In 4631, was similar to that of

In
this controlled randomized clinical trial as animal model, 150 male Sprague
Dawley rats weighted 240 ± 20 g and aged 10-12 weeks, were obtained from Center
of Comparative and Experimental Medicine, Shiraz University of Medical Sciences
and randomly divided into three animals per cage in controlled temperature room
(22 ± 2 ºC), 12 hr light and 12 hr dark cycle. They were fed with commercial
pellet diet and water, ad libitum. During the study, ethical consideration as
number 4631, was similar to that of Animal Care Committee of Shiraz University
of Medical Sciences.

Plant
materials and extraction

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The
leaves of EC (pm 780 Eucalyptus camldulensis Dehnh., family: Myrtaceae) and
roots of licorice (pm 779 Glycyrrhizaglabra L., family: Papilionaceae) were
cultivated in Fars province, Iran. In the extraction process, the leaves and
roots were air-dried and ground into a fine powder. Aqueous ethanol (75%) was
added to the powdered materials (500 g) and stirred for one hour. The mixture
was kept at room temperature for 48 hours. Following filtration, ethanol was
evaporated under reduced pressure at 40ºC. The remained water extract was dried
at oven temperature of 50ºC. Finally, the 1% EE and 10% gel of licorice were
prepared in pharmacy laboratory.

Burn
injury

To
induce burn, the rats were anaesthetized with ketamine (100 mg/kg) and xylazine
(10 mg/kg). Consequently, a standard 3rd degree burn wound was produced on the
back of the shaved rats using a hotplate with similar size, about 20% of the
total body surface area and kept for 10 sec (Manafi et al. 2009). Then, 108 colony forming units of
toxigenic strains of P. aeruginosa (PA 103) that had been isolated from burn
patients of Shiraz Burn Hospital, were inoculated subcutaneously into the burn
area. The animals were then divided randomly into five groups in separated
cages. After 48 hr, the cultures were performed from the burn sites of rats to
confirm the existence of infection. Treatment of burn was started as follows:
Groups 1 and 2 received no treatment and gel base, respectively. Group 3
(positive control) were treated with SSD. Treatment groups (4 and 5) include EE
1% and a mixture of EE 1% with hydroalcoholic licorice extract 10%,
respectively. The samples were harvested on days 3, 7, 14, 21, and 28.

Histopathological
examination

Skin
samples were fixed in 10% neutral-buffered formalin and then embedded in
paraffin. Sections of 5µm thickness were stained using hematoxylin and eosin (H
& E) and studied by Olympus DP12 Digital Camera system (Olympus Optical,
Tokyo, Japan). Histopathological examinations were performed in double blind
method according to modified wound-healing histopathological scoring system (Abramov et al. 2007).

Skin
stretch

The
skin specimens were harvested immediately after euthanasia and placed between
sterile sponges soaked with 0.9 % saline to preserve the normal tissue
hydration after harvesting. Then, they were placed in occlusive bags and stored
at -70 °C prior to examination. On the day of the biomechanical test, specimens
were retrieved from the freezer, allowed to thaw at room temperature before
being measured for thickness and length with a digital caliper. Specimens were
mounted in a material testing machine that used two clamps with a rough
surface, with the wound in the middle of the free surface. The jaw space which
was the distance between the edges of the clamps was 30 mm. Biomechanical testing
was performed according to Trudel method by testing machine (Tensile Testing
Machine Hounsfield, England) (Trudel et al. 2009). Each sample was loaded by
elongation at a displacement rate of 20 mm.s?1. The ultimate tensile strength,
yield strength, stress and stiffness were measured. The stress value (N.mm-2)
was calculated by dividing the ultimate strength (N) by the cross-sectional
area (mm2). Stiffness (N.mm?1) was calculated by fitting a linear regression
line to the load-deformation data from 30 % to 90 % of the ultimate TS on the
deformation curve.

Statistical
analysis

The
results are expressed as mean ± standard error of mean (SEM). The wound TS 28
days after treatment between experimental and control groups was compared using
one way ANOVA followed by post-hoc Tukey test and histopathological results
were analyzed by Mann-Whitney U test in SPSS version 18 (SPSS, Chicago, IL,
USA). A P-value

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