leishmanicidal, cytotoxicity and wound healing potential of
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RESEARCH ARTICLE Open Access
Leishmanicidal, cytotoxicity and woundhealing potential of Arrabidaea chica VerlotJoicy Cortez de S1, Fernando Almeida-Souza2, Renata Mondgo-Oliveira3, Iara dos Santos da Silva Oliveira1,Lyah Lamarck3, Isadora de Ftima Braga Magalhes3, Aaro Filipe Atades-Lima4, Higor da Silva Ferreira4
and Ana Lucia Abreu-Silva1,3*
Background: Leishmaniasis includes a wide complex of diseases that affect humans and other mammals, and canrange from a mild cutaneous form to a severe visceral type. The safety of the standard treatment using pentavalentantimony is a concern due to its toxic effects. The search for alternative, effective and less toxic treatments has ledto the testing of natural products. The present study aimed to evaluate the cytotoxic, leishmanicidal and healingpotential of Arrabidaea chica.
Methods: The crude ethanolic extract, as well as the chloroform, methanol and ethyl acetate fractions of A. chica wereprepared and phytochemical analysis was performed. Cytotoxic evaluation was carried out through MTT colorimetricassay, and the 50 % cellular cytotoxicity was determined. After that, the effect of the extract and fractions againstLeishmania amazonensis promastigotes, at intervals of 24, 48 and 72 h, was analyzed, and 50 % inhibitory concentrationwas determined. The healing effect of the plant was also tested in surgical lesions in Swiss mice skin.
Results: Phytochemical screening showed that the crude extracts contained flavonoids, tannins, anthocyanidinsand chalcones. The leishmanicidal potential of A. chica produced satisfactory results in concentrations ofbetween 60 and 155.9 g/mL. Cytotoxic assay revealed a 50 % reduction in viable cells at a concentration of189.9 g/mL. The healing results indicated that the treated group exhibited more pronounced signs of lesionresolution in the early period, but this pattern did not persist throughout the treatment.
Conclusions: The results of the present study demonstrate that A. chica has cytotoxic and leishmanicidalpotential but its healing effect must be better studied.
Keywords: Arrabidaea chica, Cytotoxicity, Healing, Leishmanicidal activity
BackgroundLeishmaniasis is a group of diseases that affect severalspecies of mammals that live in tropical and subtropicalregions. Literature describes more than 20 species of theprotozoan Leishmania that causes leishmaniasis, includ-ing cutaneous and visceral leishmaniasis [1, 2]. Theseclinical forms differ in clinical presentation, morbidityand mortality .
According to the World Health Organization, neglecteddiseases are those associated with low social indicators, andare thus more frequently found in developing countrieswhere there is a lack of resources aimed at control and treat-ment and little research into finding an effective solution .Among the clinical forms of the disease, the cutane-
ous, characterized by skin lesions, chiefly in exposedbody parts, is the most common . Depending on thespecies of Leishmania, in some patients leishmaniasiscan heal without treatment, while in others diffuse cuta-neous or mucocutaneous forms can develop .In all clinical forms, pentavalent antimonials have been
used as a mainstay therapy: the available drug for the treat-ment of leishmaniasis in the United States and Europe issodium stibogluconate (Pentostam), while meglumine
* Correspondence: email@example.comEqual contributors1Rede Nordeste de Biotecnologia, Universidade Estadual do Maranho, SoLus, Brazil3Programa de Ps-Graduao Mestrado em Cincia Animal, UniversidadeEstadual do Maranho, So Lus, BrazilFull list of author information is available at the end of the article
2016 Cortez de S et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Cortez de S et al. BMC Complementary and Alternative Medicine (2016) 16:1 DOI 10.1186/s12906-015-0973-0
antimonate (Glucantime) is more used in South Americaand Africa . Despite this therapy having been widelyemployed since 1912, the mechanism of action is poorlyunderstood and adverse effects, which include renal andhepatic toxicity, are severe . Other limitations of thistherapy are the necessity of daily parenteral administrationand the resistance of the drug .These limitations, the vast Brazilian biodiversity and
traditional knowledge has led to the establishment ofresearch networks to evaluate the leishmanicidal effectof plants from various biomes, such as the Cerrado andAmazon. It is therefore very important to perform ex-periments using natural products in order to identifynew, less toxic, compounds which could be taken orally.In addition, the Brazilian government has created theRENISUS (Rede nacional de plantas medicinais de inter-esse do sistema nico de sade), a list of plants that canbe used in the Unified Health Service, and Arrabidaeachica is included in this list.Arrabidaea chica Verlot, popularly known as pariri,
belongs to the Bignoniaceae Family, popularly known aspariri. It is endemic in almost all of Brazil but is mostfrequently found in the Amazon Forest  where it isused to treat skin disease, mulligrubs, anemia, jaundiceand inflammatory reaction . Widespread use bynative populations lead researchers from different coun-tries to evaluate the medicinal properties of this plant,including its anti-inflammatory ; astringent, antioxi-dant [14, 15]; anti-ulcer ; antimicrobial, antifungal[17, 18] and wound healing [19, 20] effects.The aim of the present study was to evaluate the
phytochemical effects, wound healing capacity and cyto-toxicity of Arrabidaea chica, as well to evaluate the ac-tion of the crude extract and fractions of these plants onpromastigotes of L. amazonensis.
