Antimicrobial analysis of copaiba oil extract from Passiflora cincinnata and endodontic substances

The aim of this study was to evaluate the antimicrobial activity and minimum inhibitory concentration (MIC) of copaiba oil, extracts of Passiflora cincinnata and substances commonly used against endodontic infections of bacterial strains certified by the American Type Culture Collection (ATCC) and against clinical isolates (CI). The methodology involved the preparation of crude extracts of the plants, the selection of copaiba oil and the standardization of samples. The antibacterial activity of these substances was tested against Enterococcus faecalis (ATCC), Escherichia coli (CI) and Pseudomonas aeruginosa (ATCC and CI). The MIC was determined by broth dilution and the nitroblue tetrazolium chloride dye reduction test. The data were statistically analyzed by the Kolmogorov-Smirnov normality test and by the Kruskal-Wallis test with a confidence level of 95%. The analysis of the antimicrobial activity showed that the ethanol extracts of P. cincinnata and the combination of calcium hydroxide with polyethylene glycol with and without camphorated monochlorophenol showed no antimicrobial activity. However, the copaiba oil and other substances evaluated showed some antimicrobial activity against the microorganisms used (p <0.01), exhibiting a MIC ranging from <0.3 to >400 μL/mL (p <0.05). Copaiba oil showed antimicrobial activity and could represent a potential phytotherapeutic agent to be used against microorganisms causing endodontic infections.


INTRODUCTION
The microflora of endodontic infections is polymicrobial, and there is generally a prevalence of anaerobic bacteria.Microorganisms such as Enterococcus faecalis and Escherichia coli, but Pseudomonas aeruginosa, an aerobic bacterium, can also be present (Dianat et al., 2015).Mechanical cleaning of the root canal individually cannot disinfect the canal precisely, because the bacteria can hide in out-of-reach areas, isthmi and dentinal tubules.In addition to the mechanical action of the rotary or manual instrumentation, treatments frequently include *Corresponding author.E-mail: marcos.silva@foufal.ufal.br.
Author(s) agree that this article remains permanently open access under the terms of the Creativ e Commons Attribution License 4.0 International License  (Dianat et al., 2015) and the intra-canal administration of calcium hydroxide, camphorated paramonochlorophenol-(CMCP), formocresol, tricresol formalin and/or a combination of corticosteroid and antibiotics.Given these limitations and based on the traditional use and relative ease of the isolation of active components, plants may provide potential sources of new drugs for the safe and effective treatment of diseases (Santos et al., 2008;Pimenta et al., 2015).
Among the plants that have been found to have pharmacological properties, Copaifera spp.are known to have anti-inflammatory effects on the airway and on the skin and to promote the healing of wounds and intrauterine ulcers, in addition to exhibiting antibacterial actions.Copaiba is an oil-resin produced by the exudation of the trunks of trees from the Copaifera genus.The material excreted is a transparent liquid, bright, with a coloration ranging from yellow to brown (Veiga et al., 2001).Among these plants, Passiflora cincinnata has been used to treat hypotension, as an anti-inflammatory agent, as an anti-anxiety agent and as an antitussive agent (Wolfman et al., 1994).
The aim of this study was to evaluate the antibacterial activity of preparations of copaiba oil and the extracts of P. cincinnata (passion flower) against P. aeruginosa, E. faecalis and E. coli.These effects were also compared with the antimicrobial effects of substances commonly used in endodontic practice.

Plant and extraction procedure
Tw o fractions of ethanolic extracts of the flow ers and leaves of P. cincinnata obtained from the Laboratory of Chemistry and Biochemistry, Federal University of Sergipe w ere used.The samples w ere placed in an oven (Model MA -037) at 37°C w ith continuous circulation of air for 48 h until they w ere completely dehydrated.The dried leaves and flow ers w ere crushed and pulverized for 72 h to obtain a pow der.The pow der w as subjected to extraction by maceration in 70% ethanol for 5 days.After concentration of the solvent in a rotary evaporator under reduced pressure, a crude ethanolic extract of the flow ers and leaves w ere

Com m ercial substances
Commercial formulations of substances commonly used in conventional endodontic therapy w ere used, as show n in Table 1.

