Antibacterial and cytotoxic antibacterial potential of ethanol extract and fractions from Aristolochia galeata Mart. ex Zucc

Antibacterial and cytotoxic antibacterial potential of ethanol extract and fractions from Aristolochia galeata Mart. ex Zucc Álan Alex Aleixo, Vidyleison Neves Camargos, Ana Cláudia dos Santos Pereira Andrade, Karina Marjorie Silva Herrera, Rosy Iara Maciel de Azambuja Ribeiro, Kamilla Monteiro dos Santos, Juliana Teixeira de Magalhães, José Carlos Magalhães, Luciana Alves Rodrigues dos Santos Lima and Jaqueline Maria Siqueira Ferreira*

Although there are no registers of antimicrobial activity of A. galeata, some compounds have already been isolated, and among them, stand out clerodane and labdane diter-penoids (Lopes and Bolzani, 1988).Compounds of these classes isolated from other species, present diverse biological properties, such as antiviral, antifungal and antibacterial (Salah et al., 2003;Vidal et al., 2011;Porto et al., 2012).
Studies on new therapeutic options from herbal products as antimicrobials are necessary, since bacterial infections have grown significantly, contributing to increased morbidity and mortality, especially in hospitalized and immunocompromised patients (Zhong et al., 2012;Schmitt et al., 2012;Pandey et al., 2012).Allied to the increased infections, treatment is becoming increasingly difficult in view of the notable ability of these pathogens to acquire new mechanisms of selective resistance to antibiotics (Tenover, 2006).
Therefore, in front of the lack of studies on phytochemistry, antimicrobial and cytotoxicity activity of A. galeata with the potential use of medicinal plants in the treatment of diseases caused by Gram-negative andpositive bacteria.The current investigation carried out a screning of ethanol extract and hexane, dichloromethane, ethyl acetate and hydroethanolic fractions of A. galeata against important pathogenic bacteria and evaluates its cytotoxicity potential to develop new antibacterial therapy.

Bacterial strains and antimicrobial tests
The minimum inhibitory concentration (MIC) of A. galeata ethanol extract and their fractions were determined using a broth microdilution method as described by Clinical and Laboratory Standards Institute (CLSI, 2003) with modifications.Nine reference bacterial strains of American Type Culture Collection (ATCC) were chosen due to their ability of present multi resistant to the drugs as follows: Gram-negative Escherichia coli EHEC (ATCC 43895), Pseudomonas aeruginosa (ATCC 27853), Klebsiella pneumoniae (ATCC 27736), Salmonella typhi (ATCC 19430)  The ethanol extract and fractions were dissolved in sterile dimethylsulfoxide 2% (DMSO) (Synth, Brazil) and were used in serial dilution from of 1250 until 125 µg/ml.An inoculum of 125 µl of cell culture was added to 25 µl of each concentration of samples in Mueller-Hinton broth (MH) (Himedia, India) in 96-well plates.For negative controls, wells containing MH medium or sterile DMSO 2% were used and for positive control, MH plus bacteria and the antimicrobial agent streptomycin 100 µg/ml (Sigma-Aldrich, USA) were used (growth inhibition).Plates were incubated at 35 ± 1°C for 24 h.
The MIC was assessed based on the lowest concentration of sample required to inhibit the microbial growth and was determined by measuring the absorbance at 490 nm (Powder Wave XS2, Biotec, USA).The experiments were performed in triplicate.
For assays to determine the minimum lethal concentration (MLC), aliquots of 25 µl were removed from wells without visible turbidity and placed on Agar Plate-count by a Pour-Plate Method (Costa et al., 2010).After incubation at 37°C for 24 h, colonies were counted.The concentration of sample that resulted in a growth 0.1% of initial inoculum (1.5 × 10 6 UFC/ml) was determined as the MLC.

Cytotoxicity analysis by the MTT assay
Human cervix carcinoma cell line (HeLa) was grew in Dulbecco's Modified Eagle Medium (DMEM) with 2% of Fetal Bovine Serum (FBS), at 37°C, 5% of CO2 atmosphere and in 96-well microplate, until it reaches 95% of confluence.After 72 h exposure at dosages from 1000 to 0.025 µg/ml, 20 µl (2 mg/ml) of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Bio Basic INC, Canada) in phosphate buffered saline (PBS) were added on each well and the plate was incubated at 37°C for 3 h.The medium was removed and 130 µl of DMSO were added and after incubation at37°C for 10 min, the absorbance was read at 540 nm in ELISA spectrophotometer (Powder Wave XS2, Biotec, USA) to determine the concentration that killed 50% of cells (CC50) ( Luscombe, 1987).The cytotoxicity was calculated after comparing with the control (treated with 0.1% DMSO).

Statistical analyses
All tests were made in triplicate in three independent experiments.When appropriate, mean ± standard deviation were used to describe the results.The mean effective concentration CC50 values (concentration that reduces 50% of control group response) were determined by non-linear regression using Graph Pad Prism, 5.0 (GraphPad Software Inc., San Diego, CA, USA).

Phytochemical analysis
Phytochemical analysis of A. galeata (Table 1) showed the presence of steroids/triterpenoids, flavonoids, coumarins and alkaloids in the ethanol extract.Majority of secondary metabolites found in the fractions ethyl acetate and dichloromethane were flavonoids and steroids/ triterpenoids, respectively.Alkaloids, coumarins and a minimal presence of steroids were detected in the hexane fraction.Saponins and tannins were not observed.

