Effect of Glycolic Extract of Dillenia indica L. Combined With Microcurrent Stimulation on Experimental Lesions in Wistar Rats
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Total alkaloids were assayed using the reagents of Dragendorff, Bouchardat, Mayer, and Bertrand.
Preparation of an emulsion containing the glycolic extract of Dillenia indica. The lyophilized D indica extract was solubilized in propyleneglycol:water (1:1) and incorporated into an emulsion containing the following: Butylated hydroxytoluene (BHT [0.05%]), Ethylenediamine tetraacetic acid (EDTA [0.1%]), Lanaxan® (2%), Polawax® (14%), Phenonip® (0.5%), propyleneglycol (3%), D indica glycolic extract (5%), and distilled water qsp (30%).
Animals. Male Wistar rats (Rattus norvegicus) weighing 250 g–350 g were housed individually in cages at a constant temperature (23˚C ± 2˚C). The rats had free access to food and water and were subjected to a 12-hour light/dark cycle. The average weight and behavior during this experiment did not differ significantly at the end of the study.
Linear incision wound model. A trichotomy was performed on the back of the animal 48 hours before surgical intervention. After local asepsis with 0.4% chlorhexidine digluconate, the animals were anesthetized by intraperitoneal injection of xylazine hydrochloride (20 mg/kg body weight) and ketamine hydrochloride (50 mg/kg). After the position was marked with a dermographic pen and pachymeter, a 2-cm long and 0.2-cm deep surgical incision was made in the craniocaudal direction. The incision was not sutured. In view of the similar genetic background of the animals28,29 and according to the Ethics Committee of Uniararas (protocol number 809/2006), nine animals were used per group: (A) negative control group receiving sterile saline; (B) group receiving microcurrent application (MC; [10 mA/2 min]); (C) group treated with the glycolic extract of D indica (GED); (D) group treated with the emulsion containing GED; (E) group treated with the GED and MC (10 mA/2 min), and (F) group treated with the emulsion containing GED and MC (10 mA/2 min), according to the protocol of Mendonça et al.16 A transcutaneous electrical stimulator (Physiotonus Microcurrent, Bioset, Rio Claro, São Paulo, Brazil) was used for electrical stimulation. The device was set to microgalvanic-continuous mode with the intensity at 10 mA/2, frequency 0.3 Hz, and was used for 2 minutes. The applications were performed using two metal electrodes with a spherical tip (10 mm) positioned on the wound. The treatments were started 24 hours after surgical intervention and were continued daily for 10 days.
Collection and preparation of wound samples for structural analysis. At days 2, 6, and 10 after the injury, three animals in each group were killed under anesthesia. The total area (approximately 120 mm2–160 mm2) of the wound was removed and submitted for structural and morphometric analysis. Each sample was removed and fixed in 10% formalin in Millonig buffer (pH 7.4) for 24 hours at room temperature. Next, the specimens were washed in buffer and processed for embedding in Paraplast®. Longitudinal sections (7 µm) were stained with hematoxylin & eosin for routine histology and with picrosirius-hematoxylin in order to view collagen fibers. The specimens were examined and documented using a Leica DM 2000 photomicroscope at the Laboratory of Micromorphology, Hermínio Ometto University Center, Uniararas.