Powdered bark of Endopleura uchi (Huber) (Humiriaceae) was acquired from the Sítio da Mata company (lot: Uxi03/01; N°Inscr. Prod.: 024.308.176; crop: 01/09/2005; expiry: 30/09/2008), located on the Cajuru highway, Cassia dos Coqueiros (SP), Brazil.

Plant extracts were prepared in the concentrations of 5, 10 and 20% (w/v), by various procedures: (1) maceration (M), (2) turbo-extraction (T), (3) percolation (P), (4) infusion (I), (5) decoction (D). In the first three procedures, 50% ethanol (EtOH 50%) was used as solvent and in the last two, distilled water. The extracts were dried and concentrated under reduced pressure in rotaevaporator at 40 °C, the yields obtained (w/v) were: 4.56–11.54% to 5% extracts; 5.59–14.17% to 10% extracts and 4.37–23.87% to 20% extracts. The extracts were preserved in a desiccator to avoid humidity incorporation.

The total tannin content was estimated by a colorimetric assay based on procedures described by Glasl (1983) [31] and Farmacopéia Brasileira IV (1996) [32], with slight modifications. To determine the total tannins of a plant, it is recommended to use a sample of 0.750 g [32]. However, for the extracts, it was necessary to make a small correction [23]. Thus, the amount of plant sample weighed for each extract was calculated as: M_extract = (%) extractive content of extract (EC) × 0.750/100, where EC is the percent of dry residue weight extracted from the sample and M_extract is the mass of extracts.

Briefly, the samples were dissolved in 250 mL water to give the mother liquor (ML). A 5 mL aliquot of ML was diluted in water to 25 mL and 2 mL of this solution, were transferred to a 25 mL vial with Folin-Ciocalteau phenol reagent 2 N (1 mL) and Milli-Q^® water (10 mL) and made up to volume with a 10.6% sodium carbonate solution. After 15 minutes, the absorbance was read at 730 nm. Water was used as the blank. To determine the non-adsorbent polyphenols (NAP), 10 mL ML was mixed with hide powder (Merck^®) (100 mg) and shacked for 60 minutes. A 2 mL aliquot of this solution was assayed for polyphenolics as above. The absorbance wavelength (730 nm) was previously selected by spectrophotometric scanning of samples of extract and gallic acid. The percentage of total phenolics and tannins were determined as follows: TP (%) = [15625 × Abs]/[1000 × m], NAP (%) = [15625 × Abs]/[1000 × m], TT (%) = TP − NAP, where TP = total polyphenolics (%); NAP = non-adsorvent polyphenolics (%); Abs = Absorbance; m = mass (g) of samples; TT = total tannins (%).

The agar disk diffusion technique (both with “templates” and “paper discs”) was employed to test for antimicrobial activity against strains of Bacillus subtilis (ATCC 9372), Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 27853), Escherichia coli (ATCC 25922), Shigella sonnei (clinical isolate) and Candida albicans (ATCC 64548). A colony of each bacterial strain used, or 150 μL of a previously prepared bacterial suspension was inoculated in Brain Heart Infusion (BHI) broth and incubated at 37 °C for 24 hours. The turbidity of each bacterial cell suspension was then adjusted with saline to match the 0.5 McFarland scale (∼1.5 × 10⁸ CFU/mL). Five colonies of Candida albicans were inoculated in Sabouraud broth and incubated at 35 °C for 24–48 hours. The turbidity of the yeast cell suspension was then adjusted to 0.5 on the McFarland scale (1× 10⁶–5 × 10⁶ CFU/mL) with sterile saline. Ampicillin (50 μg/mL) was used as a positive control for the bacterial strains and amphotericin B for the Candida albicans strain. The later was prepared by dissolving 16 mg of amphotericin B in 10 mL of dimethylsulfoxide (DMSO) and diluting this, twice, in the proportion 1:5 (w/v), obtaining the stock solution 64 μg/mL.

The agar diffusion tests were performed as in the approved standards M2-A8 and M44-A of the Clinical and Laboratory Standards Institute, with modifications [33,34]. Bacterial and yeast inoculums were prepared as described previously adjusting the turbidity to the McFarland scale. Müller-Hinton agar (MHA) for bacteria, or MHA supplemented with 2% glucose and 0.5 μg/mL methylene blue for yeast, were poured into sterilized Petri dishes, having been seeded with previously prepared inocula (150 μL of bacterial suspension or 100 μL of yeast suspension). Disk diffusion templates (6 wells of 6 mm internal diameter) or paper discs (6 mm diameter) were placed on the seeded plates. To each well, 50 μL of ampicillin/amphotericin B solution, 50 μL of each E. uchi extract (10 mg/mL, resuspended in DMSO) and 50 μL of DMSO:BHI solution (1:1 v/v) as a negative control were added. Sterilized filter paper discs (Whatman^® grade No. 1) were individually impregnated with 40 μL of each 10 mg/mL extracts solution (resuspended in DMSO), 40 μL of DMSO:BHI (1:1, v/v) as negative control and 40 μL of ampicillin or amphotericin B as positive controls. The plates were incubated at 37 °C for 24–48 hours. The inhibition of the bacterial and/or fungal growth was determined by measuring the haloes around of the wells and discs with the aid of a digital calliper, and expressed as the average of three independent experimental determinations.

