The Gastroprotective Effects of Eugenia Dysenterica (Myrtaceae) Leaf Extract: the Possible Role of Condensed Tannins

722 Regular Article Biol. Pharm. Bull. 37(5) 722–730 (2014) Vol. 37, No. 5 The Gastroprotective Effects of Eugenia dysenterica (Myrtaceae) Leaf Extract: The Possible Role of Condensed Tannins Ligia 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/ionization (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 desorption/ionization (MALDI) Numerous plant-derived extracts are gastroprotective and proach to plant collection relies on the premise that related represent a great source of bioactive compounds with the ther- taxa have inherited the genetic ability to produce similar apeutic potential to treat gastric and duodenal ulcers, diseases pharmacologically active secondary metabolites.14,15) There- that are considered to be worldwide heath issues.1) Thus, stud- fore, considering that many species of the Myrtaceae family ies of this subject are important because they provide data that including those from the Eugenia genus have been reported to assist governmental institutions in their policies concerning possess gastroprotective activities, we applied the taxonomic the use of plants in the phytotherapeutic industry, the sustain- approach to select E. dysenterica and study its cytoprotective able use of the biodiversity and consequently, the application actions on the stomach. In a pilot experiment published only of phytotherapeutics to health promotion programs. as part of an academic report, our co-author (Canabrava) and Many plants exhibiting gastroprotective actions that have his colleagues observed that E. dysenterica leaf extract indeed been studied belong to the Myrtaceae family and include protects the gastric mucosa of rats from damage caused by the Campomanesia lineatifolia,2) Campomanesia xanthocarpa,3) administration of indomethacin, a cyclooxygenase inhibitor.16) Eugenia jambolana,4,5) Myrtus communis,6) Plinia edulis,7) To study this subject further and to pharmacologically char- Syzygium aromaticum,8,9) and Syzygium cumini.10) Eugenia dy- acterize the E. dysenterica leaf extract, we investigated the senterica DC. (Myrtaceae), also known in Brazil as “cagaita” mechanisms involved in its gastroprotective effect on ethanol/ or “cagaiteira,” is a shrubby ornamental and melliferous tree HCl-induced ulcers in mice; moreover, a group of secondary that is widely distributed throughout the second largest Bra- compounds that are putatively responsible for this effect were zilian biome, the Cerrado or upland savannah.11) The fruit is analyzed and identified by matrix assisted laser desorption/ edible and has traditionally been used as a cathartic agent; ionization (MALDI)-MS and MS/MS. the leaves, in contrast, have been used to treat diarrheic diseases.11) Recently published data provide scientific support, at MATERIALS AND METHODS least in part, for these folk uses.12,13) In considering the selection of a plant for pharmacological Animals The experiments were performed using male studies many approaches can be used. The taxonomic ap- 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 The authors declare no conflict of interest. to tap water and standard chow. All the protocols used were * To whom correspondence should be addressed. e-mail: [email protected]; © 2014 The Pharmaceutical Society of Japan [email protected] May 2014 723 reviewed and approved by the Ethics Committee on Animal mixed carefully. After 15 min, the samples were centrifuged at Use of the Federal University of Uberlândia (CEUA/UFU; 3000 rpm for 15 min. The supernatant was removed and used process n° 022/11, addendum 175/11). to treat mice (GD-ED group) that had been fasted for 24 h Plant Material Eugenia dysenterica DC. (Myrta- (1.0 mL/40 g, i.e., 1000 mg/kg) 30 min before the administra- ceae) leaves were collected at the Santa Rita farm located in tion of the ethanol/HCl solution. A control extract (ED group) Lassance city, state of Minas Gerais, Brazil (17°59′14.23″S; was prepared by adding 1.5 mL of the acetate buffer without 44°44′38.00″W) in September, 2011. The species was identi- gelatin to the same volume of the extract (80 mg/mL). To fied by Dr. Adriana Arantes, and a voucher specimen was de- verify the effectiveness of this method in removing polyphe- posited at the Herbarium of the Federal University of Uberlân- nolic compounds from the extract, 2.980 µL of FeCl3 (0.01 M dia (Uberlândia, MG, Brazil) with the number HUFU-45956. in 0.01 M HCl) was added to 20 µL of the supernatant from Preparation of the Leaf Extract The leaves were washed the gelatin-treated, control extract or distilled water (blank) (immersed for 10 min in 70% ethanol, 5 min in 0.2% hypochlo- to determine its absorbance at 510 nm 45 min later. The data rite and 10 min in running water; this procedure was applied from both groups were expressed as the absorbance units/mg to disinfect the surface of the leaves) and dried in an oven at of the extract. 40°C for 48 h. The powdered dried leaves were extracted with Determination of the Ability of the Extract to Bind to distilled water (20%, w/v) for 48 h at room temperature. The the Gastric Mucosa and Stain with Ferric Chloride This extract was lyophilized and then stored at −20°C and protect- experiment was conducted as previously described20) with ed from light until use (yield: 0.8%). The surface disinfection some modifications. Briefly, animals without any treatment procedure neither altered the biological effect of the extract on were sacrificed by cervical dislocation under anesthesia the gastric tissue, nor changed the structure of its condensed (sodium thiopental, 60 mg/kg, i.p.); the stomachs were then tannins when compared to extract obtained with leaves that removed, fixed in phosphate-buffered 4% formalin, embed- were washed only with water (data not shown). ded in paraffin and further sectioned in 5 µm slices. Depar- Qualitative Determination of the Tannins in the Extract affinized and hydrated sections were then treated with a 5% The presence of tannins was screened by reacting the extract solution of E. dysenterica leaf extract for 1 h, rapidly rinsed samples with gelatin or FeCl3 according to the method de- in water (3 times), treated with 2% FeCl3 for 15 min, washed scribed by Costa (2000), with some modifications as follows: in water, alcohol and xylene and mounted in gum damar. No (I) 1 drop of HCl (10%) was added to 1 mL of the extract counterstain was used, and the slices were observed under a solution (1 mg/mL), and gelatin solution (2.5% in 10% NaCl) light microscope. was added dropwise; the formation of a precipitate indicated a Effect of the Extract on Gastric Secretion The assay 21) positive reaction. (II) FeCl3 (1%) was added dropwise to 5 mL was performed according to the method of Shay et al., of the extract solution (1.0 mg/mL); the solution was monitored with some modifications as follows: the mice were fasted for for a brownish green or a blue-black color and/or precipitation. 36 h with free access to water; immediately after laparotomy Evaluation of the Gastroprotective Effect of the Extract and pylorus ligature under anesthesia (10 mg/kg xylazine The acidified ethanol-induced gastric lesion model17) was used and 100 mg/kg ketamine, i.p.), E. dysenterica leaf extract to evaluate the gastroprotective effect of the E. dysenterica (1000 mg/kg), cimetidine hydrochloride (100 mg/kg)9) or saline leaf extract. Mice were divided into 6 groups (n=6–10) and were administered intraduodenally, and the abdomen was fasted for 24 h prior to the oral administration (total volume: sutured.

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