Plant material and extract preparation
The extract of S. adstringens was prepared according to the Brazilian Pharmacopoeia, 4th Edition. Briefly, 200 g of S. adstringens bark that was purchased from Florien e Cia Ltda, Brazil, was macerated for 7 days in 800 mL alcohol 70°GL. After that, this extract was filtered and concentrated to dryness, under reduced pressure and a temperature lower than 40 °C, yielding 45.8 g (18.3%, w/w). The extract was kept in vacuum desiccators to produce a completely dry solid mass.
Phytochemical analysis
The S. adstringens extract was subjected to qualitative chemical screening for the identification of the major classes of the active constituents. The phytochemical profile of the S. adstringens extract was determined through the method that consists of colorimetric reactions for the qualitative detection of flavonoids, tannins, anthraquinones, alkaloids, saponins, and coumarins [13, 14]. The thin-layer chromatography analysis was performed as described by Wagner and Bladt [15].
Total contents of Phenolics in the S. adstringens extract
The total contents of the phenolic compounds in the S. adstringens extract were determined by the Folin-Ciocalteu method. The ethanolic solutions of gallic acid were mixed with 5 mL of Folin-Ciocalteu reagent and 4 mL (75 g/L) of sodium carbonate. After 30 min, the absorption was read at 765 nm and the calibration curve was drawn [15]. Following this, 1.0 mL of the extract was mixed with the same reagents as described above, and after 1 h, the absorption was measured to determine the total phenol solution (0.1 mg/mL). The total phenol contents were expressed as gallic acid (Sigma, St. Louis, MO, USA) equivalents (GAE) in mg/g of extract [16]. All of the determinations were performed in triplicate.
Total contents of tannins in the S. adstringens extract
The total contents of tannins were determined by using the casein precipitation assay. The tannins were also analyzed according to the Folin-Ciocalteu method and the solution was shaken for 1 h. After that, the solution was filtered, and 1 mL of this solution was mixed with 5 mL Folin-Ciocalteu reagent and 4 mL sodium carbonate; the absorptions were measured after 1 h. The entire assay was performed in triplicate and the total contents of the tannins were expressed as gallic acid (Sigma, St. Louis, MO, USA) equivalents (GAE) in mg/g of extract.
Determination of the Total flavonoid contents in the S. adstringens extract
The total flavonoid contents of the S. adstringens extract were determined by a previously described colorimetric method when using aluminum chloride [17]. The solutions were prepared with varying concentrations of quercetin (Sigma, St. Louis, MO, USA), in order to establish the calibration curve (2–12 μg/mL).
After 30 min, the absorption was measured at 425 nm for each solution by a Shimadzu spectrophotometer (UV-1602PC, Kyoto, Japan). The analyses were carried out in triplicate and the total samples were expressed as quercetin equivalents (QE) in mg/g of extract.
HPLC analysis of the phenolic composition from the S. adstringens extract
The chromatographic analyses were carried out by a validated method by using the Reversed-phase HPLC (Waters 2695 Alliance Separation Module) technique. This was equipped with a Waters 2487 l Dual Wavelength UV Detector and controlled by an IEEE-488 interface module. As a stationary phase, a Waters Spherisorb ODS2 Reversed-Phase Column (250 × 4.6 μm - 5 mm diameter particles) was used. A constant flow-rate of 1 mL.min− 1 was used during the analyses. HPLC grade solvents and Milli-Q® water were used in the chromatographic studies. The phenolic acids and the flavonoids were quantitatively determined at 254 nm when using pure acetonitrile (A) and 0.1% phosphoric acid (H3PO4) (B) as the mobile phases. The gradient system was adjusted to 0/95, 20/86, and 70/65 (min/%B). Phenolic standards were used as an external standard. The analytical curves were constructed for all of the phenolic standards that were used, by plotting the peak area of the compound versus the concentration, while using five standard solution spots in the concentrations range. The analytical generated curve was obtained by linear regression (peak area versus the standard concentration). The correlation of the chromatographic peaks was achieved by comparing the experimental retention times to those used as an external standard. Their amounts were quantified (mg/mL) by a standard curve. The accuracy of the results was evaluated by the performance parameters (limit of detection (LOD), limit of quantification (LOQ), linear equation, and linear regression). All of the chromatographic operations were carried out in triplicate at room temperature.
