Cedrus deodara: In Vivo Investigation of Burn Wound Healing Properties

Objective Cedrus deodara (Roxb. Ex Lamb.) G. Don possesses various biological activities, which have been documented in modern and traditional medicine. In this study, burn wound healing activity of the methanol extract of C. deodara wood was evaluated via a burn wound model in Wistar rats. Methods The methanol extract of C. deodara was evaluated for the contents of phenolic compounds, flavonoids, and tannins. Also, its antioxidant activity was determined using the DPPH assay. Then, a topical ointment containing the methanol extract of C. deodara (10%) was used to evaluate the healing effects on a model of second-degree thermal burn in 4 groups of 7 rats within 21 days. In this respect, average wound surface area, wound closure, and various histological features were examined. Results Our findings revealed that the wounds treated with the methanol extract of C. deodara showed higher wound contraction (33.6, 87.1, and 93.4% on days 7, 14, and 21, respectively) compared with the positive control (27.6, 80.7, and 88.3% on days 7, 14, and 21, respectively) and the negative control (20.1, 77.9, and 80.2% on days 7, 14, and 21, respectively). According to the results from epitheliogenesis score, the number of inflammatory cells, neovascularization, and collagen density, good burn wound healing activity of the methanol extract of C. deodarawas demonstrated. Conclusion Using the methanol extract of C. deodara in an ointment formulation can be developed to prevent or reduce burn injury progression.


Introduction
Burns are skin injuries usually caused by excessive heat, electricity, radioactivity, or corrosive chemicals, which need careful monitoring at each step of healing. Tey have been a global public issue, leading to an estimated 180,000 deaths annually. Te destructive outcomes of burns include physical disabilities as well as mental and emotional disorders, which impose high costs on societies [1]. Burn wound healing is a complex process consisting of several phases: infammation, re-epithelialization, granulation tissue formation, and remodeling of extracellular matrix. Infections have been found as one of the main concerns delaying wound healing process and despite the discovery of a vast spectrum of antiseptics, it still remains a challenge to modern medicine [2]. Adverse efects of topical antibacterial agents and disinfectants include allergic reactions, skin irritations as well as the possibility of antimicrobial resistance, which can reduce the rate of skin repair and increase the rehabilitation period [3].
Cedrus deodara (Roxb. ex Lamb.) G. Don., commonly known as deodar, is a species of western Himalayan cypress tree belonging to the Pinaceae family. It locally grows in eastern Afghanistan, northern Pakistan, northwestern India, southwestern Tibet, and western Nepal [12]. Diferent parts of the plant have been traditionally used for the treatment of various diseases such as arthritis, asthma, gastric disturbances, infammation, microbial infections, and neurological and skin disorders. Te leaf and resin paste of the plant has been topically used for wound healing [13]. Phytochemistry studies of C. deodara extracts and oils have indicated more than one hundred constituents including favonoids, lignans, sterols, terpenoids, terpenes, sesquiterpene, and hydrocarbons [13]. In this work, burn wound healing of the methanol extract of C. deodara wood was evaluated in a topical formulation (ointment) via an in vivo burn model.

Materials and Methods
All solvents and reagents were obtained from Merck and Aldrich. S. aureus ATCC 6538 and P. aeruginosa ATCC 9027 were provided by the Iranian Research Organization for Science and Technology (IROST). Xylazine, ketamine, silver sulfadiazine (1%) ointment, and eucerin 400 were obtained from the pharmacy of Imam Khomeini Hospital Complex.

Plant Material.
Four-to fve-year-old branches of Cedrus deodara (Roxb. ex Lamb.) G. Don. (Figure 1) were obtained from the Nowshahr Botanical Garden, Iran. Tey were identifed and voucher specimen of 7079-TEH was deposited in the Herbarium of the Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.

Preparation of the Methanol Extract.
Dried branches of C. deodara (1000 g) were cut and powdered. Te methanol (80%) extract was prepared by maceration of powder in three 72 h rounds at room temperature. Te extract was concentrated under vacuum (Heidolph, Heizbad Hei-VAP, Germany) at 25°C and then lyophilized by a laboratory freeze dryer (LTE Science LTD, England) to obtain a dry and brittle powder (137 g).

Phytochemical Analysis.
Te methanol extract of C. deodara was phytochemically analyzed by measurement of the total phenolic, favonoid, and tannin contents. Also, the antioxidant activity of the extract was evaluated by DPPH radical scavenging assay.

Determination of Total Phenolic Content.
Te total phenolic content was determined using the Folin-Ciocalteu reagent and expressed as gallic acid equivalent [14].

