Chemical Characterization and Antimicrobial Activity of Hydroethanolic Crude Extract of Eugenia Florida Dc (Myrtaceae) Leaves
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International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 8, Issue 6, 2016 Original Article CHEMICAL CHARACTERIZATION AND ANTIMICROBIAL ACTIVITY OF HYDROETHANOLIC CRUDE EXTRACT OF EUGENIA FLORIDA DC (MYRTACEAE) LEAVES RENAN G. BASTOSa, CARLA P. ROSAa, JOSIDEL C. OLIVERb, NAIARA C. SILVAb, AMANDA L. T. DIASb, CLAUDIA Q. DA ROCHAc, WAGNER VILEGASc, GERALDO A. DA SILVAa, MARCELO A. DA SILVAa* aDepartment of Foods and Drugs, Faculty of Pharmaceutical Sciences, Laboratory of Medicinal Plants and Herbal Medicines, Federal University of Alfenas, UNIFAL-MG, Gabriel Monteiro da Silva Street, 700, 37130-000, Alfenas, Minas Gerais, Brazil, bDepartament of Microbiology and Immunology, Institute of Biomedical Sciences, Laboratory of Research in Microbiology, Federal University of Alfenas, UNIFAL-MG, Gabriel Monteiro da Silva Street, 700, 37130-000, Alfenas, Minas Gerais, Brazil, cInstitute of Biosciences, Laboratory of Bioprospecting of Natural Products (LBPN), São Paulo State University, UNESP, Coastal Campus-IB-CLP, Infante Dom Henrique Square, s/n°, 11380-972, São Vicente, São Paulo, Brazil Email: [email protected] Received: 25 Jan 2016 Revised and Accepted: 20 Apr 2016 ABSTRACT Objective: The present study aimed to characterize and quantify the total phenolics, flavonoids and tannins in a hydroethanolic crude extract (70% (v/v) (EB)) of the leaves of E. florida DC, as well as to evaluate the antimicrobial activity of the extract against different species of micro-organisms. Methods: EB was characterized using a mass spectrometer equipped with a direct insertion device for in-stream injection (FIA). Quantitative analyses of major compounds were carried out by spectrophotometry. In addition, we evaluated the sensitivity profiles of different strains of yeast and bacteria against different concentrations of EB. Results: The classes found were in agreement with those described in the literature: flavonoids, tannins, phenolic acids and saponins. EB showed levels of phenolic compounds, flavonoids and tannins equal to 25.82 mg gallic acid equivalents per gram of extract (EAG/g), 8.42 mg quercetin equivalents per gram of extract (EQ/g) and 7.30 mg tannic acid equivalents per gram of extract (AT/g), respectively. In the analysis of antimicrobial activity, EB was more active against yeasts but was not effective against the bacteria used in the test. Conclusion: We can conclude that E. florida DC has antimicrobial potential, due to the presence of bioactive secondary metabolites. Keywords: Phenolics, Micro-organisms, Mass spectrometer, Yeasts, Bacteria, Potential © 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) INTRODUCTION hypotensive, hypoglycemic, and antipyretic; for reducing triglycerides and cholesterol levels; and in the treatment of infections, jaundice, Medicinal plants are used in some of the oldest strategies employed heart disease and gastrointestinal disorders [6, 7]. by man in the treatment of diseases of all kinds, and their use in the prevention or cure of diseases is a habit that has always existed in Previous chemical studies of the genus indicate high levels of the history of humanity [1]. In recent decades in Brazil, there has phenolic compounds such as flavonoids [8, 9], phenolic acids [10] been a growing interest in the use of medicinal plants and their and tannins [11, 12], as well as other classes of compounds, respective extracts for treatments, which can in certain including saponins [13, 14] and terpenoids [15]. circumstances, be of assistance in primary health care compatible with conventional medicine [2]. The phytotherapy is intended to However, there are still no detailed chemical and biological studies provide useful information for the development of pharmacological on the species E. florida DC in the literature. Thus, chemical and phytochemicals studies on plant species, as well as providing characterization and elucidation of the antimicrobial activity of the therapeutic resources, conserving biodiversity, promoting social plant extract in question, to verify its potential as a medicinal agent, development, and fostering permanent education [2]. is justified due to the popular use of the plant by rural communities in the treatment of diseases. In contrast to synthetic drugs, therapeutic use of herbal products was materialized from the ancient texts and passed on through MATERIALS AND METHODS generations without scientific experimental evidence. For this reason, modern research on the pharmacology of herbal medicine is Plant material aimed to validate herbal medicine and to ensure rational use, safety Leaves of the species