Determination of phenolic profile by HPLC-ESI-MS / MS and antibacterial activity of Eugenia platysema against mollicutes strains

Article history: Received on: 08/12/2016 Accepted on: 11/03/2017 Available online: 30/05/2017 This study evaluated the phenolic composition of the extract and fractions from E. platysema leaves, in addition to antibacterial activity against different types of bacteria, like mollicutes and cel wall bacteria. The crude extract (CHE) was partitioned with solvents to yield fractions of different polarities (dichloromethane, ethyl acetate, butanol and water). The chemical composition of CHE and its fractions were evaluated by HPLC-LCESI-MS/MS together with 76 standard phenolic compounds. Nine known compounds were identified for the first time from this species: hydroxybenzoic-O-hexoside acid [1], gallocatechin [2], syringic acid [3], 4hydroxycinammic acid [4], salycilic acid [5], isoquercetin [6], isorhamnetin [7], luteolin-3’-O-glucuronide [8] and luteolin [9]. The evaluation of antibacterial activity was performed using the broth dilution method. The CHE and fractions of E. platysema were active against mollicutes strains, being the best result presented by the dichloromethane fraction against U. urealyticum, M. mycoides subsp. Capri and M. hominis (125 μg mL -1 ).


INTRODUCTION
Microorganisms are one of the most frequent causes of diseases, representing as a serious public health problem for a large portion of the population.The mollicutes are organisms responsible for several diseases such as pneumonia and pyelonephritis, both in humans and in animals.Some strains like Mycoplasma hominis and Ureaplasma urealyticum are a common commensal of the female genital tract and have been associated with pyelonephritis, bacterial vaginosis, cervicitis, endometritis, and postpartum septicemia.U. urealyticum is considered as the main cause of nonchlamydial, non-gonococcal urethritis, chorioamnionitis, preterm delivery, abortion, preterm birth, bacterial vaginosis and cervicitis (Amirmozafari et al., 2009;Falk et al., 2005;Naessens et al., 1989;Taylor et al., 2013).The mollicutes have the peculiarity of the fact they do not have cell walls, and contain cholesterol in the composition of their cell membrane (Cordova et al., 2010).The lack of a rigid cell wall renders genital Mycoplasmas innately resistant to antimicrobial agents, such as β-lactam antibiotics and vancomycin, which act in this target.
Tetracyclines, macrolides, and quinolones are the major antibiotics used in the treatment of infections caused by mycoplasmas, however, their therapeutic efficacy may be unpredictable due to increasing resistance (Bayraktar et al., 2010).Therefore, there is an importance in researching new active compounds against these microorganisms from various sources, including natural products (Benfatti et al., 2010).
These compounds are a large class within polyphenols, where there are more than 4000 structures with have important medicinal and biological activity, especially antimicrobial, antioxidant and to prevent cancer effects (Shafaghat et al., 2014).
Eugenia platysema, popularly known as "Guamirim", is a perennial tree of the Eugenia genus (Myrtaceae) native to South America (Reitz et al., 1969).There are few studies on this species related in the scientific literature.Apel et al., (2002), evaluated the chemical composition of the essential oil from leaves of E. platysema, which showed β-selinene (17.9%) and alloaromadendrene (12.6%) as major compounds.Tenfen et al., (2016) also analyzed the essential oil of E. platysema leaves and presented it as major compound the diterpene (6-E, 10-Z) pseudo phytol (65.55% ± 0.81).
Nevertheless, this is the first study that evaluates chemically and biologically the extract and fractions of E. platysema.Thus, the aim of this study was to identificate the phenolic compounds presents in the crude extract and fractions of E. platysema leaves by HPLC-ESI-MS/MS and evaluate their antibacterial activities against cell wall and mollicutes strains.
The dried and ground plant material was macerated in hydroalcoholic solution (70 %) at room temperature for 7 days.The crude extract resulting (CHE) from maceration was concentrated under reduced pressure to completely dry.After concentration, the crude extract was resuspended in water and partitioned by liquid-liquid fractionation using solvents of different polarities yielding dichloromethane (FDCM), ethyl acetate (FAE) n-butanol (FBu) and aqueous fractions (FAq).

