Assessment of Different Biological Capacities of Aspidosperma Excelsum Benth

Journal of Multidisciplinary Engineering Science and Technology (JMEST) ISSN: 2458-9403 Vol. 6 Issue 1, January - 2019 Assessment Of Different Biological Capacities Of Aspidosperma Excelsum Benth Nathália Araújo Rochaa, Débora Brandão Barbosaa, Francisco José Raposoa, Alexandre Augusto Barros Latalizaa, Cristine Bastos do Amaranteb, Nádia Rezende Barbosa Raposoa* aNúcleo de Pesquisa e Inovação em Ciências da Saúde (NUPICS), Faculdade de Farmácia, Universidade Federal de Juiz de Fora, Rua José Lourenço Kelmer, s/n, 36036-330 Juiz de Fora-MG, Brazil bMuseu Paraense Emílio Goeldi – Centro de Pesquisa, Avenida Perimetral, 1901, 66077-830 Belém-PA, Brazil *Tel.: +55 32 2102 3809 E-mail address: [email protected] Abstract—Species of the genus Aspidosperma is development of drugs from elements of nature [1,2] popularly used as a medicinal plant in the Such interest may be related to the fact that 30% of treatment of various conditions, such as asthma, medicine on the market are derived from natural diabetes, and malaria, considering they have products, mostly plants and microorganisms [3]. antimicrobial, anti-inflammatory and wound Brazil has approximately 20% of plants and healing activity. Studies relate their biological microorganisms present on the planet [4]. Due to this activities to the production of alkaloids in their country great biodiversity, Brazilian researchers have secondary metabolism. The objective of this study shown an increasing interest in the study of native was to analyze the antioxidant, antimicrobial and plants, aiming the discovery of its active principles toxicity of the hydroethanolic extract of [5,6]. Despite the crescent number of researches, only Aspidosperma excelsum, in view of the biological 5% of the Brazilian pharmaceutical market is importance of the species of this genus. The represented by medications developed from medicinal antioxidant activity was analyzed by the reduction plants [3,7]. of the 2,2-diphenyl-1-picrylhydrazyl radical The genus Aspidosperma Mart. is one of the (DPPH). The antifungal action in vitro was most important of the family Apocynaceae, and it is evaluated against four Candida strains (Candida used for healing purpose in Brazil. There are high albicans ATCC 10231, C. glabrata CCT 0728, C. concentrations of their species in the Amazon, and krusei CCT 1517 and C. guilliermondii CCT 1890). they are popularly known as carapanaúba, pau- Finally, the toxicity potential of the extract of A. pereiro, guatambus, amargoso and quina [8–10]. excelsum was tested by the MTT method 3-[4,5- Aspidosperma excelsum stands out amid the other dimethylthiazole-2-yl]-2,5-diphenyltetrazolium species of Aspidosperma. It is widely used in bromide in murine fibroblasts (L929), and through traditional medicine, and generally known as the bioassay using Artemia salina Leach. The carapanaúba [4]. In Peru, it is used by Shipibo-Conibo results indicated a good antioxidant activity for Indians for the treatment of hepatitis and malaria, and the tested extract, resulting in IC50 of 5.89 μg/mL. by other natives in order to treat bronchitis as well as The extract was not effective against any Candida a vasodilator drug, antiseptic, antimicrobial, and sp. strain, showing MIC > 5,000 μg/mL. Regarding wound healing [9]. In Brazil, tea made from the stem the toxicity, the extract showed cytotoxic activity bark and leaves of A. excelsum is utilized in the against the L929 cell line, resulting in 40% to 85% treatment diabetes and malaria. It is also used in reduction of cell viability in the concentrations blood pressure control, likewise anti-inflammatory, tested and, it was also considered toxic to Artemia wound healing and contraceptive [11,12]. Its biological salina, LD50 of 273.52 μg/mL. The species studied activities are principally attributed to the presence of has important biological activities. This fact may indole alkaloids in its structure, of which 18 serve as an incentive for future studies, in the compounds have already been isolated [13]. search for new properties not yet explored. Despite the various therapeutic indications and its application as a potent antimalarial agent [14], few Keywords—Aspidosperma excelsum, biological researches have been conducted toward antimicrobial, antioxidant, toxicity. this species [15]. Aware of its potential, this paper intended to study its antioxidant and antifungal actions, in addition 1. INTRODUCTION to its toxic potential in vitro. The use of plants for a medicinal purpose can be observed from the earliest civilizations. In recent years, there has been growing interest in the www.jmest.org JMESTN42352806 9384 Journal of Multidisciplinary Engineering Science and Technology (JMEST) ISSN: 2458-9403 Vol. 6 Issue 1, January - 2019 2. MATERIAL AND METHODS The procedures for determination of the Minimum Inhibitory Concentration (MIC) were A. Plant material performed according to the M27-A3 and M27-S4 protocols of the