E. florida DC were collected in April 2014, the and efficacy [3]. Thus, due to the large diversity of Brazilian plants with therapeutic potential and considering the history and cultures southern state of Minas Gerais, near the “Fazenda Cachoeirinha”, of the population, some species of plants have aroused great interest W045°56'07.8'', S21°33’51.7’’, Alfenas, Minas Gerais, Brazil. The details of the data collection and the coordinates of the site were among researchers [4]. provided by Dr. Flávio Nunes Ramos (Institute of Natural Sciences, The species Eugenia florida DC (Myrtaceae) is a small tree, UNIFAL-MG) and the species was identified by Dr. Geraldo Alves da approximately 3.5 m tall, that blooms in October and fruits at the Silva (Faculty of Pharmaceutical Sciences, UNIFAL-MG). A specimen end of November to mid-December. It is distributed throughout was deposited and recorded in the Herbarium of the UNIFAL-MG South America, from the Colombia to Bolivia, and from Guyana to the under the registration number 2562. The leaves were dried in an South of Brazil, growing in various ecological landscapes. It is oven with air renewal at 45oC for 72 h. After drying, the plant popularly known as black cherry or guamirim cherry. Its fruits have material was sprayed onto a knife mill. The powder obtained was a characteristic odor, and are edible and very tasty [5]. The subjected to exhaustive percolation using ethanol (70%) as a liquid extensive therapeutic use of the leaves of the species by rural extractor. The extraction solution was concentrated in a rotary populations has been highlighted. It is used primarily as a evaporator under reduced pressure and subsequently lyophilized Bastos et al. Int J Pharm Pharm Sci, Vol 8, Issue 6, 110-115 and stored, finally obtaining the crude hydroethanolic extract 70% µg/ml), in order to obtain the analytical curve A = 0.0059C+0.0014 (v/v) (EB). (R2 = 0.9846), where A is the absorbance at 510 nm and C is the concentration of tannic acid. The values obtained for the sample Chemical characterization by mass spectrometry were interpolated on the calibration curve and the concentration For analysis by mass spectrometry, EB of E. florida DC (5.0 mg) was values were expressed as milligrams of tannic acid equivalents per dissolved in methanol (3.0 ml), and filtered in a Sepak RP-18® gram of extract (mg EAT/g). The experiment was performed in cartridge and then through a Nylon membrane (Flow Supply®) with triplicate. 22.25 mm diameter and 0.22 µm pore size. The extract (5 ppm) was Evaluation of antimicrobial activity against yeasts and bacteria analyzed online by the LCQ Fleet, Thermo Scientific® mass spectrometer, equipped with a direct sample insertion device for The extract was evaluated in vitro for antifungal and antibacterial streaming injection analysis (FIA). The sample was ionized by activities using Mueller-Hinton Broth and the microdilution method electro spray (ESI) and fragmentations into multiple stages (MSn) following the methodology and interpretative criteria proposed by were held in an ion-trap (IT) interface. The negative mode was document M27-S4 for yeasts and document M7A6 for bacteria from chosen for the generation and analysis of all the spectra, and the the Clinical and Laboratory Standards Institute [19,20], with some experimental conditions were as follows: capillary voltage-35 V, modifications. The stock solutions of the extract were prepared in spray voltage-5000 V, capillary temperature 350oC, drag gas (N2) /ml): 1000; and flow rate 60 (arbitrary units). The acquisition track was m/z 500; 250; 125; 62.5; 31.25; 15.63; 7.81; 3.91; 1,95. The standard 100-2000, with two or more scan events held simultaneously in the drugs1% DMSO chloramphenicol and tested at andthe followingfluconazole concentrations were applied (μg as controls of spectrum. The experiment was performed by Laboratory of bacteriostatic and fungistatic actions, respectively, at the following Bioprospecting of Natural Products (LBPN), UNESP, Coastal Campus- /ml): 64; 32; 16; 8; 4; 2; 1; 0.5; 0.25; 0.125. IB-CLP. Theconcentrations tests were (μg performed with opportunist and pathogenic yeast Determination of total phenol content and bacterial standards from the American Type Culture Collection The determination of total phenols was performed according to the (ATCC): Candida albicans ATCC 10231, C. krusei ATCC 6258, C. colorimetric method based on the formation of complexes with glabrata ATCC 90030, C. parapsilosis ATCC 22019, Staphylococcus molybdenum and tungsten [16]. Aliquots of the dry extract were aureus ATCC 6538, and Escherichia coli ATCC 25922. The diluted in absolute ethanol, to obtain a concentration of 250 µg/ml. microorganism strains were obtained by Drª. Amanda Latércia 0.5 ml of the sample was added to 2.5 ml of