1. Introduction
Artemisia is a genus of annual, perennial, and biennial herbs in the Asteraceae (Compositae) family [
1]. The plants of the genus
Artemisia are frequently used in traditional medicine as remedies for human and animal ailments. For instance,
Artemisia species have been used in traditional medicine for respiratory disorders, including coughs and phlegm, as a pain killer, worm expelling agent, diaphoretic and diuretic agent, and for the treatment of wounds, hypertension, and allergies [
2]. In addition, some of the
Artemisia plants are traditionally used to treat seizures, and the activity is confirmed through in vivo animal experiments [
3,
4,
5].
Artemisia species have been reported in in vitro and in vivo experiments and in clinical trials evaluating their anticancer, antimalarial, antimicrobial, and antiviral activities [
6,
7].
Furthermore, several side effects and misuses have also been reported for some of the genus’ plants. For instance,
A. monosperma leaves are not recommended in pregnancy and are used to induce abortion in Jordan [
8]. However, this plant, in addition to other plants of
Artemisia, e.g.,
A. vulgaris, has been used in folklore medicine for labor induction [
8,
9,
10]. Besides abortion, vomiting, diarrhea, headache, pruritus, and rashes have been reported among young children and pregnant women who used
A. annua to treat malaria [
11]. The
Artemisia plants’ biological activities were attributed to the presence of essential oils, sesquiterpene lactones, flavonoids, bitter principles, coumarins, and phenolic acids [
1,
2,
12,
13]. Several
Artemisia species grow wildly or as cultivated plants for their use as medication and as a herbal tea preparation in the Mediterranean region [
9,
14,
15].
Artemisia judaica L. (ArJ) is widely grown in the Mediterranean region, including Algeria, Libya, Egypt, Jordan, and Saudi Arabia [
16,
17,
18,
19,
20]. In Saudi Arabia, ArJ grows in the kingdom’s northern region, including the border area of the Hail-Qassim regions [
21]. ArJ has been reported for several traditional uses, e.g., healing external wounds and repairing snake and scorpion bites [
22]. In addition, ArJ is traditionally used to treat gastrointestinal disorders, sexual inability, hyperglycemia, heart diseases, inflammatory disorders, arthritis, cancers [
1,
20], skin diseases, atherosclerosis, and enhance vision and immunity [
23,
24]. The Bedouins in Egypt (Sinai) and Saudi Arabia also use the plant as a herbal tea in treating GIT disorders [
16]. Biologically, ArJ demonstrated antidiabetic, antioxidant, hepatoprotective, and anti-inflammatory activities in experimental animals [
22,
25,
26] due to the properties inherent in the chemical structure of the compounds it contains [
27]. The plant also exhibited weak antimicrobial activity against Gram-positive and Gram-negative bacteria [
28,
29]. In vitro studies reported the plant extract’s potential antioxidant and anticancer activities [
28,
29].
ArJ chemical analysis revealed the presence of flavonoids, e.g., glycosides and aglycones of apigenin, luteolin, and quercetin [
22]. Other natural classes, such as phenolics, triterpenes, bitter principles, and sesquiterpene lactones, i.e., judaicin, have also been reported from the plant [
22,
30]. Additionally, ArJ is an aromatic plant. Its essential constituents have been identified from the plant species growing in different areas and climatic regions [
18,
20,
23,
24,
31]; as well known as the anthropogenic factors, environmental conditions primarily affect the composition of the plant [
32]. The overall analysis of the essential oil constituents of ArJ indicated that the monoterpene, i.e., piperitone, is the major chemotypic constituent in the plant from different genotypes [
1,
24]. In addition, other essential constituents of the plants, such as camphor, ethyl cinnamate, and spathulenol, have also been identified in relatively high concentrations in individual plant genotypes [
24]. In addition, environmental conditions and the geographical locations of the plant growing areas have been reported to affect the major chemotypic constituents of ArJ essential oils.
Table 1 demonstrates the major constituents of the plant essential oils from different locations.
The methods used for essential oil production from aromatic plants vary and mostly depend on the nature of the volatile constituents, the amount of the essential oils, and the nature of the plant samples [
36]. Thereby, distillation procedures are primarily used for the plants containing a considerable amount of the thermostable volatile constituents; however, volatile (e.g., diethyl ether) and non-volatile (e.g., lard) solvent extraction processes are used for the extraction of the highly delicate aromatic plants which contain heat-sensitive and small quantities of the essential oils [
37]. In addition, modern extraction techniques, such as CO
2, supercritical CO
2 extraction and microwave-assisted extraction techniques, are used for the industrial-scale production of the essential oils with specific advantages, e.g., time- and quantity-based efficiency and environmentally friendly properties [
38,
39,
40].