Antibacterial activity
The MIC and MLC values from crude extract of A. galeata are shown in Table 2.This extract showed bacteriostatic activity in Gram-positive bacteria, and S. aureus strain appeared to be the most sensitive bacteria with MIC of 250 µg/ml.However, no Gram-negative bacteria showed sensitivity to the extract.Following the antibacterial evaluation of crude extracts, the fractions were tested.Like the crude extract, Grampositive bacteria showed sensitivity (Table 2).The hexane and dichloromethane had MIC ranging from 500 to 1000 μg/ml.The most sensitive bacterial samples were S. epidermidis and S. aureus, with MIC of 500 μg/ml.The MLC was only observed for the fraction of dichlorometane of 1250 μg/ml against S. aureus and S. saprohyticus.The ethyl acetate and hydroethanolic fractions showed no bactericidal or bacteriostatic activity.
The DMSO used as a negative control, showed no bacteriostatic or bactericidal activity, as expected (data not show) (Table 3).

Cytotoxicity activity
Analyzing the A. galeata cytotoxicity in vitro, the results show that the dichloromethane fraction presented moderate cytotoxic effect with CC 50 = 90 µg/ml (Table 3).However, the crude extract and other fractions showed little cytotoxicity activity with the CC 50 ranging between 1620 and 369 µg/ml.

DISCUSSION
Many studies have demonstrated the antibacterial activity of plants commonly used in traditional medicine (Rakholiya and Chanda, 2012;Tekwu et al., 2012, Mishra et al., 2013a. Rios and Recio (2005) suggested that MIC greater than 1 mg/ml for crude extracts or 0.1 mg/ml for isolated compounds should be avoided and proposed that activity would be very interesting in MICs of 0.1 and 0.01 mg/ml for extracts and isolated compounds, respectively.On the other hand, Fabry et al. (1998) defined active crude extracts as those having MIC values <8 mg/ml.In this study, however, MIC and MFC values of less than 1 mg/ml were considered to be of good activity.
Our results showed that the crude extracts of A. galeata, and their fractions presented selective antibacterial activity against Gram-positive bacteria.It is not known exactly why Gram-negative bacteria typically have lower sensitivity to components of plant extracts, but this may be related to the chemically more complex cell wall with the presence of additional membrane that can act as a selective barrier compared to the Gram-positive bacteria (Deans and Ritchie, 1987;Srinivasan et al., 2001;Nikaido, 2003).
Gram-positive bacteria belonging to the genera Staphylococcus and Enterococcus, with clinical relevance, showed sensitivity to A. galeata.These genera are the most frequent cause of nosocomial infections by Gram-positive strains, and the species S. aureus, S. epidermidis, S. saprophyticus and E. faecalis are the most common in this scenario (Boneca and Chiosis, 2003;Kuroda et al., 2005).These microorganisms may acquire resistance against antibacterial drugs by a variety of mechanisms that includes drug modification or destruction, alteration of binding sites, altered metabolism, and prevention of drug entry into the cell (Tenover, 2006;Barie, 2012).
The bacterial samples analyzed presented great sensitivity to aminoglycoside streptomycin used in this study.However, many studies have reported the resistance of both Gram-positive and Gram-negative bacteria to this class of antibiotics.This fact justifies the search for new molecules with antibacterial properties (Josephson, 2006;Coutinho et al., 2010;Zhong et al., 2012).
The Aristolochiaceae genus has revealed its potential as antimicrobial agent.Aristolochia species have also been described as getting antibacterial properties against strains of medical importance (Shafi et al., 2002).The A. esperanzae species showed antibacterial activity against samples of S. aureus, E. coli, S. typhimurium, B. cereus, C. freundii and L. monocytogenes (Pacheco et al., 2010).Some species of this genus stood out due to the potential cytotoxicity of cancer cell lines (Yu et al., 2007).In our study, A. galeata presented low cytotoxicity in HeLa cells, except for dichloromethane fraction.The presence of large amounts of steroids/triterpenoids on the dichloromethane fraction may be related with the antimicrobial activity.These results corroborate with literature data that showed antistaphylococcal activity of terpenes (Gibbons, 2004) and antifungal of steroids (Johann et al., 2010).However, the moderate cytotoxicity shown for this fraction (CC 50 = 90 µg/ml) may be related to compounds belonging to this class, such as sesquiterpenes, very common in species of this genus (Yu et al., 2007).
Coumarins and alkaloids were the main secondary metabolites found in the hexane fraction, which also demonstrated antibacterial activity.These classes of compounds are also described in the literature as natural antimicrobial agents (Cottiglia et al., 2001;Gibbons, 2004;Mishra et al., 2009;Mishra et al., 2013b).The hexane fraction has been promising, because besides having good antibacterial activity it also showed low cytotoxicity (CC 50 = 0.726 mg/ml) against the cell line studied.Moreover, the hydrophobicity of this fraction determines the minimal presence of triterpenoids compounds and aristolochic acid, which is expected, because these substances have cytotoxic and nephrotoxic effects.

Conclusion
The results of the present study revealed for the first time evidence for prospect in A. galeata to new molecules with antibacterial activity, especially in the hexane fraction.It is relevant in front of the growing resistance that bacterial strains have shown to actual drugs.São João Del Rei (UFSJ) and Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG) for financial support and fellowships; and to Prof. Dr. Alexandre Salino for botanical identification of this plant.

Table 3 .
Effects of A. galeata and their fractions on HeLa cells by MTT colorimetric assay.