The qualitative phytochemical analysis of the B. trimera aqueous extract was performed to evaluate the presence of chemical compounds. The antioxidant capacity was also evaluated. After that, the anti-inflammatory effect of the aqueous extract was evaluated in vivo in the acute inflammatory pain model (Formalin test), in order to proceed to the next evaluation of the chronic inflammatory model (CFA into the paw).
Collection of the plant material
The aerial parts of B. trimera were collected in July 2013 in the city of Bagé, Rio Grande do Sul, Brazil. The samples collected were identified and a specimen voucher (Naresuan University of the Campanha Region, under number 00014) was deposited at the Nicanor Risch Herbarium in the University of the Campanha Region (URCAMP). The access to Brazilian biodiversity was registered in the National System for Management of the Genetic Heritage and the Associated Traditional Knowledge (SISGEN), under protocol A02A526.
Sample preparation
The aerial parts were dried at room temperature and protected against the sunlight for 7 days. Afterward, the dried plant (250 g) was finely ground and the aqueous extracts were prepared by infusion at 80 °C (1/10 plant/water solvent). The infusion was kept at room temperature for 30 min. After cooling and filtration, the extract was frozen and then concentrated by lyophilization for 5 days overnight, in order to obtain the aqueous extract of B. trimera (29.25 g, yield: 11.7%, w/w). This extract was kept in a vacuum desiccator to avoid changes in its chemical profile.
Chemicals
The solvents that were used for the HLPC analyses, together with the other necessary chemicals, were purchased from Merck (Darmstadt, Germany). These were Quercetin (QE) (Sigma, St. Louis, MO, USA); Gallic Acid (GA) (Sigma, St. Louis, MO, USA); 3-(2,3-dihydroxyphenyl) propionate (DPPH) (Sigma, St. Louis, MO, USA); Freund’s Complete Adjuvant (FCA) (Sigma Chemical Co., St. Louis, MO, USA); Formaldehyde (Vetec, Rio de Janeiro, Brazil); and Diclofenac Sodium (Medley, São Paulo, Brazil).
Phytochemical screening
The crude B. trimera aqueous extract was submitted to qualitative phytochemical screening, as described by Harborne (1998) [18]. The method consists of colorimetric reactions for the qualitative detection of flavonoids, tannins, anthraquinones, alkaloids, saponins, coumarins, and cardiac glycosides. The thin-layer chromatography analyses were performed according to Wagner and Bladt (1996) [19].
High-performance liquid chromatography (HPLC) analyses
The HPLC analyses were performed on a Waters Alliance 2695 Separation Module (Milford, Massachusetts, USA), with a double wavelength UV detector (Waters 2487) that was controlled by an interface module (IEEE-488). For the stationary phase, a reverse phase (250 × 4.6 mm - 5 one particle diameter Waters Spherisorb ODS2 HPLC Column was used. Reference standards were used as the external standards. A constant flow of 1 mL.min− 1 was used during the analyses. HPLC-grade solvents and Milli-Q® water were used in the chromatographic studies. The mobile phase was prepared daily and degassed by sonication before use. The phenolic acids and flavonoids were quantitatively determined at 254 nm when using pure acetonitrile (A) and 0.1% phosphoric acid (H3PO4) (B) as the mobile phase. The gradient system was adjusted to 0/95, 20/86, 70/65 (min /% B). The correlation of the chromatographic peaks was obtained by comparing the experimental retention times with the reference standards and by the co-injection of the sample and the authentic samples. The standard solutions were prepared in different concentrations. The quantitative analyses of the phenolic compounds were performed by constructing five-point calibration curves for each standard solution, which showed the linearity of the detector response in the range of 8.0 μg.min− 1 to 225 μg.min− 1. The correlation coefficient value was greater than 0.9985.
The LOD detection (≤ 0.64 μg.min− 1) and the quantification limits (LOQ ≤ 2.15 μg.min− 1) were calculated by using the parameters of the calibration curves, being defined as 3.3 and 10 times the value of the regression error, divided by the slope, respectively. The LOD and LOQ values were always lower than the lowest tested standard concentration in the dynamic range of the calibration curves, indicating a satisfactory sensitivity for each phenolic standard. All of the chromatographic operations were performed in triplicate at room temperature [20].
Total phenolic compound content
The content of the total phenolic compounds in B. trimera was determined by the Folin-Ciocalteu method. For the calibration curves, aliquots of 1 mL of gallic ethanolic acid solutions at concentrations of 0.015, 0.024, 0.075, and 0.105 mg.min− 1 were mixed with 5 mL of Folin-Ciocalteu reagent (diluted ten times) and 4 mL (75 g.L− 1) of sodium carbonate. The absorption was detected after 30 min at 765 nm and the calibration curve was drawn [21]. One (1) mL of a solution of B. trimera (0.1 mg.mL− 1) was mixed with the same reagents as described above, and after 1 h, the absorption was measured to determine the total amount of the phenols. All of the determinations were performed in triplicate. The total phenol content was expressed as a gallic acid (GAE) equivalent in mg.g− 1 of extract.
Total flavonoid content
The total flavonoid content of the samples was determined by a colorimetric method, as previously described using aluminum chloride [22]. Solutions containing different concentrations of quercetin were prepared to establish a calibration curve (2–12 μg.mL− 1). After 30 min, the absorption at 425 nm was measured for each solution in a Shimadzu Spectrophotometer (UV-1602PC, Kyoto, Japan). The analyses were performed in triplicate, and the total flavonoid content in the samples was expressed as quercetin equivalents (QE) in mg.g− 1 extract.