Burn injury traumas occur by friction, cold, heat, radiation, chemical, or electric sources, but hot liquids, solids, and fire contribute significantly towards burn injuries [
41]. In Saudi Arabia, 52% of all burns occur in young children, and males are more prone to burns than females (1.42:1). Burn wounds require immediate attention to avoid hypovolemic shock and sepsis [
42]. New approaches and drugs are being researched to facilitate faster burn wound healing [
43], thereby minimizing adverse reactions, like allergy or irritation, due to topical agents that increase the rehabilitation period [
44]. In addition to their general availability, herbal medicines have demonstrated a promising role in wound healing compared to silver sulfadiazine (SS) [
45,
46,
47]. Nevertheless, modern approaches and methodologies are required to validate claims for herbal compounds [
48].
The current study is designed to demonstrate the wound healing properties of ArJ essential oils as part of the plant’s use in traditional medicine. Therefore, a phytochemical analysis of the ArJ essential oil for the species growing in the Northern Qassim region of Saudi Arabia was conducted. The study also investigated the antioxidant, antimicrobial, and antibiofilm activities of the plant essential oil as associated analyses related to the wound healing potential of the plant.
2. Materials and Methods
2.1. Plant Materials and Distillation Procedure
The aerial plant parts were collected during March 2020, in the morning, from the Northern Qassim region of Saudi Arabia and identified as Artemisia judaica L. by the taxonomists in the Department of Plant Production and Protection, College of Agriculture, Qassim University (Buraydah, Saudi Arabia). A sample of the plant with the registered number #090 was kept at the herbarium of the College of Pharmacy, Qassim University (Buraydah, Saudi Arabia). The plant was dried in the shade at room temperature for ten days before the distillation process. The plant materials, 200 g, were reduced to coarse powder form, backed to a 2 L conical flask with a stopper, and thoroughly mixed with 700 mL of distilled water. The flask was connected to the Clevenger apparatus and fixed over the heating mantel. The flask contents were allowed to boil for a continuous 5 h. The distillate essential oil was collected over anhydrous sodium sulfate and stored in an opaque glass vial in a −20 °C freezer.
2.2. GC-FID Analysis of the Essential Oil
A gas chromatography (Perkin Elmer Auto System XL, Waltham, MA, USA) equipped flame ionization detector (GC-FID) was used to analyze the essential oil of ArJ. The chromatographic separation of the oil samples was achieved on a fused silica capillary column ZB5 (60 m × 0.32 mm i.d. × 0.25 µm film thickness). The oven temperature was maintained initially at 50 °C and programmed from 50 to 240 °C at a rate of 3 °C/min. ArJ essential oil sample was dissolved in analytical grade diethyl ether (2.9 mg of the oil in 100 µL of the solvent). Then, 1 µL of the mixture was injected with a 1/20 split ratio. The helium was used as the carrier gas at a 1.1 mL/min flow rate. The injector and detector temperatures were 220 and 250 °C, respectively.
2.3. Gas Chromatography–Mass Spectroscopy Analysis of the Volatile Oil
The GC–MS analysis of ArJ essential oil was conducted using an Agilent 8890 GC system attached to a PAL RTC 120 auto-sampler and equipped with a mass detector, Agilent 9977B GC/MSD mass spectrometer (Agilent technology, Santa Clara, CA, USA). An HP-5 capillary column (30 m, 250 µm i.d., 0.25 µm film thickness) was used to separate target molecules. The initial column temperature (50 °C for 2 min, isothermal) was programmed up to 220 °C at a rate of 5 °C/min, and then 10 °C/min up to 280 °C and kept constant at 280 °C for 10 min (isothermal). The injector temperature was 230 °C. Helium was used as a carrier gas at 1 mL/min flow rate. All the mass spectra were recorded using the following conditions. The run time was about 65 min. The transfer line was set at 280 °C, and the ionization source and the mass analyzer temperatures were set at 230 and 150 °C, respectively. Diluted samples (1% v/v) were injected with split mode (split ratio 1:15).