Determination of Total Flavonoid Content.
Total favonoid content was measured using the aluminum chloride method and expressed in terms of catechin equivalent, based on the described method in our previous work [15].

Experimental Animals.
Twenty-eight Wistar male rats (200-220 g) of approximately eight weeks of age were studied and randomly divided into four groups of seven rats. Te animals were housed in the standard environmental conditions (temperature: 22 ± 3°C, humidity: 60 ± 5%, and a 12 h light/dark cycle). During the experiment, the rats were fed a standard pellet diet (Pastor Institute, Iran) and water ad libitum. All procedures were carried out according to the institutional guidelines for animal care and use.

Preparation of Ointment.
Te topical ointment formulation (10%) was accordingly prepared: 20 g of the methanol extract of C. deodara was dissolved in 20 mL distilled water, and eucerin 400% was gradually added to the resulting solution to obtain 200 g of ointment, which was then well mixed to aford a homogenous product (fnal concentration of extract: 10%). Also, an ointment base (eucerin 400%) with no extract was used as a placebo (negative control).

Burn Wound Model.
A burn wound model was used to evaluate the healing activity of the methanol extract of C. deodara. After inducing anesthesia via the intraperitoneal injection of 2% xylazine (5 mg/kg) and 10% ketamine (100 mg/kg), the rats were fxed in a ventral posture on a surgery table. Te hair on the backs of the rats was covered in shaving cream and washed of after 10 minutes, so that no wounds were created in these areas. Deep second-degree burns were created by a circular probe with a diameter of 1.5 cm using a standard burn device (110°C for 10 seconds). Burn wounds were washed with physiological saline.

Grouping Animals.
Te animals were numbered, weighed, and randomly divided into four groups of seven rats: O group: rats treated with ointment base with no extract as placebo (negative control).
P group: rats treated with a topical ointment containing 10% (w/w) of the methanol extract of C. deodara.
NC group: rats treated with nothing and only dressed in sterile gauze to assess "normal" wound healing (Normal healing group).
PC group: rats treated with "silver sulfadiazine (1%) ointment" as the positive control.
After creating burns on the rats' backs, all groups were frst treated with only sterile gauze for 24 h to allow the wounds to expand. Ten, topical ointments (extract and placebo) were applied to the wound surface using sterile gauze. For the positive control group (PC), the ointment was also applied to the wound using sterile gauze. Te dressings were changed every 24 h. All rats were monitored daily for 21 days. Wound size was calculated using Adobe Photoshop CC 2018 (Adobe Systems Inc.).

Statistical Analysis.
For the parametric data, the difference between groups were evaluated by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test for multiple comparisons. P values less than 0.05 were considered signifcant.

Histopathological Studies.
Animals from each group were euthanized at 7-, 14-, and 21-days postinjury (DPI), and the skin tissues were harvested and immediately fxed in the 10% neutral bufered formalin (pH = 7.26) for 48 h. Te fxed tissue samples were then processed, embedded in parafn, and sectioned to 5 μm thickness. Finally, the sections were stained with haematoxylin and eosin (H&E). Te histological slides were evaluated by two independent reviewers, using light microscopy (Olympus BX51; Olympus, Tokyo, Japan) in a double-blind fashion. Angiogenesis, infammatory cell infltration, and fbroplasia (collagen content) were assessed in diferent groups, comparatively. Magnifcation × 400 was employed to count diferent cells and calculations were repeated in fve felds. Finally, the average number of each criterion for these felds was recorded. Epithelialization was assessed at 7, 14 and 21 DPI, semiquantitatively on 5-point scores: 0 (without new epithelialization), 1 (25%), 2 (50%), 3 (75%), and 4 (100%) epithelialization. For these parameters, the results were validated by a comparative analysis of two independent observers blinded to the treatment groups.

Statistical Analysis.
For parametric data, the diference between groups were evaluated by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test for multiple comparisons. P values less than 0.05 were considered signifcant.

Extraction Yield and Phytochemical
Analysis. Te yield of methanol extract of C. deodara was calculated as 13.7%. Also, the total phenolic, favonoid, and tannin contents were assayed as reported in Table 1.

Antibacterial Assay.
Antibacterial activity of the methanol extract of C. deodara was evaluated against two Gram-positive and Gram-negative strains, Staphylococcus  (Table 2). It showed pronounced antibacterial activity against S. aureus.