Gel preparation
Three gels were prepared, using respectively, saline 0.9% (Hydrogel), saline 0.9% associated with 5% S. adstringens extract (H + SAHE), and Carbopol (negative control). The carbomer, 1% Carbopol 940, was utilized, in order to provide viscosity to the gels and its pH was adjusted to 7.0 with AMP 95 (Aminomethyl Propanol, Mapric Produtos Farmacocosméticos Ltda, São Paulo, Brazil).
Antioxidant activity by the DPPH assay
The ability of SAHE to extinguish the stable radical, di (phenyl) - (2,4,6-trinitrophenyl) iminoazanium (DPPH) (Sigma, St. Louis, MO, USA), was measured according to the method as described by Mensor et al., [18]. Methanol was used as a blank, DPPH/methanol was used as a negative control, and gallic acid was used as a positive control sample. 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:
$$ \%\mathrm{inhibition}\ \mathrm{of}\ \mathrm{DPPH}=\left[\left(\mathrm{Abscontrol}\left(+\right)-\mathrm{Abssample}\right)\ \mathrm{x}\ 100\right]/\mathrm{Abscontrol}\left(+\right). $$
Cell culture conditions and treatment
The Mus musculus fibroblasts L929 cell line (RJCB Collection, Rio de Janeiro, Brazil), was routinely maintained in medium DMEM (Invitrogen Corporation, Carlsbad, CA, USA) suplemented with 10% fetal bovine serum (v/v; Cultilab, Campinas, SP, Brazil) at 37 °C and with 5% CO2, and humidity of at least 95%.
A pilot study was conducted, in order to define the concentration to be tested in the in vitro assays. Concentrations of 0.5, 1.0, 4.0, and 8.0 mg/mL were tested. The 4.0 mg/mL concentration was the one chosen since it was the maximum dose that was capable of being dissolved in the culture medium.
Cytotoxicity and proliferation assays
The cytotoxicity was evaluated by using the colorimetric assay of MTT (3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide, Life Technologies™, Oregon, USA). The quadruplicate cultures were exposed to the gels for 24 h. After the treatments, the cells were incubated with 100 μL of MTT solution (0.1 mg/mL MTT) in the culture medium, without FCS, and without phenol. They were cultured at 37 °C for 4 h. After the incubation, the supernatant was carefully removed, and the purple formazan crystals were solubilized in 200 μL of DMSO (10%) [19]. The assay was performed by a microplate reader (Multiskan, Uniscience, Sao Paulo, SP, Brazil), with an optical density of 540 nm. The cytotoxic effects were determined based upon ISO10993-52009. A reduction of cell viability by over 30% was considered cytotoxic.
For the cell proliferation study, the L929 cells were seeded in 24-well flat-bottom plates at a density of 2 × 104 cells/well and they were stabilized for 24 h. Afterward, the cells were exposed to the gels for another 24 h. The evaluation of the proliferative effects was performed as described by Sigalas et al. [20]. The cells were counted in four random fields by using a Neubauer chamber. Finally, the average number of cells per plate-well was calculated.
Scratch wound assay
The L929 cell line were seeded in quadruplicate in 24-well plates at a density of 105 cells/well. The plates were maintained at 37 °C and with 5% CO2 in a moist atmosphere overnight, in order to obtain a confluent cell monolayer. The confluent monolayers were scratched with a pipette tip and the culture medium was immediately removed and was replaced by a non-supplemented fresh medium [21]. The cell migration was analyzed by photographs after 0 h and 24 h, following the creation of the lesions. The images were analyzed by using ImageJ software (version 1:48), and the results of the migration were expressed in the form of a percentage. Time 0 was considered as being equivalent to 100% of the width of the risk measure. The results were expressed in percentages of the wound closure.