Antim icrobial activity
10 mg of the leaves and flow ers w ere solubilized in 2 mL of distilled w ater to obtain a standard solution w ith a concentration of 5.0 mg/mL of each extract.2.4 mg of calcium hydroxide paste (CALEN, SS WHITE, Rio de Janeiro, Brazil) and calcium hydroxide paste w ith camphorated paramonochlorophenol-CMCP (CALEN w ith CMCP, SS WHITE, Rio de Janeiro, Brazil) w ere used, w hich w ere each diluted in 2 mL of distilled w ater to yield a concentration of 1.2 mg/mL of each aliquot.
The follow ing substances w ere used in the antimicrobial tests: Ciprofloxacin, formulations of the copaiba oil I, II and III, formocresol, tricresol formalin, camphorated paramonochlorophenol, 1% sodium hypochlorite and hydrocortisone + neomycin sulfate + polymyxin B sulfate.The bacteria from the American Type Culture Collection (ATCC), E. faecalis and P. aeruginosa, w ere received as lyophilized samples and w ere placed on maintenance medium, w hich w as prepared w ith Miller-Hinton agar and Brain Heart Infusion (BHI) broth.The clinical isolates of E. coli and P. aeruginosa w ere derived from pre-cultures of the bacteria grow n for 18 h at 37°C on Miller-Hinton agar.
The inoculum w as diluted in saline to achieve an opacity optical density (OD) equivalent to 0.5 on the McFarland scale, w hich corresponds to 10 8 colony forming units (CFU/mL).The antimicrobial activity w as assessed according to the method reported by Bauer et al. (1966) (disk agar dilution method).Previously sterilized paper discs w ere soaked in the solutions of standardized extracts and medications and w ere placed at pre-dish and incubated at 37°C for 24 h.The readings w ere performed by measuring the inhibition halo around the disc in millimeters, using a calibrated caliper.

Minim um inhibitory concentration (MIC)
The plants and substances used for the determination of the minimum inhibitory concentration w ere copaiba oils, formocresol, tricresol formalin, camphorated paramonochlorophenol, sodium hypochlorite 2.5%, hydrocortisone + neomycin sulfate + polymyxin B sulfate and ciprofloxacin.A total of 15 µg of ciprofloxacin w as diluted in 300 µL of saline to a concentration of 5 µg/100 µL.The other substances used w ere applied as commercial formulations.
To determine the minimum inhibitory concentration, 96-w ell microdilution plates (Andrew s, 2001) w ere used.Ciprofloxacin w as used at concentrations ranging from 0.3 to 50 µg/mL, and the other substances w ere used in concentrations ranging from 3.12 to 400 µL/mL.For the negative controls, the w ells w ith saline and brain heart infusion (BHI) broth w e treated.Each w ell containing culture medium (BHI) and the substance (100 µL) w as inoculated w ith 10 µL of a microbial strain.On the other hand, Calen w as diluted in the same manner as in antimicrobial activity test and used in the evaluation of MIC.Then, the plates w ere sealed and incubated at 37°C for 24 h.After this incubation period, 10 µL of p-Iodonitrotetrazolium w as added to reveal w hether microbial grow th w as present, and the minimum inhibitory concentration of eac h substance w as determined in triplicate.
The data w ere analyzed regarding the normal distribution by the Kolmogorov-Smirnov test.Since the normal distribution w as not detected, the nonparametric Kruskal-Wallis test w as used and the data w ere analyzed.The softw are ASSISTAT 7.0 softw are (UFCG, Campina Grande, Brazil) w as used to apply the statistical tests w hich w ere set at a confidence level of 95%.

RESULTS
The ethanolic extracts of P. cincinnata and the combination of calcium hydroxide and polyethylene glycol with and without camphorated paramonochlorophenol showed no antimicrobial activity.However, the opaiba oils and the other substances evaluated showed antimicrobial activity against the target microorganisms (p <0.05) (Table 2).