Identification of phenolic compounds by HPLC-LC-ESI-MS/MS
The profile of crude extract, FDCM, FAE, FBu and FAq fractions were analysed by HPLC-ESI-MS/MS in LABEC/INCT-Catálise at Federal University of Santa Catarina (UFSC).Analysis were conducted in an Agilent® 1200 chromatograph, with TurbolonSpray ® as ionization source, coupled to a Qtrap ® 3200 mass spectrometer, with a Phenomenex ® Synergi 4μ Polar-RP 80A (150 mm x 2 mm ID, particle size of 4 μm) at the temperature of 30 ºC.The eluents were formed by mixing solvents A (MeOH/H 2 O in ratio of 95:5, v v -1 ) and B (H 2 O ultrapure/formic acid (0,1 %) as follows: 1 st stage -10 % solvent A and 90 % B (isocratic mode) for 5 minutes; 2 nd stagelinear gradient of solvents A and B (from 10 to 90 % of A) for 2 minutes; 3 rd stage -90 % solvent A and 10 % B (isocratic mode) for 3 minutes; 4 th stagelinear gradient of solvents A and B (from 90 to 10 % of A) for 7 minutes with a flow rate of 250 μL min -1 of mobile phase.In all analyses, the injected volume was 5 uL at a concentration of 300 mg L -1 .
The liquid chromatograph was coupled to a mass spectrometer with electrospray ionization source using negative ionization mode with the following source parameters: ion spray interface at 400 °C; ion spray voltage of 4500 V; curtain gas, 10 psi; nebulizer gas, 45 psi; auxiliary gas, 45 psi; collision gas, medium.The Analyst® (version 1.5.1)software was used for recording and processing the data.Pairs of ions were monitored in MRM (Multiple Reaction Monitoring) mode.

Determination of minimum inhibitory concentration (MIC)
The antibacterial activity of the crude extract and fractions from E. platysema was evaluated by determination of the minimum inhibitory concentration (MIC).The microdilution broth assay was performed in sterile 96-well microplates, as recommended by the Clinical and Laboratory Standards Institute (CLSI, 2012) for cell-wall bacteria and Bebear and Roberteson (1996) for mollicutes.
The samples were properly prepared and transferred to each microplate well with the appropriate culture medium, in order to obtain a twofold serial dilution of the original extract in a 10 % H 2 O/dimethyl sulfoxide (DMSO) solution, obtaining sample concentrations ranging between 1000 μg mL -1 to 7.81 μg mL -1 .The inoculum containing 10 4 to 10 5 microorganisms per mL were then added to each well.
A number of wells were reserved in each plate to test for sterility control (no inoculum added), positive control (gentamycin or ciprofloxacin), inoculum viability (no extract added), and the DMSO inhibitory effect.
The microplates were incubated at 37 °C ± 1 °C for 24 hours to cell-wall bacteria and 48 hours for mollicutes.Thereafter, growth of mollicutes strains was detected by observing the colour change in the medium.For cell-wall a methanolic solution of triphenyl tetrazolium chloride (5 mg mL -1 ) was added into each well, and the presence of a reddish bacterial "button" observed at the bottom of each well.The MIC was defined as the lowest concentration of the sample able to inhibit bacterial growth.

Identification of phenolic compounds by HPLC-ESI-MS/MS
Chemical profiling of 76 phenolic compounds from 5 samples using the HPLC-ESI-MS/MS method were carried out.The results are shown on Table 1.These results indicated that in the crude extract four phenolic compounds were identified: gallocatechin The skeletons of identified compounds are characteristics from the Eugenia genus (Baliga et al., 2010;Einbond et al., 2004;Oliveira et al., 2014).However, this is the first report of identification of salycilic acid and hydroxybenzoic-O-hexoside acid on leaves of Eugenia genus.Futhermore, this is the first report about the presence of these compounds in Eugenia platysema.The flavonoids isoquercetin and gallocatechin have been previously described in another plants of Eugenia genus, as E. brasiliensis (Pietrovski et al., 2008), E. jambolana (Baliga et al., 2011) and E. uniflora (Lee et al., 2000).Syringic acid were described in E. pyriformis and E. jambolana (Sharma et al., 2016); luteolin were identified in E. cariophyllata (Gülçin et al., 2004).