Animals
Forty (40) male Wistar rats, with ages ranging between 60 and 80 days, at 200 – 250 g weight, were obtained from the Lutheran University of Brazil (ULBRA) vivarium. They were individually maintained in standard cages, with a 12-h light/dark cycle, and in a temperature-controlled room (23 ± 2 °C). Food and water were allowed ad libitum. All of the in vivo experiments were approved by the Ethics and Animal Experimentation Committee of the Center for the Lutheran University of Brazil (ULBRA) (CEUA), Protocol N° 2014-2P. This study followed the guidelines for animal research, in accordance with the Guide for Laboratory Animal Care in Research Experiments of the National Council for Animal Experimentation Control (CONCEA), in accordance with the ARRIVE Guidelines [22] and conducted in accordance with the internationally accepted principles for laboratory animal use and care as found in for example the European Community guidelines (EEC Directive of 1986; 86/609/EEC).
Diabetes mellitus model
The DM model was induced by a single intraperitoneal injection of 80 mg/kg of the diabetogenic drug streptozotocin (Sigma Chemical Company, St. Louis, MO, USA), which was dissolved in a 0.1 M citrate buffer (pH 4.5) [23]. Hyperglycemia was confirmed when the glucose levels exceeded 200 mg/dL. This was confirmed by an Accu-Check® (Roche Diagnostics) on day 3 after the inductions.
Wound creation and treatments
For the surgical procedures, the rats were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg) by an intraperitoneal route. The dorsum of the rats was previously shaved and was disinfected with 70% ethylic alcohol, in order to excise a full-thickness wound of 1.5 × 1.5 cm2, using a pair of sharp scissors and a scalpel. The non-diabetic rats and the diabetic rats were randomly divided into five groups (n = 08). The animals received treatments according to the following groups: Hydrogel; H + SAHE; SAHE; Collagenase with Chloramphenicol; and Carbopol. Each wound was treated twice a day, with a rich layer (± 2 g) of the corresponding treatment, for 16 days.
Morphological analysis of the injury
The wounds were photographed on the 4th, 8th, 12th, and 16th day, by using a digital camera from a standard distance of 30 cm. The images were analyzed with ImageJ Image-Processing Software (NIH, Bethesda, MD, USA), in order to estimate the area of the wounds. On the same days, the appearance and the exudate production of the wounds were also evaluated. For the morphological analyses, which were conducted at different times, the study used the method of evaluation of the healing process, PUSH (Pressure Ulcer Scale for Healing) [23], which considers the parameters of the presence of a crust, the reepithelialization, the granulation, and the slough. The appearance of the wound was defined as being necrotic tissue, slough, epithelial tissue, or as a closed wound. These designations were matched with scores of 0 (wound closed), 1 (epithelial tissue), 2 (granulation tissue), 3 (slough), and 4 (necrotic tissue). The amount of the exudate that was present in the wound was assessed before applying any topical agent and it was classified as absent (0), small (1), moderate (2), and large (3).
Histological analysis
One specimen from each group was collected on the 8th, 12th, and 16th day after surgery for the histological analyses (all of the remaining animals were sacrificed on the 16th day by an overdose inhalation of isoflurane). The specimens were fixed in 10% formalin for 24 h. After conventional ethanol gradient dehydration, the tissues were embedded in paraffin and they were sectioned at 5 μm. For the histological analyses, the sections were stained with Hematoxylin and Eosin (H&E) and Masson’s Trichrome (MT). The histological examination of the tissues was performed by a pathologist who was blinded to the experiment. The histological changes were evaluated by considering the density of the inflammatory cells, the thickness of the granulation tissues, and the thickness of the epithelial layers. At least six areas of each slide were analyzed.
In the tissues that were stained with HE, the presence of a crust and a reepithelialization in the epidermis were evaluated, as well as the dermal hemorrhage and neovascularization. The data that was obtained for these parameters was graded according to the intensity by: absent - up to 15% (0), very little - 20% (1) low - 50% (2) moderate - 80% (3), and very high - up to 100% (4). At least two fields of each blade were analyzed. MT was used to assess collagen deposition. The collagen area percentages were evaluated by using ImageJ Software Version 1:48, with the Color Deconvolution Plugin.
Statistical analysis
The analysis results of the lesion contractions and the morphological analysis of the lesions were studied by Two-Way Analysis of Variance (ANOVA), followed by Bonferroni’s test when using GraphPad 4 Software (USA). For the cell proliferation and the cell migration study, One-Way ANOVA, followed by the Student–Newman–Keuls (SNK) post hoc test, were used. All of the data was expressed as mean ± standard error and it was considered a significant value if p < 0.05.