DISCUSSION
To test the antimicrobial properties of plants and chemicals, testing was performed using agar diffusion, as advocated by Bauer et al. (1966) which is a reliable, inexpensive, reproducible and simple method that is widely used in microbiological research (Ohara et al., 1993;Estrela et al., 2000;Gomes et al., 2002;Drumond et al., 2004;Silva et al., 2006;Costa et al., 2008).
It was shown that the copaiba oil preparations (copaiba oil I and II) were effective against E. faecalis (ATCC) and P. aeruginosa (CI), with MIC values < 3.12 and > 400 μL/mL, respectively, while P. aeruginosa (ATCC) and E. coli (CI) were resistant to all the three oil preparations tested.However, in a study by Mendonça et al. (2009), the copaiba oil was effective against P. aeruginosa and E. coli, with MIC values of 12.5 and 1.56%, respectively, which may be explained by differences in bacterial resistance, such as the removal of proteins by an efflux pump, the differences in the expression of outer membrane proteins and differences in the protein production of DNA gyrase.
In another study, Lima et al. (2006) stated that the presence of the terpenoid in copaiba oil was responsible for its activity against Gram-positive bacteria, while it had no effects against E. coli, which is a Gram-negative bacterium.The present study and previous work by Packer and Luz (2007) showed similar results regarding the effects of copaiba oil on E. coli.However, Pieri et al. (2012) suggested that copaiba oil exerts an antimicrobial action that is only bacteriostatic in nature, because no concentration led to 99.9% destruction of bacteria in his studies performed to determine the bacteriological concentration maximum.In addition to these microorganisms, copaiba oil has shown antimicrobial activity against cariogenic agents such as Streptococcus mutans and some biofilm-forming agents, such as Streptococcus salivarius, Streptococcus pyogenes and E. faecalis (Pieri et al., 2012).
In the present work, the analysis of antimicrobial activity showed that the extracts of the leaves and flowers of P. cincinnata demonstrated no activity against any of the bacteria tested.However, Nicolls et al. (1973) assessed the antimicrobial activity of the bark extract of the fruit of P. mollissima and observed antimicrobial activity against E. coli, which may be attributed to the presence of polyacetylenes occurring in these antimicrobial plants (Birner et al., 1973;Nicolls et al., 1973), which were absent from the ethanol extracts of P, cincinnata tested in the present study.
Similar results were found for calcium hydroxide + polyethylene glycol with and without CMCP, which showed no zone of inhibition for any of the test organisms.However, the analysis was performed after 24 h, while Estrela et al. (1998) found that calcium hydroxide exerted antibacterial effects against bacteria after 48 h; moreover, in the work by Gomes et al. (2002), antimicrobial effects were observed after 7 days.Notably, in another study by Estrela et al. (2003a), complete inhibition of microorganisms occurred after 60 days, showing that the timing of studies is fundamental and that it may take a long time for the calcium hydroxide to exert its full activity, likely because the antimicrobial activity is directly proportional to the rate of diffusion of hydroxyl ions.
On the other hand, formocresol was effective against most of the bacteria tested in the present study, except E. faecalis, with MIC values of 50 μL/mL for P. aeruginosa (ATCC and CI) and 200 μL/mL for E. coli.However, Ferreira et al. (2007) also found that formocresol had antibacterial effects against E. faecalis.Similarly, an antimicrobial action of formocresol against anaerobic bacteria was observed by (Ohara et al., 1993).
Tricresol formalin showed an antibacterial front for P. aeruginosa (ATCC and CI), with MIC values of 50 and 100 μL/mL, respectively.However, the other bacteria tested were resistant.This result was in agreement with a previous study by (Melo et al., 2004), in which tricresol formalin's bactericidal actions were observed only against P. aeruginosa, and with a study by Silva et al. (2006), in which tricresol formalin did not inhibit the growth of E. faecalis.
In the current study, camphorated paramonochlorophenol was only effective against P. aeruginosa (ATCC), with a MIC of 200 μL/mL.In contrast, Costa et al. (2008) showed that CMCP had antibacterial effects against E. faecalis.Moreover, the work of Orstavik and Haapasalo (1990) showed that CMCP eliminated P. aeruginosa within four hours; however, there was also an effective front for E. faecalis (24 h) and E. coli (20 min).
The sodium hypochlorite solution (2.5%) was effective against P. aeruginosa (ATCC and CI), with a MIC > 400 μL/mL for both.In another study by Estrela et al. (2003b), a similar result was found for P. aeruginosa when they tested the effects of 2% alkaline hypochlorite by direct exposure and agar diffusion.Those authors observed antimicrobial effects against E. faecalis and P. aeruginosa for both methods, but better performance was observed with the direct exposure for sodium hypochlorite.
The combination of hydrocortisone + neomycin sulfate + polymyxin B sulfate was the only treatment tested that showed activity against all strains tested, with a MIC of < 3.12 µL/mL for E. faecalis and > 400 μL/mL for the other microorganisms.Similarly, Siqueira Jr. and Lopes (1999) tested the effectiveness of this combination treatment on the bacteria present in saliva under aerobic and anaerobic conditions, and found that this drug only exhibited inhibitory effects in the presence of oxygen.The authors suggested that this phenomenon could be explained by the fact that the antibiotics (neomycin sulfate and polymyxin B sulfate) are effective against both aerobic and facultative anaerobic bacteria.Ciprofloxacin showed activity against E. faecalis and P. aeruginosa (ATCC and CI), with MIC values of 0.03, 2.5 and > 5 μg/mL, respectively, because the strain of E. coli was resistant.Likewise, the work of Sader et al. (2001) showed that ciprofloxacin eliminated 61.4% of P. aeruginosa strains isolated from clinical infections, with a MIC of up to 0.5 µg/mL.Similar results were obtained in a study by Gales et al. (1997), who observed inhibition of the growth of P. aeruginosa (ATCC) by ciprofloxacin.

Conclusion
Based on the results of this study, copaiba oil exerted antimicrobial activity against E. faecalis and P. aeruginosa; however, the combination of hydrocortisone + neomycin sulfate + polymyxin B sulfate was the best antimicrobial treatment.Our study also showed that E. coli is the most resistant to the various substances tested.
obtained.The copaiba oil fractions w ere prepared and donated by the Laboratory of Natural Products ICB (Institute of Chemistry and Biotechnology -UFAL).Due to the difficulty in the synthesis of the products, the fractions w ere acquired in 2007, 2008 and 2009.These fractions w ere denominate as samples I, II and III, used in their pure form.Microorganism s E. faecalis (ATCC 4083) and P. aeruginosa (ATCC 9027) from the National Institute of Quality Control and Health -INCQS Osw aldo Cruz Foundation -FIOCRUZ -RJ, and also clinical isolates of Pseudomonas aeruginosa (204 C) and Escherichia coli (73 C) obtained from the Tropical Disease Hospital -HDT-AL w ere used.

Table 1 .
Substances used and their respective trademarks.

Table 2 .
Distribution of the halo of inhibition of antibacterial action (in mm) on the microorganisms tested (p <0.01).