Burn Wound
Healing Activity of C. deodara. Te wound area was measured on the 0, 1st, 7th, 14th, and 21st days after burn in all groups using Adobe Photoshop CC 2017. Wound surface reduction was recorded in Table 3. Te percentage of wound contraction (wound closure) was also calculated according to equation (1) ( Table 4).
Wound closure (%) � (wound surface area on day n − wound surface area on day 1) wound surface area on day 1 * 100.
As shown in Table 3, group P containing the methanol extract of C. deodara showed remarkable decrease in wound surface area compared with groups O, NC, and PC especially at 21-day postinjury.
According to Table 4, the percentage of wound closure within 21 days was signifcantly increased for group P compared with the other groups (P < 0.05). On both days 14 and 21, group P demonstrated signifcantly higher wound contraction compared to groups O, NC, and PC (P < 0.001, P ≤ 0.001, and P < 0.05, respectively).

Histopathological Studies.
All groups were studied for histopathological changes using light microscopy as shown in Figure 2. Te histopathologic evaluation of lesions in the NC group showed the granulation tissue formation and infltration of numerous infammatory cells for 21 days. Moreover, the re-epithelialization was not initiated until 21 DPI. Micrographs of sections in the PC group showed ulcerated surfaces on the 7th day. At the early stage of the healing processes (7 days), the wounds in this group displayed evident infammatory cell infltration and granulation tissue formation. However, the infammatory cells and granulation tissue disappeared quickly for 21 days, and new blood vessels began to grow in this group on the 14th day. Finally, the reepithelialization process was almost completed at 21 DPI.
Te evaluation of group O was like that of the group NC on the 7th to 14th days with the presence of the granulation tissue formation and infltration of numerous infammatory cells. However, the epithelial layer was started to form on the 21th day as shown in Figure 2. Te group P showed the greatest resemblance to normal skin, with less hypertrophic scarring, a thin epidermis, and rejuvenation of skin appendages. Moreover, the re-epithelialization process was initiated on the 14th day ( Figure 2).
Overall, the P-treated wounds showed the best results when compared to other experimental groups at 7, 14, and 21 DPI.
Te analysis of histological features was performed at 7, 14, and 21 DPI ( Table 5). As can be seen in Table 5, group P showed similar epitheliogenesis score with the group PC on the 21th day. However, it demonstrated higher score on the 14th day. Comparison of the results obtained from group P with groups NC and O indicated the higher potency of P than others to induce re-epithelialization.
Te infammation phase is essential for initiating the healing process; however, its prolongation is unwanted. Te sharp decrease in the number of infammatory cells for group P (Figure 3) indicated anti-infammatory activity of the methanol extract compared with groups PC, O, and NC. In group P, the number of infammatory cells was signifcantly decreased from day 7 to day 21 (P < 0.01). However, the downward trend showed a steeper slope in the positive control (PC) from day 7 to day 21 (P < 0.001). Comparison of the number of infammatory cells on the 21th day in groups P and O depicted signifcant decrease of infammation in group P (P < 0.001).
In the case of neovascularization (blood vessels/5HPF), groups P and PC showed desirable changes. For group PC, high neovascularization occurred on the 14th day which was signifcantly higher than day 7 (P < 0.001) and it decreased on the 21th day comparing with the 14th day (P < 0.001). However, a downward trend was observed for group P from day 7 to 21 (P < 0.001). It seems that the increase of neovascularization occurred before day 7 indicating early proliferative phase. On the other hand, groups NC and O showed increased neovascularization on the day 21 which showed delayed proliferative phase or disrupted remodeling stage.
Analysis of histological features also indicated increase of mature collagen type I in groups P and PC. However, increase of collagen density in group P was signifcantly higher than the corresponding value for group PC on the same days (P < 0.01, P < 0.001, P < 0.001 for days 7, 14, and 21, respectively). However, the changes in groups O and NC were not signifcant and no defnite pattern was seen.