2.4. Identification of the Essential Oil Constituents
The constituents of the oil were identified based on the experimental retention index (RI) calculated with references to a series of standard n-alkenes series (C8–C40) and the retention indexes reported for the ArJ essential constituents besides the reported retention indexes obtained for the analysis of different essential oils under similar GC experimental conditions. In addition, the National Institute of Standards and Technology (NIST-11) and mass fragmentation patterns of the peaks were also used to identify the compounds. The relative percentages of the constituents were calculated from the area under the peak obtained from the GC-FID chromatogram.
2.5. Antioxidant Activity of ArJ Essential Oil
2.5.1. Total Antioxidant Capacity (TAC)
The method described by Aroua et al. [
49] was followed to conduct this experiment. In brief, sulfuric acid (0.6 M) and ammonium molybdate (4 mM) in sodium phosphate buffer (28 mM) were mixed to prepare the molybdate reagent. Then, 3.6 mL of the molybdate reagent was mixed with 0.4 mL of ArJ essential oil (containing 200 µg of the oil) in a stoppered glass test tube. The tube was vortexed and warmed for 30 min at 90 °C in a water bath. After cooling, the absorbance of the developed blue color was recorded at 695 nm using a spectrophotometer against a blank prepared essential oil. The TAC of ArJ essential oil was calculated equivalent to the Trolox using the standard calibration curve.
2.5.2. DPPH (2,2-Diphenyl-1-Picrylhydrazyl) Scavenging Activity (DPPH-SA)
The method was conducted according to Shimada et al. [
50]: 1 mL of the diluted ArJ essential oil (containing 200 µg of the oil in methanol) was mixed with 1 mL of DPPH (prepared by dissolving 6 mg of the DPPH in 50 mL of methanol). The mixture absorbance was measured at 517 nm after 30 min of standing at room temperature in a dark place. The DPPH-SA was calculated equivalent to Trolox from three independent measurements.
2.5.3. Ferric Reducing Antioxidant Power (FRAP) Assay
Minor modifications to the method of Benzie and Strain [
51] were carried out to measure the FRAP of ArJ essential oil. FRAP working reagent was freshly prepared by adding TPTZ (2,4,6-Tris(2-pyridyl)-s-triazine, 10 mM prepared in 40 mM HCl) to FeCl
3·6H
2O (20 mM) and acetate buffer (300 mM, pH 3.6) in a ratio 1:1:10. Then, 2 mL of the FRAP reagent was added to 0.1 mL of the ArJ essential oil (containing 200 µg the oil), the mixtures were incubated for 30 min at room temperature, and the absorbance was recorded at 593 nm. The procedure was conducted in triplicate, and the prepared FRAP–Trolox calibration curve was used to calculate the extract activity as mg Trolox equivalent per gram of the used plant’s dried extract.
2.5.4. Metal Chelating Activity Assay (MCA)
The ArJ essential oil ability to chelate iron compared to the EDTA was evaluated using Zengin et al.’s method [
52]. Briefly, a mixture of the ArJ essential oil (2 mL of ethanol containing 200 µg of the oil) and ferrous chloride (25 µL, 2 mM) was added to 100 µL of ferrozine to inchoate the color. The mixture’s absorbance was recorded at 562 nm against a blank (2 mL of the ArJ essential oil plus 200 µL of the ferrous chloride without ferrozine). The standard calibration curve of EDTA was prepared, and the chelating activity of the ArJ essential oil was calculated in equivalents of the EDTA.
2.6. Antimicrobial Activity of ArJ Essential Oil
2.6.1. Preliminary Antimicrobial Activity
The preliminary antimicrobial activity of ArJ essential oil was determined by the disc diffusion method [
53]. Modified Mueller–Hinton agar (MMHA) and potato dextrose agar (PDA) were used as test media. MMHA plates were prepared according to the protocol mentioned in the literature [
54]. The sterile paper discs (6 mm in diameter) were impregnated with 20 μL of pure ArJ essential oil and then used to evaluate the antimicrobial potential of ArJ essential oil against the selected human pathogens. Levofloxacin (5 µg/disc) and clotrimazole (50 µg/disc) were used as antibacterial and antifungal control (C) drugs. Each test organism’s inoculum was prepared in sterile tryptic soy broth (TSB), and the turbidity of each suspension was adjusted equal to 0.5 MacFarland standard, which is equal to 1.5 × 10
8 colony forming units (CFU/mL) for bacteria, 1 × 10
6–5 × 10
6 CFU/mL for yeast and 4 × 10
5 to 5 × 10
6 CFU/mL for mold. Following that, 100 µL suspensions of each adjusted inoculum were poured individually over the surface of the test agar plates and then uniformly spread using sterile swabs. The prepared discs of ArJ and control drugs were then put on the inoculated plates. The plates were incubated at 35 °C for 24 h for bacteria and 48 h for fungi. After incubation, the diameters of inhibitory zones were calculated on a millimeter (mm) scale. Each test was performed in triplicate. The results are expressed in mm ± standard deviation (SD).
