Accelerated Burn Wound Closure in Mice with a New Formula Based on Traditional Medicine.

BACKGROUND: A combination of the oils of sesame, hemp, wild pistachio, and walnut has been used for treatment of skin disorders, including wound burns, in some parts of Kerman, Iran. Evaluation of this remedy in the form of a pharmaceutical formulation in animal models can pave the way for its future application in wound burn healing in humans.
OBJECTIVES: This experimental study investigated the healing potential of a new formula (NF) based on folk medicine from Iran for the treatment of third degree burns in mice. The formula was a combination of the oils of four plants: sesame (Sesamum indicum L.), wild pistachio (Pistacia atlantica Desf.), hemp (Cannabis sativa L.), and walnut (Juglans regia L.).
METHODS: Twenty-four mice were selected based on simple random sampling. Twenty-five percent of the total body surface area was burned by exposure to boiling water, according to the Walker-Mason method. NF and silver sulfadiazine (the positive control) were applied topically twice a day for 21 days. The burned area in the negative control group was left untreated. Epithelialization time and the percent of wound contraction were measured during the treatment period. The process of wound repairing was evaluated using histological (H and E and trichrome staining) and immunohistological (anti-pancytokeratin) methods.
RESULTS: When compared to the controls, NF significantly improved wound contraction after day 10. Epithelialization time in the NF group was significantly faster than in the other groups (20 vs. 25.5 days) (P < 0.001). Histopathological and immunohistochemical findings confirmed the efficacy of the NF.
CONCLUSIONS: A new therapeutic remedy was introduced for the treatment of burn wounds. Further clinical and molecular studies are suggested to determine the exact mechanism(s) involved in the burn wound healing effect of NF.

1. Background

Thermal burn injury is a major cause of death and disability in both developed and developing countries and results in high health care costs (1). Burn management comprises different stages, such as hospitalization for a significant duration, expensive medication, and multiple operative procedures (2, 3). Many therapeutics have been introduced, but healing of burn wounds remains a challenge in modern medicine.

The third-degree type of burn leads to severe damage to the epidermis, dermis, and deeper tissues, including muscles and connective tissues, and it heals with thick scars (4). Healing of any wound, including burn wounds, is a collaborative process involving extracellular and intracellular signals that drive separate inflammatory, proliferative, and remodeling stages (4, 5). Hemostasis is an early event in the inflammatory stage. During this step, growth factors secreted from platelets attract macrophages and neutrophils. The neutrophil influx results in clearance of both cellular debris and bacteria at the damaged area. The macrophages, in turn, secrete factors that promote angiogenesis during wound healing (4, 6-9). Most of these cells that appear at this stage disappear in the proliferative stage, and the wound area becomes filled with new blood vessels (4). These processes of angiogenesis and neo-vascularization are critical determinants of the wound healing outcomes for deep burn injuries (10, 11). The final remodeling stage occurs when fibroblasts migrate into the wound and induce collagen formation. Afterward, fibroblast differentiation into myofibroblasts causes tissue contraction (12).

The use of traditional and alternative medicine in wound therapy is reported in many folklore accounts, including those from Iran. Some recent clinical trials have confirmed the effectiveness of some of these traditional medicines (13). The use of traditional medicine is also viewed as a promising way to reduce the financial burden of wound treatments.

2. Objectives

The aim of this study was to evaluate a new formula (NF) as a topical remedy for the treatment of burn injury. The formulation for NF was based on the information derived from one of the most important Iranian traditional medicine sources: Avicenna’s Canon of medicine (14). NF is a combination of the oils of four plants: sesame (Sesamum indicum L.), wild pistachio (Pistacia atlantica Desf.), hemp (Cannabis sativa L.), and walnut (Juglans regia L.). The criteria for selecting these plants for inclusion in NF were their use in folk medicine and their known beneficial attributes for wound healing, such as their antifungal (15), anti-inflammatory (16, 17), antioxidant (18-20), antimicrobial (15, 21, 22), and healing potential (23-26) properties.

