Plant collection and extraction
P. ruderale leaves were collected, early in the morning period in the month of in September (2011), in the Medicinal Plant Garden of Hermínio Ometto University Center, UNIARARAS, Araras, São Paulo, Brazil. A voucher specimen was deposited at the herbarium of the Luiz de Queiroz College of Agriculture (Escola Superior de Agricultura Luiz de Queiroz—ESALQ-USP) (no ESA115686).
After collection, the leaves (100 g) were selected and properly cleaned under running water to remove impurities. The hydroalcoholic extract of P. ruderale was prepared by maceration of the leaves in a hydroalcoholic solution of 70 % (v/v solution of ethanol in water) for 7 days at room temperature, followed by vacuum filtration using a qualitative paper filter and evaporation in a rotary evaporator (Fisatom, model 803) at 40 °C. The evaporation period was 1 h. The resulting samples were subjected to lyophilization [14] and the yield of the lyophilized extract was from 10 %, ie, after all proceedings of extraction, the 100 g of leaves yield 10 g of the lyophilized. Obtaining this extract was based on popular culture [15].
Phytochemical screening method
The qualitative identification of chemical constituents was carried out in the same extract as that used in the wound repair test using chemical methods [14] and thin-layer chromatography [16]. The chemical groups analysed were polyphenolic components, flavonoids, tannins, alkaloids, saponins, fatty acid, triterpenes, volatile oils, coumarins and anthraquinones.
The presence of polyphenol compounds was analyzed with a solution of 1 % ferric chloride. Tannins have been identified using the dried extract dissolved in water, 2 ml of sodium chloride (2 %); filtered and mixed with 5 ml of 1 % gelatin. The presence of flavonoids was determined using aluminum chloride solution 1 % in methanol, concentrated hydrochloric acid, magnesium and potassium hydroxide. Dragendorff reagent was used to evaluate the presence of total alkaloids. Saponins were analyzed based on its ability to produce foam. For detection of triterpenes extract was mixed with 5 ml of chloroform was heated to 80 °C for 30 min and then treated with a small volume of concentrated sulfuric acid. Moreover, the extract was analyzed by thin layer chromatography on silica gel using chloroform: methanol (98:2) and hexane:ethyl acetate (80:20) as eluent. The components were first visualized under UV light and then by spraying the chromatographic plates and each containing different specific solutions, followed by incubation at 100 °C for 5 min.
The method of Chandra and Mejia Gonzalez [17] was used for quantitative analysis of total polyphenols. The hydroalcoholic extract (0.1 ml) was added to 1 ml Folin-Ciocalteau reagent and the mixture was left to stand for 5 min. Next, 2 ml 20 % sodium carbonate was added. After incubation for 10 min at room temperature, absorbance of the mixture was read in a spectrophotometer at 730 nm. The amount of total polyphenols is expressed as catechin equivalents.
For the analysis of flavonoids, the hydroalcoholic extract was incubated with 10 % aluminum chloride, 95 % ethanol, 1 mol/l sodium acetate, and distilled water for 30 min at room temperature. Absorbance was read in a spectrophotometer at 425 nm and samples were analyzed in triplicate. The amount of flavonols and flavones is expressed as quercetin equivalents [18].
Animals
Seventy-two (Rattus norvegicus albinus) male Wistar rats aged approximately 120 days and weighing on average 250 g were obtained from the “Prof. Dr. Luiz Edmundo de Magalhães” Animal Experimentation Center, of Hermínio Ometto University Center, UNIARARAS. The animals were housed in individual polycarbonate cages at a constant temperature (23 ± 2 °C) and humidity (55 %) under a 12-h light/dark cycle, with free access to standard chow and drinking water. All surgical and experimental procedures were approved by the Ethics Committee of Hermínio Ometto University Center, UNIARARAS (Protocol number 092/2011) and were conducted in accordance with the ethical guidelines of the Brazilian College of Animal Experimentation (COBEA) and of Guide for the Care and Use of Laboratory Animals (NIH).
Experimental design and burn wound creation
The animals were divided randomly into four groups of 18 animals each: group C, untreated control; group L, treated with 670-nm InGaP laser; group P, treated with the hydroalcoholic P. ruderale extract; group PL, treated with the hydroalcoholic P. ruderale extract and 670-nm InGaP laser (extract applied prior to laser therapy).
For experimental wounding, the back of the animals was shaved 48 h before the procedure under general anesthesia induced by the intraperitoneal administration of ketamine (1.0 ml/kg) and xylazine (0.2 mg/kg). Skin burns were produced on the backs of all animals, after the same anesthetic procedure, by applying an aluminum plate measuring 2 cm in diameter adapted to an apparatus that maintained a constant temperature of 120 °C. The plate was pressed on the animal’s skin for 20 s for the creation of a second-degree burn [19]. After induction of burns, the animals received pain killers: sodium dipyrone, one drop in the postoperative, after 12 and 24 h and they were placed in individual cages. Besides the treatments for repair of burns was not carried out any specific care except individualize the animals throughout the experimental procedure. The treatments were started 24 h after injury and were applied daily at the same time for 21 days. The animals were immobilized without sedation for treatment. The control (untreated) animals were subjected to sham treatments to control for effects of immobilization without treatment.
