears to be related to the molecule size; thus, procyani-din hexamers and pentamers show higher protein-binding and precipitation ability than tetramers, trimers show little precipi-tation ability, and as mentioned above, procyanidin dimers and monomers (as catechins) are unable to precipitate proteins.25) These reported data and the fact that many condensed tan-nins were identified in the E. dysenterica leaf extract strongly suggest that condensed tannins are the main compounds responsible for the powerful gastroprotective effect of the E.

Fig. 5. General Structure of the Tannins Presented in the Aqueous Eugenia dysenterica Leaf ExtractTypical polymer structure of the condensed tannins repeating unit: procyanidin (PCY) and prodelphinidin (PDE). Typical linear condensed tannins type B with C4–C8

(1) and C4–C6 (2), linkages unit substituted by galloyl (3), catechin (4) and procyanidin B-1 (5).

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dysenterica leaf extract.Ethanol/HCl induced-ulcers involve the formation of free

radicals34) that may induce lipid peroxidation and cell dam-age.35) Moreover, ethanol decreases the gastric levels of glu-tathione, a well-known antioxidant compound.36) Therefore, based on our observation that E. dysenterica leaf extract possesses antioxidant activity, a property that is usually found in plants containing polyphenolic compounds,37) it is pos-sible that this effect is part of the mechanism by which the extract protects the gastric mucosa from injury. To investigate this possibility, we analyzed whether blocking endogenous SH groups with the SH alkylator NEM38) could decrease E.

dysenterica leaf extract-mediated gastroprotection, as SH groups possess electron-donating ability and are thus capable of binding free radicals.36) NEM administration greatly re-duced the protective effect of the extract, thus highlighting the participation of endogenous SH groups in the gastropro-tective effect. This observation suggests that the antioxidant activity of the extract alone is not sufficient to fully protect the stomach from ethanol/HCl-induced damage. Therefore, there may be a cooperative relationship between the activity of the extract and free-radical scavenging by the endogenous SH-bearing molecules; alternatively or additionally, the extract may augment the bioavailability of the endogenous SH groups.

Fig. 6. Mass Spectra of the Aqueous Eugenia dysenterica Leaf ExtractMass spectra recorded in positive ionization mode: series 1–2 (A) and 3–4 (B).

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Several lines of evidence suggested that NO is an impor-tant endogenous gastroprotective mediator. The mechanisms involved in NO-mediated gastroprotection include the main-tenance of mucosal integrity, the inhibition of leukocyte mi-gration and platelet adherence, and the increase in mucosal blood flow.39) L-NAME, a nonselective inhibitor of the nitric oxide synthase enzymes that produce NO, did not significantly compromise the extract’s gastroprotective effect, suggesting that this mediator is not involved in the cytoprotective actions of E. dysenterica. Finally, the extract inhibited the secretion of HCl similar to the standard treatment (cimetidine); this effect may also contribute to the decrease in mucosal injury induced by ethanol/HCl administration.

It has been suggested that the extrapolation of animal dose to human dose is correctly performed through normalization to body surface area.40) Accordingly to this approach, the human equivalent doses calculated for E. dysenterica extract are approximately 45 mg/kg and 81 mg/kg (take into account 550 and 1000 mg/kg as active doses in mice, respectively).

Despite these doses appear even to be high for humans, it should be noted that the actually gastroprotective dose of some extract used in clinical studies41) represents only a frac-tion (ca. 28%) of that can be calculated by the body surface area normalization method.42) Therefore, the effective human equivalent doses of E. dysenterica leaf extract concerning its gastroprotective effect may be lower than that necessary to protect mice. Moreover, as pointed out by Souza-Formigoni and his colleagues, it is possible that lower doses of the ex-tract given chronically may also have therapeutic effects against gastric lesions.42)

In summary, the present study demonstrates that E. dysen-terica leaf extract strongly protects the gastric mucosa from ethanol/HCl-induced injury and highlights the biopharma-ceutical potential of this species. The gastroprotective effect is linked to the inhibition of HCl production, to the extract’s free-radical-scavenging activity (antioxidant property) and to the presence of endogenous SH-containing compounds, but does not involve NO-mediated cytoprotection. These pleio-tropic actions appear to be primarily related to the condensed tannins in E. dysenterica, which are predominantly composed of procyanidin followed by prodelphinidin, in addition to the presence of the galloyl group in one polymeric series. Moreover, tannins present in the extract appear to bind to epithelial mucins in the gastric mucosa forming a protective coating against damaging agents.

Acknowledgments We thank Débora Cristina de Oliveira Nunes and Simone Ramos Deconte for technical assistance. This work was supported by a master fellowship by the Co-ordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for LCSP. We are also indebted to Pró-Reitoria de Pós-Graduação e Pesquisa da Universidade Federal de Uber-lândia (PROPP-UFU) and to Fundação de Amparo à Pes-quisa do Estado de São Paulo (FAPESP) for financial support. Finally, we would like to thank Dr. Norberto Peporine and the Mass Spectrometry Facility of Physics and Chemistry from FCFRP-USP for the excellent customer service.

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Table 3. Identification of Condensed Tannins by MALDI-TOF from Aqueous Extract of E. dysenterica

[M+Na]+ (error) MF Compound

Series 1 889.1905 (5.7 ppm) C45H38O18 3 PCY1177.2586 (0.3 ppm) C60H50O24 4 PCY1465.3279 (3.8 ppm) C75H62O30 5 PCY1753.3888 (1.7 ppm) C90H74O36 6 PCY2041.4441 (2.5 ppm) C105H86O42 7 PCY2329.5no C120H98O48 8 PCY2617.6no C135H110O54 9 PCY2905.6no C150H122O60 10 PCY3193.7no C165H134O66 11 PCY

Series 2 905.1897 (0.9 ppm) C45H38O19 2 PCY–1 PDE1193.2517 (1.8 ppm) C60H50O25 2 PCY–1 PDE–1 PCY1481.3120 (3.5 ppm) C75H62O31 2 PCY–1 PDE–2 PCY1769.3835 (1.6 ppm) C90H74O37 2 PCY–1 PDE–3 PCY2057.4293 (7.2 ppm) C105H86O43 2 PCY–1 PDE–4 PCY2345.5no C120H98O49 2 PCY–1 PDE–5 PCY2633.6no C135H110O55 2 PCY–1 PDE–6 PCY2921.6no C150H122O61 2 PCY–1 PDE–7 PCY3209.7no C165H134O67 2 PCY–1 PDE–8 PCY

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Series 4 753.1418 (1.8 ppm) C37H30O16 PCYG–PCY1041.1998 (6.5 ppm) C52H42O22 PCYG–2 PCY1329.2725 (1.9 ppm) C67H54O28 PCYG–3 PCY1617.3297 (2.2 ppm) C82H66O34 PCYG–4 PCY1905.4042 (3.9 ppm) C97H78O40 PCYG–5 PCY2193.4679 (3.5 ppm) C112H90O46 PCYG–6 PCY2481.5177 (2.3 ppm) C127H102O52 PCYG–7 PCY2769.6no C142H114O58 PCYG–8 PCY3057.6no C157H126O64 PCYG–9 PCY3345.7no C172H138O70 PCYG–10 PCY

MF: molecular formula, PCY: procyanidin unit, PDE: prodelphinidin unit, PCYG: procyanidin O-gallate unit, no: not observed with internal calibrant (low intensity).

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