3. Methods

3.1. Plant Material

The plant materials were collected from Jiroft province, Iran, in 2012 and were authenticated by a pharmacognosist (Dr. Mehrabani, herbal and traditional research center, Kerman University of Medical Sciences, Iran). The voucher specimens presented in Table 1 were preserved in the herbarium of the department of pharmacognosy of the School of pharmacy, Kerman University of Medical Science, Kerman, Iran.

Table 1.

Plant Species Used in the Present Study

Plant Samples, Species	Voucher Number	Common Name	Plant Used
Pistacia atlantica Desf.	1824 - 1	Wild pistachio	Fruit
Sesamum indicum L.	1823 - 1	Sesame	Seed
Cannabis sativa L.	1369 - 4	Hemp	Fruit
Juglans regia L.	1844 - 1	Walnut	Seed

3.2. Preparation and Formulation of the Traditional Remedy

NF is a combination of four plant oils at specific ratios: 60% sesame (S. indicum) oil, 20% wild pistachio (P. atlantica) oil, 12 % hemp (C. sativa) oil, and 8% walnut (J. regia) oil. The fixed oils of sesame, wild pistachio, hemp, and walnut were obtained by pressing 3 kilograms of seeds or fruits in a cold press machine (PR500, Germany). The resulting fixed oils were stored in universal dark bottles and refrigerated at 4°C until use. Our experimental study and formulation preparation were carried out under sterile conditions and fine particles were removed from the oil by filtration.

3.3. Animal Study

The male albino mice used in the experimental study weighed 22 to 25g and were 6 to 8 weeks old. The animals were allowed to adapt to their new housing for one week prior to the beginning of the experiment. Animals were housed in a 12:12 light-dark cycle, 25°C room temperature, and 45% humidity. Food and fresh water was provided ad libitum. The research was approved by the ethical committee of Kerman University of Medical Sciences and was conducted according to the international guidelines for laboratory animal use and care (EEC Directive of 1986; 86/609/EEC). Mice were randomly divided into 3 groups, with 8 mice per group, and treated with NF or silver sulfadiazine (SSD; 200 mg/kg/day, as a positive control) or left untreated (as a negative control). Mice were sacrificed at various intervals after thermal injury for histopathological examinations.

3.4. TBSA Calculating

Evaluation of the total body surface area (TBSA) in the experimental mice is essential for the modeling of burn injuries. The relationship between the two-dimensional surface area and three-dimensional body volume can be assessed from the body mass. The burn surface was calculated using the Meeh formula: A = KW2/3, where A= body surface area, K = 10 and W = weight in grams (27). In this study, the burn area covered over 25% of the TBSA.

3.5. Burn Induction

The method was based on that described by Walker-Mason (28). Briefly, the mice were intraperitoneally (i.p.) anesthetized by injection of a cocktail of ketamine (80 mg/kg) and xylazine (1.2 mg/kg), and then the dorsal surface of animals was shaved. The mice were transferred into separated restrainers and 25% of the TBSA was exposed to boiling water (100°C) for 10 seconds (28). All animals were dried immediately and resuscitated via IP injection of 1.0 mL physiological saline. The NF (100 mg/mouse) was applied topically twice a day from wound induction until complete healing. The rate of wound healing was determined by measuring the size of the lesions once every three days. The wound area on day one was considered as 100% and the wound areas on subsequent days were compared with this initial wound area.

3.6. Wound Planimetry

Epithelialization time and wound contraction were measured to assess wound healing. Epithelialization time was monitored until the scars that covered the burn wound surface fell off. Wound contraction was calculated using the following formula:

Concentration (%) = (wound area of day 0 - wound area of day x) / wound area of day 0 × 100

Where x = 0, 4, 7, 10, 14, 18, or 21 (29)

The wound area was measured in mm2 by tracing the wound boundaries using image J software (http://rsbweb.nih.gov/ij) once every three days.

