In Vitro Antimicrobial Activity of Embothrium coccineum Used as Traditional Medicine in Patagonia against Multiresistant Bacteria

Embothrium coccineum J.R. Forst. & G. Forst is an evergreen tree that has been used as a folk remedy for the treatment of neuralgia, tooth pains, wound healing, and glandular conditions, as well as an antiseptic agent against bacterial infection. The antibacterial activities of sequential extracts (hexane, dichloromethane, ethyl acetate, and ethanol) from the leaves of E. coccineum were evaluated by means of the micro-dilution assay against six (Escherichia coli; Klebsiella pneumoniae; Proteus mirabilis; Pseudomonas aeruginosa; Staphylococcus aureus and Streptococcus pyogenes) multiresistant bacteria strains. Ethyl acetate extract showed the best spectra of antibacterial activity against all tested bacteria, and was analyzed by gas chromatography–mass spectrometry (GC-MS) for its composition. The results of the present work provide useful baseline information for the potential development and use of nanoparticles and/or nanofibers doped with extracts of E. coccineum in the fight against multiresistant bacteria, which would allow the validation of the traditional use of E. coccineum by native peoples of Patagonia as an antimicrobial agent in the biomedical Field.

people [3,4]. An earlier study by Mølgaard suggested that the ethanol extract of E. coccineum does not exhibit significant antibacterial activity against E. coli and P. aeruginosa [5], while in other studies it has been shown that extracts of E. coccineum have antioxidant activity because of their contents of phenols, flavonoids and anthraquinones, and molecules which were related to antimicrobial activity [6]. However, the available information concerning E. coccineum is particularly limited. Therefore, in view of the fact that E. coccineum has been widely used as a dermatologic drug [7], and therefore is likely safe and effective, and the importance of finding new anti-infective extracts to be loaded into or coated onto biomaterials, the aims of this study were to evaluate the antibacterial activities of sequential extracts from the leaves of E. coccineum against Gram-positive and Gram-negative multidrug-resistant strains and determine the composition of the most active extract by GC-MS.

Chemical Composition of AcOEt Extract
Results of the gas chromatographic analysis of ethyl acetate extract from the leaves of E. coccineum are summarized in Table 1. Twenty-six components were identified in the ethyl acetate extract: 31.12% were hydroxybenzoic acids, 13.10% were terpenes, 13.06% were fatty acids and derivatives, 5.86% were aldehydes and ketones, 5.41% were linear hydrocarbons, 2.98% were lactones, 2.88% were aromatic hydrocarbons and 25.59% were unknown compounds. The ethyl acetate extract was mainly characterized by gentisic acid (21.17%), β-resorcylic acid (8.06%), phytol (7.19%), myristic acid (4.72%), and ethyl palmitate (3.89%). In addition, this is the first report made on the composition of leaves from E. coccineum in which hydroxybenzoic acids were the predominant portion.

In Vitro Antimicrobial Assay
A previous study showed that the chemical components of E. coccineum include anthraquinones, flavonoids and phenols [6]. It is known that secondary metabolites such as those described in E. coccineum have good antimicrobial properties. As shown in Tables 2 and 3, the sequential extracts of the leaves showed antibacterial activity against all bacteria with minimum inhibitory concentration (MIC) values of 31.125-500 µg/mL and minimum bactericidal concentration (MBC) values of 62.5-500 µg/mL.

Discussion
The results revealed that the ethyl acetate extract showed higher activity against S. pyogenes with MIC and MBC values of 31.125-62.5 µg/mL; these values are comparable with those reported [8]. In addition, ethyl acetate extract was potently active against Gram-negative bacteria K. pneumonia with MIC and MBC values of 125 µg/mL in the clinical setting; it is the most significant member of the Klebsiella genus of Enterobacteriaceae [9]. The dichloromethane extract showed potent activity against E. coli with MIC and MBC values ranging from 31.125-62.5 µg/mL. Ethanol extract showed the same antibacterial activity against all bacteria with MIC and MBC values of 250-500 µg/mL. Similar results have been reported where the ethanol extract had low antimicrobial activity [10].
However, this study showed that the MIC and MBC values of E. coccineum extracts found highest against S. aureus were 250-500 µg/mL. Moreover, due to the activity shown by the ethyl acetate, it was subjected to GC-MS to identify the components in the extract responsible for the antibacterial activities observed in the study. Gentisic acid and β-resorcylic acid were the main metabolites in the extract; these hydroxybenzoic acids are found in many plants and fruits, and these compounds show a wide spectrum of action involving antitumor, antiviral, antibacterial, cardioprotective, pro-oxidant and antimutagenic activity [11]. The presence of the hydroxyl group and a system of delocalized electrons might be responsible for the antimicrobial activity of these compounds [12]. Furthermore, there is phytol in the extract, which is an important member of branched-chain unsaturated terpene and is a product of the chlorophyll metabolism in plants. It has also been reported that phytol can inhibit microorganisms [13]. In addition, the presence in the extract of fatty acids as myristic acid and ethyl palmitate, which are in natural fats and dietary oils, and are known to have antibacterial and antifungal properties [14], justifies the potential antimicrobial activity of this extract.
The results of the present work provide useful baseline information for the potential development and use of nanoparticles and/or nanofibers doped with extracts of E. coccineum in the fight against multiresistant bacteria, which would allow the validation of the traditional use of E. coccineum by native peoples of Patagonia as antimicrobial agents in the biomedical field.