MethodsEthics committeeThe experimental protocol for this study was ap-proved by the ethics committee for animal researchof the Universidade Estadual do Maranho (CEUA-UEMAprotocol 26/2009) and by the ethics committee in animalexperimentation of Instituto Oswaldo Cruz - FIOCRUZ/RJ (CEUA-FIOCRUZ protocol LW72/12).
PlantLeaf and stem samples of Arrabidaea chica Verlot. werecultivated and collected in the Atico Seabra Herbarium,Departmento de Farmcia, Universidade Federal doMaranho (So Lus, Maranho, Brazil). The plantmaterial was identified by Dr. Ana Zlia Silva and avoucher specimen was deposited under the number1067. The plant material was collected between Augustand November 2011, which represents the dry season in
this region, with a rainfall rate of 900 mm and an averagetemperature of 28 C.
Preparation of crude extract and fractionsTo obtain the crude ethanolic extract, the samples weredried at 40 C with forced air circulation. The dried mater-ial from each part of A. chica was macerated and immersedin ethyl alcohol, in a proportion of 3:1 (alcohol and plantmaterial, respectively). In order to obtain the ethanolic ex-tracts solvent removal was carried out by rotoevaporation,under reduced pressure at 30 to 40 C, until the desiredconcentrations were achieved. The samples were storedunder refrigeration and protected from light until analysis.To determine the dry weight of the samples three ali-
quots of 0.5 mL of crude ethanolic extract were used.The yield of samples was calculated using the totalweight of the powder, the dry weight obtained and thetotal final volume of the concentrated extract.Additionally, the crude ethanolic extracts were fraction-
ated by sequential partition using solvents of increasingpolarity: chloroform, ethyl acetate and methanol. Extrac-tion was carried out in the solid phase using silica gel,grail, pistil and approximately 500 mL of each solvent.Once obtained, the fractions were submitted to leishmani-cidal and cytotoxic tests. Those that produced satisfactoryresults were sent to the Analytical Center of the Universi-dade de So Paulo (So Paulo, Brazil), to identify the bio-active compounds through gas chromatography and massspectrometry.
Phytochemical analysisTo evaluate the presence of secondary metabolites (sapo-nins, anthocyanidins, chalcones, phenols, tannins, flavo-noids, steroids, triterpenoids and anthraquinones), crudeethanolic extracts of A. chica were solubilized in methanol(in a concentration of 100 mg/5 mL), according to Costaand Matos [21, 22].
In vitro cytotoxic assayObtaining of peritoneal macrophages and cell culturePeritoneal macrophages were obtained from 4-week-oldfemale BALB/c mice, from the Laboratory Animal Breed-ing Center (CECAL-FIOCRUZ) and kept in the Hlio andPeggy Pereira Pavilion animal facilitiy, located at theOswaldo Cruz Foundation (FIOCRUZ, Rio de Janeiro,Brazil).Three mL of 3 % sodium thioglycollate was inoculated
into the peritoneal cavity of the animals and, after 72 h,were euthanized in a CO2 chamber. For macrophage re-covery, 5 mL of a cold and sterile pH 7, 2 PBS solutionwas injected into the abdominal cavity, followed by mas-sage and collection of lavage, which was kept in a falcontube, placed in an icebox. After collection, the lavagewas analyzed under an optical microscope to verify the
Cortez de S et al. BMC Complementary and Alternative Medi