Discussion
Wound healing based on natural products has attracted lots of attention both in folk and modern medicine because they are rich in phytochemicals possessing potent anti-  [18]. Te role of phenolic compounds and tannins in the wound healing process has been fully discussed in the literature. Flavonoids play an important role in angiogenesis, collagen deposition, epithelialization, and wound contraction in the proliferative stage [19]. Also, they have shown antioxidant, antiviral, antimicrobial, anti-infammatory, and antiproliferative activities, which can accelerate wound healing by the reduction of complications of the process [20]. Tannins also have been documented for their potent antibacterial [21,22] and antioxidant activities [23], which can improve the wound healing process [24]. Tey not only heal burn wounds, but they also stop infection along with the healing process. Moreover, they are able to construct a layer over the exposed injury, protecting the tissue from infection [25].
Based on the DPPH radical scavenging assay, the methanol extract of C. deodara showed good anti-oxidant activity. As high levels of reactive oxygen species (ROS) complicate the healing process, the healing activity of the plant can be considered from the aspect of anti-oxidant activity [26].
Infections slow down or prevent the healing process. In this respect, antibacterial agents are necessary to stop the bacterial growth as it can lower the wound pH and decrease the oxygen levels. Furthermore, infection can also increase the levels of proinfammatory cytokines, prolonging the infammatory phase and preventing the proliferative stage from moving on to the remodeling stage [27]. Staphylococcus spp. specifcally S. aureus is a signifcant cause of infection, strongly in hospitalized patients and fghting against this bacterial strain is important in the healing process [28]. Good anti-bacterial activity of the methanol extract of C. deodara against S. aureus proves the healing ability.
Good results from phytochemical studies and in vitro assays are in good agreement with those obtained from in vivo experiment, confrming burn wound healing activity of the methanol extract of C. deodara in a topical ointment formulation.
An in vivo study within 21 days depicted that the group treated with plant extract (P) showed signifcantly higher wound closure (%) than the other groups (O, NC, and PC with P < 0.001, P < 0.001, and P < 0.05, respectively).
As reported in Table 5, group P showed much better reepithelialization than group O and was also comparable with the group PC. It seems that the high content of favonoids of the studied extract is responsible for the desirable result as the role of favonoids in enhancing re-epithelialization has been well discussed in the literature [11].
As decreasing the number of infammatory cells in the wound bed is essential for the progression of the healing process [27], the downward trend for group P from day 7 to day 21 (P < 0.01) proves its better healing activity on the 21th day, comparing with groups PC (P < 0.05) and O (P < 0.001).
Formation of new blood vessels or neovascularization is an essential step in the proliferative phase. In this phase, increasing the number of new blood vessels is vital for providing the necessary nutritional metabolites for the injured tissue. However, this number regresses to the preinjury state moving into the remodeling phase. Failing this process can prolong the proliferative stage and slow down the healing process, creating chronic skin impairments [29,30]. In this study, treating wounds with the methanol extract of C. deodara led to a signifcant reduction of neovascularization on the 21th day, compared with the 14th day (P < 0.001), while the group O demonstrated signifcant   Healing activity was investigated using a burn wound model: O � group treated with ointment base with no extract, P � group treated with topical ointment containing extract, NC � group treated with nothing and only dressed in sterile gauze, and PC � positive control. b Reported as mean ± SD. Mean values within each group were compared with PC as the positive control: ns � not signifcant, * P < 0.05, * * P < 0.01, and * * * P < 0.001.
Evidence-Based Complementary and Alternative Medicine 5 increase in neovascularization on the 21th day, compared with the 14th day (P < 0.01), which indicated delay in the proliferative phase or a disruption in the remodeling stage. It is known that increase of mature type I collagen promotes wound contraction, which was clearly achieved in group P. In this group, a signifcant early increase of type I collagen density was observed from day 7 to day 14 (P < 0.001), depicting signifcantly higher collagen content on the 14th day, compared with group PC (P < 0.001) and O (P < 0.001). A signifcant increase of collagen density was    Evidence-Based Complementary and Alternative Medicine observed from the 14th day to the 21th day in group PC (P < 0.05), however, the collagen content of group P on the 21th day was still signifcantly higher than that of group PC (P < 0.001). Tese results can be attributed to the high favonoid content since these compounds are known to stimulate biosynthesis of collagen [31].

Conclusion
Te burn wound healing activity of the methanol extract of C. deodara wood was studied in a burn wound model using an ointment formulation. It was perceived that the plant can be considered in the development of efective healing agents for treating burns as desirable antioxidant and antibacterial properties were confrmed. Furthermore, the in vivo experiment indicated satisfactory results from wound closure percentage, epitheliogenesis score, number of infammatory cells, neovascularization, and collagen density as the main factors in the wound healing process.

Data Availability
Te original datasets supporting the fndings of the present study can be obtained from the corresponding author upon reasonable request.

Ethical Approval
Te project was conducted in accordance with the ethical principal and national norms and standards for conducting medical research in Iran (IR.TUMS.VCR.REC.1398.584).

Conflicts of Interest
Te authors have declared no conficts of interest.

Authors' Contributions
AR contributed to the preparation of extract and topical ointment and analysing data. AM performed phytochemical analysis. TA supervised the wound healing activity of the plant. RH contributed to in vivo test. NS performed antimicrobial activity. MK contributed to the preparation of the manuscript. SN contributed to the preparation of topical ointment. MS designed the project, wrote the manuscript, and supervised all phases of the project. Mean values within each group were compared with PC as the positive control: ns � not signifcant; * P < 0.05, * * P < 0.01, and * * * P < 0.001. b Reported as mean ± SD.
Evidence-Based Complementary and Alternative Medicine 7