Clinical and Laboratory and The bark of Aspidosperma excelsum Benth. Standards Institute (CLSI)[17,18], adapted according was purchased at Bio Ervas Comércio market in to Almeida et al. [19]. All analyzes were done in Castanhal, Pará, Brazil. The dried bark was triplicate. The fungal suspension was prepared in macerated and extracted with 400 mL of ethanol- sterile saline (0.9% NaCl w/v), then it was diluted in water (70:30, v/v) for 72 hours at room temperature. Roswell Park Memorial Institute (RPMI) 1640 culture The residue was removed by filtration, and the medium (Sigma-Aldrich, USA), buffered with 3-(N- extract was evaporated to dryness at a lower morpholine)-propanesulphonic acid (MOPS) temperature (<40 °C) under reduced pressure in a (JTBaker, Germany). The pH was adjusted to 7.0 ± rotary evaporator (Rotavapor® R-210, Buchi, 0.2 in order to obtain final concentration of 5 x 102 to Switzerland), followed by lyophilisation (ALPHA 1-4 2.5 x 103 colony-forming units (CFU) per mL. LD plus, Christ, Germany) under 1.8 mbar pressure The dry extract of A. excelsum was diluted in and −14 °C. The material was stored protected from RPMI-1640 medium buffered with MOPS and 20 light in air-tight containers with cap at −20 °C until µL/mL Tween-80/dimethyl sulfoxide (DMSO) (1:1, v/v), use. in the concentration range of 9.76 to 5.000 μg/mL. Amphotericin B (Cristália, Brazil) and nystatin (Sigma- B. Antioxidant activity Aldrich, USA) were used as reference drug, at concentrations of 0.0313 to 16 μg/mL to amphotericin The scavenging activity of Aspidosperma B, and 0.08 to 10.6 μg/mL to nystatin. excelsum dry extract was measured according to the The assay was performed in 96-well sterile 1,1-diphenyl-2-picrylhydrazyl free radical (DPPH) microplates, to which 100 µL of analogs dilutions and method, as previously described by Sreejayan e Rao 100 µL RPMI 1640 were added, buffered with MOPS [16], with minor modifications. Briefly, the sample (50 and inoculated with a suitable number of the µL) at different extract concentrations (0.97–250 microorganism’s colony forming units. The growth μg/mL) was added to each well of a 96-well microplate control consisted of 100 µL of the same inoculated and mixed with 150 µL of 0.05 mM DPPH (Sigma- culture medium and 20 µL/mL Tween 80/DMSO (1:1, Aldrich, USA) in ethanol solution. The reaction mixture v/v) and a sufficient quantity of the uninoculated was kept for 30 minutes in the dark at room medium to make up 200 µL. The negative control was temperature. Then, the absorbance was measured in prepared by adding 200 µL of uninoculated medium. a spectrophotometer at 510 nm against the negative The microplates were incubated at 37 ± 2 ºC during control (ethanol). Resveratrol (Sigma-Aldrich, USA) 48 hours. was used as a positive control at the same The minimum inhibitory concentration (MIC) concentrations. Inhibition of DPPH radical was was established as the lowest concentration at which calculated using (1): no turbidity was observed in the culture medium. After checking the MIC, an aliquot of 20 µL was retained IC50 (%) = 100 x (A0 – As) / A0 (1) from those wells which showed no visible growth and re-incubated with 4 mL of Tryptic Soy Broth (TSB) being A0 negative control absorbance and As without the addition of an antifungal agent, for another test-sample absorbance. 48 hours at 37 ± 2 ºC. The lowest concentration at The IC50 value was calculated from the straight- which no turbidity was noticed after this period was line equation of the linear dispersion graph and considered the Minimum Fungicidal Concentration represents the extract concentration that inhibits 50% (MFC). of DPPH radical. All tests were performed in triplicate. The results were submitted to Student's t test for D. Cell viability assay independent samples and the level of significance considered was 95%. The immortalized murine fibroblasts (L929) cell line from Mus musculus murine was grown in C. Antifungal activity Dulbecco's modified Medium Minimum Media (DMEM- Gibco, USA) high glucose supplemented with 10% The standard strains used in this study were as (v/v) fetal bovine serum (FBS, Gibco, USA), 1% (w/v) follows: Candida. albicans, American Type Culture antibiotic penicillin/streptomycin, and maintained at 37 Collection (ATCC) 10231; C. glabrata (Taniwaki, ± 2 ° C in a 5% CO2 humidified atmosphere. M.H.), Collection of Tropical Cultures (CCT) 0728; C. The 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl krusei, (FTI) CCT 1517; and C. guilliermondii (CCT), tetrazolium bromide (MTT) assay was used for the 1890; from the Foundation André Tosello and the study of cell viability. Briefly, L929 cells were seeded Laboratory of Reference Microorganisms of the in a 96-well plate at a density of 5x103 cells in 100 μL National Institute for Quality Control in Health of the medium per well. After 24 hours of incubation, the Oswaldo Cruz Foundation. culture medium was replaced by fresh medium containing the treatments.

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