2.6.2. Minimum Inhibitory Concentration (MIC) and Minimum Biocidal Concentration (MBC)
MIC was determined by the resazurin-based micro-broth dilution method, while MBC was performed following the standard spot inoculation method [
53,
55]. The inocula of each test bacteria were prepared in TSB, following the Clinical and Laboratory Standards Institute (CLSI) guidelines (
https://clsi.org/, accessed on 1st December 2021), where the OD
600 value (0.08–0.12) was adjusted, resulting in ~1 × 10
8 CFU/mL. Then, adjusted inocula were further diluted by 1:100 in TSB, resulting in ~1 × 10
6 CFU/mL. In contrast, the inocula of test fungi were prepared in potato dextrose broth (PDB) following the CLSI guidelines, where the OD
600 value (0.08–0.12) was adjusted, the resulting stock suspension contained 1 × 10
6 to 5 × 10
6 CFU/mL for yeast and 4 × 10
5 to 5 × 10
6 CFU/mL for mold. A working yeast suspension was prepared by a 1:100 dilution followed by a 1:20 dilution of the stock suspension with PDB, resulting in 5.0 × 10
2 to 2.5 × 10
3 cells/mL, while a working mold suspension was prepared by a 1:50 dilution of the stock suspension with PDB, resulting in 0.8 × 10
4 to 1 × 10
5 cells/mL. The initial stock solution of ArJ essential oil was prepared in DMSO (dimethyl sulfoxide) at a 200 µL/mL concentration. Each well in column 1 was dispensed with 200 µL of stock solution of ArJ essential oil. At the same time, each well of columns 2 to 10 contained 100 µL of tryptic soy broth (TSB) for antibacterial evaluation, while for antifungal assessment, 100 µL of potato dextrose broth (PDB) was used. A two-fold serial dilution of ArJ essential oil was made from columns 1 to 10 using a multichannel micropipette, resulting in concentrations of ArJ essential oil ranging from 200–0.39 µL/mL in columns 1 to 10. Column 11 had 200 µL of standardized inoculum suspensions, which served as negative control (NC), and column 12 had 200 µL of sterile broth, which served as sterility control (SC). Each organism’s adjusted inoculum was dispensed, 100 µL into each test well in columns 1–10, respectively. The 100 µL of adjusted microbial inocula were dispensed in all the wells of columns 1 to 10, resulting in ~5 × 10
5 CFU/mL for bacteria and ~2.5 × 10
2 to 1.25 × 10
3 CFU/mL for
C. albicans, and 0.4 × 10
4 to 5 × 10
4 CFU/mL for
A. niger. At this stage, the final concentrations of ArJ essential oil were 100 to 0.195 µL/mL in columns 1 to 10. The time taken to prepare and dispense the OD-adjusted microbial inocula did not exceed 15 min. The inoculated plates were incubated at 35 °C for 24 h for bacteria and 48 h for fungi. Following incubation, 30 µL of sterile resazurin dye (0.015%
w/
v) was dispensed into each well of columns 1 to 12, and then plates were re-incubated for 1–2 h to observe color change. After incubation, columns with the lowest concentrations showing no color change (blue resazurin color stayed intact) were scored as MIC.
MBC was determined by directly plating the contents of wells with concentrations above the MIC on sterile tryptic soy agar (TSA) plates for bacteria, while potato dextrose agar (PDA) plates were used for fungi. The contents from the wells, which did not change from blue to pink, were inoculated on sterile tryptic soy agar (TSA) plates and incubated at 35 °C for 24 h for bacteria and 48 h for fungi. The lowest concentration of ArJ did not produce isolated colonies of the test organisms on inoculated agar plates considered as the MBC. The results are recorded in µL/mL.