Antibacterial Activity
For the evaluation of the antibacterial activity, a criterion established by Machado et al., (2005), was used.Samples with MIC values lower than 10 μg mL -1 were considered to have excellent antibacterial activity; between 10 and 100 μg mL -1 were considered good; values between 100 and 500 μg mL -1 were considered of moderate activity; values between 500 and 1,000 μg mL -1 of low activity, and for MIC values above 1,000 μg mL -1 , samples were considered inactive.The results for MIC of all samples are shown in Table 2.Among the tested samples, the fractions which had the highest activity was FDCM against U. urealyticum, M. hominis and M. mycoides (125 g mL -1 ) and FAE against U. urealyticum and M. mycoides subsp.capri (125 g mL -1 ).The best activity of crude extract was against U. urealyticum (125 g mL -1 ).
These results are important because the mollicutes are responsible for various diseases such as pneumonia, vaginitis, urethritis, pyelonephritis in humans (Cordova et al., 2010).Reports of resistance to these microorganisms in conventional treatments also increased (Yechouron et al., 1992), in addition these microorganisms do not have cell wall, which makes them completely resistant to β-lactam antibiotics and other drugs that act on it (Silva, 2006).For the cell-wall bacteria none of samples tested were active, showing with MIC values above 1,000 μg mL -1 .This indicates that the mechanism of action of the extract and the fractions of E. platysema to inhibit bacterial growth is not involved with the bacterial cell wall.Some of the identified phenolic compounds were report on literature to have antimicrobial properties.Luteolin, a wellknown polyphenolic compound identified in the dichloromethane fraction, has diverse biological benefits that include antimicrobial effects.Joung et al., (2016) reported that luteolin showed synergistic activity by increasing cytoplasmic membrane permeability and inhibiting ATPase.This is important on the effects of the membrane permeabilizing agent and ATP-binding cassette (ABC) transporter inhibiting agent, since most bacteria produce ABC transporter that is an essential uptake system for amino acids in the bacterial membrane, and this mechanism can be a determinant of bacterial antibiotic resistance.
Other identified phenolic compounds like syringic acid has been reported to exhibit antibacterial ability against various microorganisms (Shi et al., 2016).The syringic acid retarded bacterial growth, and caused cell membrane dysfunction, with decrease of intracellular ATP concentration, cell membrane hyperpolarization and changes in cellular morphology (Shi et al., 2016).
However, this is the first study that evaluated the antibacterial activity of samples containing the syringic acid in its composition against mollicute strains.The presence of syringic acid and luteolin in the FDCM fraction probably contributes to the antibacterial potential of this fraction.These mechanisms may help to explain some of the good antibacterial activity shown by the dichloromethane fraction.However, further studies with the isolated compounds should be conducted, together with the identification of other classes of compounds, to searching for the responsible compounds for the antibacterial activity in these fractions and to better answer these questions.

CONCLUSIONS
Present results showed that nine phenolic compounds were identified in the extract and fractions from E. platysema.All of these compounds were reported the first time in this species.Interesting antibacterial activity for the extract and fractions of this species against mollicutes strains were observed, especially in the FDCM fraction.Further phytochemical studies should be performed to isolate and identify the active constituents for this activity.

Table 2 .
Antibacterial activity of crude extract and fractions from E. platysema.MIC =Minimal Inibitory Concentration.b Positive Control= ciprofloxacin, for mollicutes; gentamicin, for cel wall bacterials. a Phenolic compounds identified in the leaves of Eugenia platysema.