Animals
Male mice (25-30 g, 2 months old) were purchased from of the animal house of the School of Pharmacy, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. The animals were housed in groups of 6 in standard laboratory conditions at 20-25 °C and 65-75% humidity, and they were kept in separated cages for 12 hours light/dark cycle and free access to standard food and tap water. All mice were observed according to the animal rights ethics protocol of Yazd Shahid Sadoughi University of Medical Sciences (IR.SSU.MEDICINE.REC.1398.055).
Preparation of Althaea officinalis Extracts
The dried Althaea officinalis flowers were obtained from Yazd medicinal plants market. The scientific name and the quality were confirmed with a quality assurance number was obtained from the Faculty of Pharmacy, Shahid Sadoughi University of Medical Sciences (SSU0042). The flowers were grinded and powdered, then the hydroalcoholic extract was performed during several week percolation. The aqueous extract was performed by infusion method for 20 minutes at 80 °C. The total hydroalcoholic and aqueous extracts were dried and standardized.
The dried aqueous and hydroalcoholic extracts were used to prepare the hydrogels (1, 2 and 4 percent) containing 2% polymeric dry matters including 40% hydroxypropyl methylcellulose, 40% methylcellulose and 20% hydroxyethyl cellulose as. Five percent propylene glycol was added to the hydrogel to improve its rheological properties.
Standardization
Total phenolic compounds were measured by folin-ciocalteu method. Three concentrations of the hydroalcoholic extract were prepared in distillated water including 0.1, 0.01 and 0.001 µg/ml. Then 200 µl of each concentration was poured into separated test tubes and 400 µl of distilled water was poured into the blank tube. Then, 3 ml and 1.5 ml of folin-ciocalteu reagent was added into the blank and test tubes respectively and all of them were incubated at 22 °C for 5 minutes. Then 3 ml and 1.5 ml sodium bicarbonate 0.6% was added into the blank and test tubes respectively and were incubated at 22 °C for 5 minutes. The absorbances were observed at 725 nm by a spectrophotometer device (Figure 1).
Total phenolic content was calculated by below formula.
Y=0.0561 X + 0.0267
Y=0.067, X=7.183 μg /ml, R₂=0.9996
Total flavonoid compounds were measured by catechin method. Three concentrations of the hydroalcoholic extract were prepared in distillated water including 0.1, 0.01 and 0.001 µg/ml in three separated tubes (1.5 ml). Then 300 µl of sodium nitrite 5% was added into the blank and test tubes. After 5 minutes, 300 µl aluminum chloride 10% and Then 2 cc NaOH 1 M was added and distilled to 100 ml. The absorbances were observed at 510 nm by a spectrophotometer device (Figure 2).
Total flavonoid content was calculated by below formula.
Y=0.0003 X +0.0129
Y=0.0137, X=26.66 μg/ml, R₂=0.9865
Burn induction
Burn induction was performed according to Shanmoga method with some modifications (9). The dorsal skin of the mice was burned by a metal plate (2 cm in diameter) under general anesthesia following i.p. injection of ketamine (50 mg/kg) and xylazine (10 mg/kg). According to the histopathological parameters, the best temperature for induction the second- and third-degree burns was determined as 120 °C for 3- and 7-seconds contact time, respectively (Figure 3).
The mice were randomly divided into two main groups including the second- and the third-degree burns groups. Each main group was randomly divided into 9 groups (n-6) including control group (received topical normal saline), topical hydroalcoholic and aqueous Althaea extract groups (1, 2 and 4 percent), topical phenytoin 1% group and topical hydrogel group. Following the burn induction, the animals were kept in separated cages with free access to food and water and treated twice daily for 20 days. They were weighted weekly. Finally, the mice were sacrificed and the entire layer of the burned skin was separated and maintained in formalin 10% solution for histopathological evaluations.
Histopathological evaluations
The tissue was fixed in Bouin’s solution (7.5 mL saturated picric acid, 2.65 mL glacial acetic acid, and 2.5 mL 7% formaldehyde), post-fixed in 70% alcohol, and embedded in paraffin blocks. A full thickness tissue section was obtained, deparaffinized, and stained with hematoxylin eosin.
The histopathological parameters including epithelialization, PMN migration, collagen formation and angiogenesis were scored under a optic microscope double blindly.
Statistical analysis
Data are expressed as mean ± S.E.M which were analyzed by one-way ANOVA followed by Tukey’s post hoc test. All statistical analyses were made by using SPSS software (version 19).