2.6.3. Minimum Biofilm Inhibitory Concentration (MBIC) and Minimum Biofilm Eradication Concentration (MBEC)
MBIC Assay
MBIC is defined as the lowest concentration of the antimicrobial agent (ArJ), preventing the biofilm formation of the tested organism. MBIC was conducted against the bacteria only. The 96-well microtiter plate was used to evaluate the anti-biofilm activity of ArJ [
54]. The inocula of the test organisms were prepared in TSB equal to 0.5 MacFarland standard (1–2 × 10
8 CFU/mL). An aliquot of 100 µL from the adjusted inocula was dispensed into each test well of a 96-well plate. Then 100 µL of different concentrations of ArJ were dispensed into test wells. Thus, the final concentrations for MBIC assessment were MIC, 2 × MIC, and 4 × MIC. The wells containing only 200 µL of TSB served as a blank control (BC), whereas those containing bacterial cultures without ArJ served as negative control (NC). The plates were incubated in a shaking water bath at 35 °C for 24 h at 100 rpm shaking speed. After incubation, the supernatants from each well were decanted gently by reversing the plates on a tissue paper bed/or removed by a pipette without disturbing the biofilms. The plates were dried in air for 30 min, stained with 0.1% (
w/
v) crystal violet at room temperature for 30 min, and then washed three times with distilled water. Subsequently, the crystal violet was solubilized by adding 200 µL of 95% ethanol to each test well. The absorbance was recorded in a microplate reader (xMark™ Microplate Absorbance Spectrophotometer-Bio-Rad, Hercules, CA, USA) at 650 nm. The lowest concentration of ArJ at which the absorbance equals or falls below the negative control is considered MBIC. Each test was performed in triplicate. The mean of three independent tests was taken. The results are expressed in µL/mL.
MBEC Assay
MBEC is defined as the minimum concentration of an antimicrobial agent (ArJ) that eradicates the biofilm of the test organism [
54]. A 200 µL (1–2 × 10
8 CFU/mL) inoculum of each test organism was inoculated into each test well of a flat-bottom 96-well microtiter plate. The plates were incubated at 35 °C for 48 h in a shaking water bath at 100 rpm shaking speed for biofilm formation. After the biofilms had formed, the contents of the test wells were decanted gently by reversing the plates on a tissue paper bed/or removed by a pipette without disturbing the biofilms. The various concentrations, i.e., MIC, 2 × MIC, and 4 × MIC of ArJ, were added to different test wells (200 µL/well). The inoculated plates were re-incubated at 35 °C for 24 h. After incubation, the contents of each test well were discarded by inverting the plates on a tissue bed. The plates were dried in air for 30 min, and then 200 µL of sterile TSB was dispensed in each test well. Then, 30 µL of 0.015%
w/v resazurin dye was added into each test well. The plates were re-incubated for 1–2 h. After re-incubation, the MBEC was recorded by observing the color change from blue to pink. The column with no color change (blue resazurin color stayed intact) was scored MBEC. Biofilm without ArJ served as a negative control (NC). Each test was performed in triplicate. The mean of three independent tests was taken. The results are expressed in µL/mL.
2.7. Preparation of Ointment Formulation Loaded ArJ Essential Oil
Ointment formulation of 5%
w/
w strength of ArJ essential oil was prepared. The simple ointment base was prepared by the fusion method according to the
British Pharmacopoeia 1988 [
56]. Briefly, 100 g of simple ointment base was prepared by melting hard paraffin (5 g) in a beaker at 61 °C. The other ingredients, i.e., cetostearyl alcohol (5 g), wool fat (5 g), and soft white paraffin (85 g), were added in descending order of melting point. The homogenous mixture was removed from the heat and stirred until cold. Then, 5%
w/
w strength ArJ essential oil ointment was prepared by incorporating 5 g of the essential oil into 95 g of a simple ointment base in small portions by mixing with trituration using an ointment mortar and pestle. Finally, the ArJ ointment was transferred to a clean container. The control ointment, 50 g of the entire base ingredients, was taken and treated in the same way to formulate without the essential oil. The prepared ArJ ointment was physically examined and was consistent, homogenous, and stable for the measured one month.
2.8. In Vivo Wound Healing Animal Experiment
Twenty healthy 3-month-old Sprague Dawley female rats weighing about 150 ± 50 g were individually maintained in the cage under 25 ± 2°, 65% humidity, 12:12 light/dark cycle. Animals were fed with a standard chow diet with water ad libitum, and the wound healing study was conducted following the guidelines of the Institutional Animal Ethics Committee (Registration # 21-04-06). The animal groups involved intact, negative control, positive control (1% silver sulfadiazine, SS), and ArJ 5% ointment.