Antioxidant DPPH assay
The ability of the crude aqueous extract of B. trimera to extinguish stable DPPH (2,2-Diphenyl-1-picrylhydrazyl) was measured according to Mensor et al., (2001) [23]. Ten milligrams of B. trimera extract were weighed and diluted in methanol to obtain six distinct concentrations (50, 100. 150, 200, 250, and 300 μg/mL). Afterward, 2.5 mL of the sample was transferred to a 3.5 mL cuvette and 1 mL of the DPPH solution (0.2 mg/mL) was added. Methanol (blank), DPPH/methanol (positive control), and quercetin (standard) were used to evaluate the antioxidant activity. After 30 min, the absorbance was measured at 518 nm when using a Shimadzu Spectrophotometer (UV-1602PC, Kyoto, Japan). The measurements were performed in triplicate, and the antioxidant activity was calculated according to the formula:
$$ \%\mathrm{DPPH}\kern0.34em \mathrm{inhibition}=\left[\left(\mathrm{Abscontrol}\kern2.77695pt \left(+\right)-\mathrm{Absample}\right)\times 100\right]/\mathrm{Abscontrol}\kern2.77695pt \left(+\right).< mathdollar> $$
Animals
105 Swiss male mice (35–40 g) were obtained from the Vivarium of the Lutheran University of Brazil (ULBRA). The animal care and experimental procedures were strictly conducted in accordance with the ARRIVE Guidelines [24]. All of the experiments were performed under the Consent and Surveillance of the Ethics Committee for Using Animals from ULBRA under Protocol n° 2013-20P. Five mice per box were maintained with food and water ad libitum in a temperature-controlled room (24 ± 2 °C), while under a 12-h light/dark cycle.
Treatments
In order to analyze the anti-inflammatory and antinociceptive effects of the aqueous extract of B. trimera, the mice were treated with doses of 20, 40, and 80 mg.kg− 1 aqueous extract, diclofenac sodium (5 mg.kg− 1, a nonsteroidal anti-inflammatory drug used to treat pain and inflammatory diseases), and water (10 mg.kg− 1) by the gavage route (p.o.).
Formalin test
The formalin test was performed according to Hunskaar and Hole (1987) [25]. To evaluate whether the B. trimera extract (20, 40, and 80 mg.kg− 1) prevented the presence of nociception, the mice received a pre-treatment with the compound orally, one hour before the intraplantar administration of 1.5% formalin, in the right hind leg (20 μL s.c.). The licking of the paw time, or its removal from the floor, was recorded as an indication of nociception. The animals were observed for 30 min, divided into two phases. The first phase was evaluated at 0–5 min after the formalin injection (neurogenic phase), and the second phase was evaluated at 15–30 min (inflammatory phase).
Chronic inflammatory model induced by Freund’s complete adjuvant (FCA)
The chronic inflammation model was induced by then intraplantar injection of FCA (20 μL) into the right-hind paw of the mice [26]. To investigate the actions of the extract of B. trimera, the mice received a daily dose of 80 mg.kg− 1, p.o., for five consecutive days, starting from the first day of the FCA injection. The effect of edema on the paw that was produced by FCA was evaluated 48 h later, and then again on the last day of treatment. Paw edema was expressed by comparing the paw thickness (mm) to the baseline measurement.
Thermal hyperalgesia
The exposure of the peripheral sensory nerve endings to elevated temperatures can evoke sensations of warmth, heat, or pain [27]. After the chronic inflammation induction by the intraplantar injection of FCA (20 μL) into the right-hind paw of the mice, one hour after the treatment (water, extract, diclofenac), the mice were placed on a hot metal plate (52 ± 0.5 °C) and the behavior was evaluated. The time of each animal when they licked or elevated their paw was recorded. A cut-off time of 30 s was established to avoid pain or damage to the animal [28].
Mechanical allodynia
Mechanical allodynia was evaluated when using Von Frey’s filaments (0.02–10 g), as described by Dixon (1980) [29], at three different moments. Firstly, one hour before the injection of FCA into the mice paw. Secondly, 48 h after the first application. Thirdly, on the last day of the treatments.
The animals were acclimatized for 1 h in individual acrylic compartments with a mesh floor. Von Frey filaments were perpendicularly directed to the right-hind paw plantar surface of the animals, with enough high pressure to bend the filament. Starting with an application of the 0.4 g filament, if the tension-response was harmful (paw withdrawal), a filament with less value was used. However, if the response was innocuous, the next filament with a higher force was tested. At least six responses around the estimated threshold were required for the optimal calculation of the 50% withdrawal threshold (PWT).
Myeloperoxidase measurement (MPO)
To investigate the involvement of neutrophils in the inflammatory response that was induced by FCA, the researchers measured the MPO activity. After the last behavioral test, the mice were euthanized by an isoflurane overdose, followed by cervical dislocation. The plantar surface of the rear paw was collected to determine the activity of the MPO as described by Suzuki et al., 1983 [30]. The values were expressed as optical densities, corrected by a gram of homogenized tissue (OD/g tissue).
White blood cell count
The white blood cell count test was performed as described by Bentley et al., 1980 [31]. Briefly, 50 μL of peripheral blood was placed on the slide and with the help of a distending slide, the smear was performed. After drying, the slides were stained with methylene blue eosinate, while going through three processes: fixation, staining, and washing. After drying again, these slides were visualized and analyzed under a microscope at 10 x and 40 x magnification. A differential count of the blood cells was then performed, with an emphasis on the segmented cells that were present with coverage in the inflammatory process.
Statistical analysis
The data is expressed as mean ± SEM. The statistical analyses were carried out by One-Way or Two-Way Analysis of Variance (ANOVA), followed by the Student-Newman-Keuls (SNK) test, or the Bonferroni test (GraphPad Prism 5.0, USA). In all of the comparisons, values of p < 0.05 were considered statistically significant.