Chemicals and Materials
Chemical reagents, culture media, solvents and positive controls were purchased from Sigma-Aldrich (Darmstadt, Germany).

Plant Material
E. coccineum was collected in November 2015 at Valdivia, Los Ríos Region, Chile. A voucher specimen (VALPL 2156) was deposited at the VALP Herbarium, Department of Biology, Universidad de Playa Ancha, Valparaíso, Chile.

Extraction
In brief, leaves of E. coccineum (500 g) were isolated manually from aerial parts and dried at room temperature for four weeks. The dry plant was powdered, and then mixed with 1.0 L hexane. The extraction was carried out by using an orbital shaker (150 rpm) at 25 • C for 72 h. The resulting mixture was filtered through Whatman No. 1 filter paper (Sigma-Aldrich, Darmstadt, Germany) and the hexane was removed from the filtrate under reduced pressure with a rotatory evaporator. The residue was further extracted with dichloromethane, ethyl acetate, and ethanol, sequentially and serially. Finally, each extract was weighed and the yield was calculated. E. coccineum extracts were kept at −4 • C prior to further analyses.

Chromatographic Analysis
The ethyl acetate extract was diluted with ethyl acetate, and analysis by gas chromatography (Hewlett Packard, Palo Alto, CA, USA) was carried out according to the method detailed elsewhere [15]. The operating conditions were as follows: on-column injection; injector temperature, 250 • C; detector temperature, 280 • C; carrier gas, He at 1.0 mL/min; oven temperature program: 40 • C increase to 260 • C at 4 • C /min, and then 260 • C for 5 min, to afford the best separation through a capillary Rtx-5MS column. The mass detector ionization employed an electron impact of 70 eV. Compounds in the chromatograms were identified by comparison of their mass spectra with those in the NIST/EPA/NIH Mass spectral Library [16]. Chromatographic peaks were considered "unknown", when their similarity index (MATCH) and reverse similarity index (RMATCH) were less than 850 and discarded in this identification process [17]. These parameters are referred to the degree the target spectrum matches the standard spectrum in the NIST Library (the value 1000 indicates a perfect fit), and by comparison of their retention index with those reported in the literature [18], for the same type of column or those of commercial standards, when available. The retention indices were determined under the same operating conditions in relation to a homologous n-alkanes series (C 8 -C 36 ) by the equation: RI = 100 × (n + Tr (unknown) − Tr (n) /Tr (N) − Tr (n) ) (1) where, n = the number of carbon atoms in the smaller n-alkane; N = the number of carbon atoms in the larger n-alkane; and Tr = the retention time. Components relative concentrations were obtained by peak area normalization.

Bacterial Strains
The bacteria were recent clinical isolates, belonging to the Facultad de Ciencias de la Salud (Universidad Central de Chile) collection. They comprised of: Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pyogenes. All isolates were identified according to the latest Clinical and Laboratory Standards Institute (CLSI) guidelines [19]. Strains were cultured in Luria-Bertani (LB) broth, Mueller-Hinton Broth (MHB) and Mueller-Hinton agar, at 37 • C.

In Vitro Antimicrobial Assay
The Minimum Inhibitory Concentration (MIC) and minimum bactericidal concentration (MBC) of plant extract was determined using the broth dilution method, following the CLSI guidelines [20]. Briefly, the test extracts were first dissolved in dimethyl sulfoxide (DMSO) and the final concentration of DMSO in each medium was 1%, which did not affect the growth of the test strain. A dilution range of the extracts from 1 mg/mL to 12.5 µg/mL was tested. Later, 100 µL of microbial suspension (1 × 10 5 CFU/mL) of each strain was inoculated in tubes with equal volume of nutrient broth and plant extracts. After, the tubes were incubated aerobically at 37 • C for 24 h. After the cultures were incubated at 37 • C for 24 h, the minimum inhibitory concentration (MIC) was determined as the lowest concentration of the test extract that demonstrated no visible growth. MBC value was determined by sub culturing the test dilution (100 µL) (which showed no visible turbidity) on to freshly prepared nutrient agar media. The plates were incubated further for 48 h at 37 • C. The highest dilution that yielded no single bacterial colony on the nutrient agar plates was taken as MBC. Rifampicin (1 mg/mL) and chloramphenicol (250 mg/mL) were used as positive control for bacterial strains and 1% DMSO was used as negative control. Each concentration of the extracts were tested in triplicate.

Statistical Analysis
Data were expressed as mean and standard deviation. A one-way variance was used to analyze data, with p < 0.05 representing a significant difference between means (Student's t-test).

Conclusions
In conclusion, the obtained data indicate that the ethyl acetate extract showed the best spectra of antibacterial activity against all tested multiresistant bacteria. The biological activity could be partly explained by the majority presence of hydroxybenzoic acids, such as gentisic acid and β-resorcylic acid. Importantly, these results also contribute to the validation of E. coccineum as a medicinal plant for the treatment of infectious diseases.