The minimum inhibitory concentration (MIC) of the extracts that presented some activity against the tested microbial strains was determined by the broth micro-dilution method, as described in the M7-A6 reference guideline of the Clinical and Laboratory Standards Institute, with modifications [35].

The test was carried out in BHI broth for bacterial strains or RPMI-1640 (adjusted to pH 7.0 with 3-(N-morpholino) propanesulfonic acid (MOPS) buffer, 0.165 mol/L) for the yeast in 96-well flat-bottomed microtitration plates, containing 0.1 mL medium in each well. The extracts were prepared in DMSO at an initial concentration of 200 mg/mL and diluted in DMSO:BHI (1:5, v/v) to obtain 10 mg/mL in test solutions. Sample solutions (0.1 mL) were two-fold serially diluted in the plates with liquid medium. The working inoculum suspension (0.1 mL) was added to give a final inoculum concentration of 1 × 10⁵–5 × 10⁵ and 0.55 × 10³–2.5 × 10³ CFU/mL, for bacteria and yeast assays, respectively. Ampicillin final dilutions ranging from 12.5 μg/mL to 0.012 μg/mL and amphotericin B final dilutions ranging from 16 μg/mL to 0.015 μg/mL were used for bacteria and yeast, respectively, as positive control. Negative contamination controls using only medium (BHI or RPMI), and with or without extract were used in the tests. The plates were incubated at 37 °C for 24 and 48 hours for bacteria and yeast, respectively. No inhibitory effects were observed in the presence of DMSO at the highest concentration used. To perform the visual reading, 20 μL of 0.01% (w/v) resazurin (Sigma^®) was added to each well. The plates were incubated for a further 30 minutes, and estimated visually for any change in color from blue to pink, indicating reduction of the dye due to microbial growth. The lowest concentration that remained blue was taken as the MIC. Experiments were performed in duplicate.

Cytotoxicity was tested on rabbit corneal fibroblasts (SIRC, CCL-60). These cells were maintained in culture bottles incubated at 37 °C with 5% CO₂ in Eagle medium (pH 7) supplemented with 15% fetal bovine serum, without sodium bicarbonate. The extract samples were prepared by dissolving 10 mg of 20% decoction, 10% infusion, 5% maceration (EtOH 50%), 10% percolation (EtOH 50%) and 20% turboextraction (EtOH 50%) in 1 mL of DMSO. These initial stock solutions were diluted (1:5, v/v) in Eagle medium, to obtain the test solutions.

For cytotoxicity evaluation, the cells were collected, centrifuged (1500 rpm, 10 minutes) counted and adjusted to the concentration 1 × 10⁵ cells/mL in Eagle medium. The cells were incubated in 96-well flat-bottomed microtitration plates at 37 °C in an atmosphere of 5% CO₂ for 72 h. E. uchi samples, diluted in DMSO:Eagle (1:10, v/v), were added. The extracts were serially diluted (1:1, v/v) across the plate resulting in an initial concentration of 200 μg/mL to a final concentration of 39 μg/mL. The plate was incubated for 24 hours at 37 °C in a humid atmosphere with 5% CO₂ [36–38]. After this, 15 μL of resazurin aqueous solution (0.1 mg/mL) was added. The plate was incubated in a humid atmosphere with 5% CO₂ at 37 °C. The wells were read visually after 3 hours by distinguishing the original blue color (absence of living cells) from pink (presence of living cells) and with a microplate fluorescence reader (Spectra Fluor Plus, Analysis program Magellin), with filters of 530 and 590 nm [39].

The assay of antioxidant activity of the extracts was based on the scavenging activity of 2.2-diphenyl-1-picrylhydrazyl solution (DPPH) [40]. Gallic acid, rutin and vitamin C were used at a concentration of 250 μg/mL (in methanol), as antioxidants [41]. A Ginkgo biloba L. extract (Santosflora^®) was also used in this test, at the concentration of 250 μg/mL (in methanol), for its recognized antioxidant activity [42]. The tests were performed on the extracts: 20% decoction; 10% infusion; 5% maceration (EtOH 50%); 10% percolation (EtOH 50%) and 20% turboextraction (EtOH 50%). To 1 mL of the sample solutions, 2.5 mL of 0.004% DPPH in methanol solution were added. The resulting solutions were homogenized by vortex and kept in the dark for 30 minutes at room temperature. A solution of DPPH in methanol (2.5:1, v/v) was used as negative control, and methanol was used as blank. Absorbance was measured at 517 nm in a Shimadzu-1603 spectrophotometer, with quartz cuvettes of 1 cm light pathway. The anti-radical activity was calculated by the equation: percent radical scavenging activity = (Abs_control − Abs_sample)/Abs_control × 100, where Abs = absorbance.

The results were expressed as the average of three determinations ± standard deviation, using Microsoft Office Excel 2007^®. When necessary, analysis of variance (ANOVA) was performed, with P < 0.05. The Spearman and Kendall-Tau tests (α = 0.05) were performed with Stat Plus Professional 2009^® to verify correlation coefficient among total tannins content, total phenols content and antioxidant activity.