Collection and identification of plant material
Samples of the bark of P. major were collected from an adult plant in the rural region of the municipality of Campo Largo, Paraná, Brazil. It was not necessary permission to carry out sampling. For the identification of the plant material, we created herbarium specimens and deposited them in the Herbarium of the Positivo University (Registry Number 100).
The steps for preparation and fractionation of extracts are presented in Fig. 1.
The plant material was dried in a forced air oven at 40 °C until the weight stabilized. Then, it was crushed to a powder, and thus, we obtained the pulverized drug for preparation of the crude ethanolic extract.
Obtaining plant extracts
Plant extracts were obtained according to Younes et al. [24]. We blended the material by using an industrial turbine (Rodrimar, São Paulo, SP, Brazil). We used 187.11 g of the dried and pulverized bark and 2000 mL of 70% ethanol (Rioquímica, São José do Rio Preto, SP, Brazil). This material was extracted for 20 min and the liquid obtained was filtered in vacuum, resulting in the crude ethanolic extract (approximately 2050 mL).
The concentration of the extract was performed in a rotary evaporator (Quimis, Diadema, SP, Brazil). Each cycle lasted for approximately 30 min, and the rotation speed was adjusted to six to obtain the CCE.
Fractionation of the CCE (liquid–liquid partition)
Liquid–liquid partition [25] was performed using the CCE and 2000 mL of ethyl acetate (Vetec, Duque de Caxias, RJ, Brazil). We placed 100 mL of the CCE in a separation funnel, and then, an equivalent volume of ethyl acetate (100 mL) was poured on it with gentle agitation. This step was repeated nine times, until the solvent was clear. Thus, we obtained the EAF, which was then concentrated in a rotary evaporator and stored in an amber flask.
Freezing and lyophilization
The extracts were frozen in autoclaved plastic containers and covered with a plastic wrap for 48 h. Microperforations were introduced in the wrap using a sterilized gingival needle to allow the sublimation of the liquid. Then, the samples were placed in a lyophilizer (Ilsinh Lab. Co., Ltd., Korea). Any residual liquid was removed, and the sample was frozen again until the sample dried completely. The lyophilization process was completed in 7 days.
Preparation of the drug for the analysis of antimicrobial activity
The samples were powdered using a glass rod. Visual inspection allowed the granulometric analysis of the preparations, which were weighed in mini Petri dishes in a precision balance. For preparing drug samples, 500 mg lyophilized powders from the CCE, EAF, and Ca(OH)2 P. A. (Biodinâmica, Ibiporã, PR, Brazil), and 0.1 mL of 2% CHX solution (chlorhexidine S 2%—FGM, Joinville, SC, Brazil), were used.
The propylene glycol was the vehicle for treatment of the powder samples and for experiments because of its harmless characteristics and the common use in endodontic treatments.
Two samples from P. major were very viscous, which hampered pipetting. Therefore, 0.05 mL of sterile saline solution were added to them, including the Ca(OH)2 paste. No vehicle was required for the 2% CHX solution.
Determination of the MIC by using the broth microdilution technique
We used the E. faecalis strain (ATCC 19433) and the Mueller–Hinton broth as the culture medium, with a pH between 7.2 and 7.4, at 25 °C. The bacterial inoculum was prepared according to standardized methods [26].
The bacterial strain was transferred from the maintenance medium into a tube containing the brain heart infusion (BHI) and kept at 35 °C for 18 h for strain activation. To isolate young colonies, we transferred aliquots into a Petri dish containing Muller-Hinton agar, and incubated it at 35 °C for 24 h. Then, 4–5 colonies were transferred into a tube containing 5 mL of sterile saline solution (0.85%) followed by homogenization in a vortex mixer for 15 s.
The turbidity of the cell suspension was adjusted to obtain an optical density similar to the 0.5 McFarland standard, which corresponds to a suspension containing approximately 1 to 2 × 108 cells mL−1. Subsequently, this solution was diluted (1:10) to obtain an inoculum containing 107 cells mL−1.
The antimicrobial activity was examined using the broth microdilution technique [26] with modifications. The method comprised preparation of successive dilutions of each treatment to be assessed—CCE, EAF, Ca(OH)2 solution, Ca(OH)2 solution with CCE, and 2% CHX—in a liquid culture medium, inoculation of the bacteria, and after incubation, interpretation of the result for determining the MIC.
The treatments were placed in 96 well microplates (TPP Cultilab; Campinas, SP, Brazil). The treatments were diluted to obtain a final concentration in each well from 166.50 to 2.6 μg mL−1 for the solutions, and from 1 to 0.01% for the CHX. Then, 100 μL (0.1 mL) of the inoculum suspension to the wells was added. The columns were filled with suspensions as follows: microdilutions of the treatments, culture media control (negative control), controls for the treatments, and control for E. faecalis viability (positive control). Five test wells were repeated in each group.
The plates were incubated at 35 °C for 24 h and were read visually to determine the MIC. When bacterial growth was confirmed, suspensions from each well were transferred into Petri dishes identified and containing nutrient agar. After incubation at 35 °C for 48 h, the presence or absence of bacterial growth was evaluated. The MIC was regarded as the lowest concentration in the series of dilutions, which did not permit the growth of the susceptible bacteria [9]. So, to determine the MIC, we compared the bacterial growth in each well with that in the positive control and that observed in the nutrient agar dishes.
The concentration of the powder products tested was determined by calculating the concentration of the stock standard solution, by using the following formula: amount of product (mg)/volume of the vehicle (mL); thus, 550 mg/1.5 mL ≈ 333 mg mL−1.
Calculation of constant concentrations and volumes was used to determine the initial concentration of the products tested (concentration of the stock standard solution). To calculate the concentration of each dilution, we used the equation of constant concentration and volume (C1 × V1 = C2 × V2). We used the same volume in all wells, and thus, the dilution in each subsequent well was half of that in the previous well; we successively filled all wells in this manner (with the lower concentration tested).