2.9. Skin Burn Induction Model
Briefly, the animals were anesthetized using xylazine 5 mg/kg and ketamine 50 mg/kg, and the rat’s dorsum was shaved with a hair trimmer (GEEPAS
®, Guangzhou, China) at a 45° angle to minimize the angle skin injury during shaving and disinfected using 70% ethanol. An aluminum cylinder (1-inch square diameter, 86 g weight) was heated using a hot water bath at 120 °C for at least 60 min to ensure thermal equilibrium with the water. The exact temperature of the cylinder and the water was measured before inducing the burns using a dual probe thermometer (UT320D Mini Contact Type Thermometer Dual Channel K/J Thermocouple, UNI-T, Dongguan, China). Second-degree burns of 1-inch square diameter were induced on the rat’s shaved dorsum by patching the aluminum cylinder on the rat’s dorsum for 10 s, allowing it to stand on its own weight to ensure symmetrical burns across all rats [
57,
58,
59]. The animals were administered with 0.9% normal saline i.p injection 10 mL/kg. The treated groups were applied topically twice daily for three weeks with ArJ 5% ointment or 1% SS cream topically on the wound area.
2.10. Biopsy
At the end of the experiment on day 21, the animals were euthanized and a biopsy measuring 1 × 1 cm diameter was collected using scissors and tweezers from the underlying tissue. One part of the biopsy was fixed in 3.7% formalin for paraffin embedding, while another part was homogenized and the supernatant isolated and stored at −20 °C for biochemistry analysis.
2.11. Histological Staining
Tissue was processed within 48 h of collection by dehydration with increasing ethanol percentages before being cleared with xylene and embedded in paraffin wax. Tissue sections of 5 microns were cut using a microtome (MEDIMEAS, Haryana, India), allowing simultaneous sectioning of the epidermis and dermis. Sections were stained using hematoxylin and eosin (H&E) and visualized under a light microscope at 40× magnification. Five fields/sections were counted for the amount of fibroblast, collagen, inflammation, and neovascularization, and the data was scored from 0–4, where 0, 1, 2, 3, and 4 represented normal, low, moderate, high, and very high, respectively, as described previously [
60].
2.12. Determination of Oxidants and Antioxidants
The catalase (CAT, Serial No. 24IF07D5A0) and superoxide dismutase (SOD, Serial No. 745402C55B) activity, and lipid peroxide (LP, malondialdehyde, Serial No. 1F4346D808) levels were determined in skin wound tissue homogenate by enzyme-linked immunosorbent assay (ELISA) kits (Cloud Clone Corp Company, Houston, TX, USA), according to the manufacturer’s instructions. The absorbance was measured at 450 nm by a microplate ELISA reader and the concentration was calculated using a standard curve.
2.13. Determination of Pro-Inflammatory and Anti-Inflammatory Cytokine Levels: Interleukins, TGF-b, and TNF-α Levels
The pro-inflammatory cytokines: interleukin 1 beta (IL-1b, Serial No. 282D397BBC), IL-6 (Serial No. 51D9580378), and TNF-α (Serial No. 3898289A45) and the anti-inflammatory cytokines (IL-10 (Serial No. 6AB644B25F), transforming growth factor beta 1 (TGF-b1, Serial No. 34997E20C3) were assayed in tissue homogenate by ELISA kits (Cloud Clone Corp Company, Houston, TX, USA) according to the manufacturer’s instructions. The microplates were measured with a 450 nm filter by a microplate reader.
2.14. Wound Area Measurement
Using a standard camera, images of skin burn for all the animals were captured on the day of burn induction and at different time points (week 1, 2, and 3), while the wound measurement was performed before the treatment and 2 weeks after the treatment using freely available Image J software (version 1.8.1, Public Domain, Madison, WI, USA). Due to hair regrowth on the wound area, wound size could not be measured accurately after 2 weeks.
2.15. Statistical Analysis
Data were expressed as the mean ± standard error of the mean (SEM) (
n = 5). Differences between groups were analyzed using one-way ANOVA, except for wound area measurement at different time-points, which was analyzed using two-way ANOVA followed by a post hoc test using Tukey’s multi-group comparison on GraphPad Prism 8.0.2 (GraphPad Software, San Diego, CA, USA). The data were considered significant if
p < 0.05 [
61]. The superscripts (A–C) describing significance among the groups in the tables were obtained using Minitab 19.1 (Minitab LLC, State College, PA, USA).