For laser therapy, an InGaP laser (Phisiolux Dual Bioset®, Indústria de Tecnologia Eletrônica Ltda., Rio Claro, São Paulo, Brazil) operating at a wavelength of 670 nm (visible red) was applied in the continuous mode using the following parameters: power output of 30 mW, energy density of 4.93 J/cm2 and total energy dose of 0.36 J, with the laser beam covering an area of 0.073 cm2. Punctual irradiation was performed by non-contact energy delivery at a distance of ± 2 mm and an angle of 90° in relation to the wound surface. The time of application (12 s) was determined by the specification of the equipment and the laser was applied to four points within the burn area. The apparatus was calibrated by the manufacturer.
The hydroalcoholic extract of P. ruderale (1.0 ml/day) was applied with a Pasteur pipette to the borders of the wound.
Structural and morphometric analyses
Wound samples were collected 7, 14 and 21 days after injury from three animals per group euthanized with an overdose of the anesthetic. An area measuring 25 mm in diameter was delimited in the center of the wound to obtain standardized samples for structural and morphometric analysis.
The tissue fragments were immersed in fixative solution containing 10 % formaldehyde in Millonig buffer, pH 7.4, for 24 h at room temperature. Next, the specimens were washed in buffer and submitted to routine procedures for embedding in Paraplast™ (Histosec®, Merck, Darmstadt, Germany). The blocks were cut into 6-μm longitudinal sections.
Longitudinal sections stained with Toluidine Blue and by the Domini method [20] were used to determine the number of fibroblasts and granulocytes and newly formed vessels in the repair area of the groups studied (n/104 μm2). Three samplings were performed from each of the five sections obtained from whole sections of the middle part of each surgical sample. The first 16 sections of the mid-section of the surgical specimen collected from each animal per group were collected. The sections were mounted on eight slides and were chosen randomly for the methods described above. After staining, three samplings of 104 μm2 (100 × 100 μm grid) were performed per section obtained in each test. Each sample was photomicrographed and digitized bright-field images were obtained with a Leica DM2000 photomicroscope at the Laboratory of Micromorphology, Hermínio Ometto University Center, UNIARARAS. Masson’s trichrome staining was used to quantify collagen fiber content in the repair area (percentage of total area). The fields were separated using the blue color distribution as a discrimination parameter. The intensity of blue represents the collagen density. The color band was adjusted by trial and error until representative areas of collagen had been separated in the image. The same parameter was then used to identify collagen fibers in all digitized fields. Next, the area occupied was calculated for each field [21].
Samples measuring 104 μm2 (100 × 100 μm grid) obtained from the area of the wound healing on days 7, 14 and 21 of treatment were examined using the Leica Image Measure™ visual grid for morphometric analysis of the following parameters: total number of fibroblasts, granulocytes, newly formed blood vessels (n/104 μm2), and collagen fiber content (% area). The Sigma Scan Pro 6.0™ program was used to evaluate the deposition of granulation tissue in the repair area. The results were entered into Excel for Windows XP™ spreadsheets and compared by ANOVA and the Tukey post-test (p < 0.05) [22].
Western blotting analysis
For analysis of protein expression by Western blotting, wound samples were collected from three animals per group after euthanasia with an overdose of the anesthetic, on days 7, 14 and 21 of treatment. For protein extraction, the samples obtained were chopped and homogenized in a Polytron homogenizer (PTA 20S model PT 10/35; Brinkmann Instruments, Westbury, NY, USA) operated at maximum speed for 40 s in buffer (10 mM EDTA, 100 mM Trizma base, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 100 mM sodium orthovanadate, 2 mM PMSF, 0.1 mg/ml aprotinin, and deionized water; Sigma Chemical Co., USA). The extract was centrifuged at 12,000 rpm for 20 min at 4 °C for removal of insoluble material. The supernatant was collected for the measurement of protein concentration in the samples by the Biuret method (Protal colorimetric method, Laborlab, São Paulo, Brazil). Aliquots of the supernatant were treated with Laemmli buffer containing 100 mM DTT (Sigma Chemical Co., St. Louis, MO, USA). Samples containing 50 μg protein were boiled for 5 min and submitted to 10 % (VEGF, 40 kDa) and 12 % (TGF-β1, 25 kDa) SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) in a mini-gel apparatus (Mini-Protean®, Bio-Rad-Richmond, CA, USA). Next, the protein bands were transferred from the gel to a nitrocellulose membrane (Hybond ECL, 0.45 μm) [23]. The membranes were washed in basal solution (1 M Trizma base, 5 M NaCl, Tween 20 a 0,005 %, and deionized water) and incubated in blocking solution (basal solution plus 5 % Molico® skim milk) for 2 h to reduce nonspecific protein binding. After washing with basal solution, the membranes were incubated overnight at 4 °C with specific antibodies (diluted 1:200) Anti-TGF-β1 (TB21, Santa Cruz Biotechnology, USA) and Anti-VEGF (VG-1, Santa Cruz Biotechnology, USA). Next, the membranes were incubated with the secondary goat anti-mouse IgG1:HRP antibody (diluted 1:1000, Santa Cruz Biotechnology, USA) for 2 h at room temperature. The reaction was developed using a chemoluminescence kit (SuperSignal® West Pico Chemiluminescent Substrate 34080, Thermoscientific, Rockford, USA). The membranes were exposed in Syngene G: Box and the bands intensity were evaluated by densitometry using the Scion Image 4.0.3.2 software (Scion Co., USA). The densitometric values of VEGF and TGF signals are expressed relative to proteins stained with Ponceau S, which were taken as 100 %. The results were analysed by ANOVA and the Tukey post-test (p < 0.05) 0.29 using the GraphPad Prism® 3.0 program.