3.7. Histopathology Study

Burned skin samples for histopathological examinations were collected from all groups on the 4th, 12th, and 21st days, after sacrificing the mice. These skin samples were fixed in 10% formaldehyde solution for 48 hours and each was embedded in a paraffin wax block for sectioning. The sections were stained with hematoxylin and eosin (H and E) and Masson trichrome (for detection of collagen fibers) (Olympus BX51). Inflammatory cell (neutrophil) numbers, re-epithelialization, angiogenesis, fibroblast numbers, necrosis, and collagen deposition were evaluated according to a semi-quantitative scoring system: - (absent), Trace ~ weak, + (minimal), ++ (mild), +++ (moderate), and ++++ (marked) (30).

3.8. Immunohistochemistry

Immunohistochemistry was performed on samples obtained at day 21 after burn injury induction. Briefly, sections were incubated overnight at 48°C with an anti-pancytokeratin monoclonal antibody (diluted 1:50 Dako).

3.9. Statistical Analysis

All data were expressed as means ± SD. One-way ANOVA with Scheffe’s post hoc test was used to compare the areas of the wounds. A P value < 0.001 was considered significant.

4. Results

4.1. Wound Contraction

As shown in Table 2, the burned area on the first day did not differ among the three groups (P = 0.34). Wound contraction on different days is shown in Table 2, which indicates that the NF treatment significantly increased wound contraction at days 10, 14, 18, and 21. Wound contraction was significantly lower in the SSD group than in the negative control (P < 0.001) beyond day 18 after burning (Table 2 and Figure 1).

Table 2.

Measurement of Wound Size at 4, 7, 10, 14, 18, and 21 Days After Burning, Presented as Wound Contraction

Groups	Percentage of Wound Contraction (%) (Mean ± SD)
	Day 4	Day 7	Day 10	Day 14	Day 18	Day 21
NF	4.6 ± 1.8	20.7 ± 5.2	65.9 ± 3.8[a]	79.9 ± 4.2[a]	97.3 ± 1.5[a]	99.5 ± 0.8[a]
SSD	5.1 ± 2.8	17.5 ± 2.9	31.5 ± 4.6	44.3 ± 3.9	61.4 ± 3.3[b]	78.0 ± 4.0[b]
Negative control	5.4 ± 1.9	18.8 ± 4.9	37.4 ± .7	56.4 ± 4.1	73.5 ± 3.7	88.4 ± 2.5

Abbreviations: NF, new formula; SSD, silver sulfadiazine.

aSignificant healing occurred in the NF group when compared the other groups (P = 0.000). Data were analyzed by one way ANOVA.

bSignificantly less healing was noted in the SSD compared with the negative control (P = 0.000). Data were analyzed by one way ANOVA.

Figure 1.

Measurement of Wound Size at 4, 7, 10, 14, 18, and 21 Days After Burning, Presented as Wound Size Contraction

*Significant healing occurred in the NF group when compared to the other groups (P < 0.001), # Significantly lower healing occurred with SSD when compared with the negative control (P < 0.001). NF, new formula; SSD, Silver sulfadiazine.

4.2. Epithelialization Time

NF significantly (P < 0.001) decreased the epithelialization time when compared with the other groups (Table 3 and Figure 2).

Table 3.

Effect of NF on Epithelialization Time of Third Degree Burns in Mice[a,b]

Groups	Epithelialization Time, Mean ± SD
NF	20.5 ± 1.37[c,d]
SSD	26.33 ± 0.81
Negative control	25.5 ± 0.83

Abbreviations: NF, new formula; SSD, silver sulfadiazine.

aValues are expressed as mean ± SD.

bNo. of mice = 6.

cP < 0.001 in comparison to the negative control (P = 0.000).

dP < 0.001 in comparison to SSD treatment (P = 0.000).

Figure 2.

Effect of NF on epithelialization time of third degree burns in mice

Values are expressed as mean ± SD, n=6 mice. ***P < 0.001 in comparison to the negative control, ### P < 0.001 in comparison to the SSD treatment. NF, new formula; SSD, Silver sulfadiazine.

Comparison of Wound Closure Among the NF, Negative Control, and SSD Groups.

Representative photographs of the burn wounds at days 1, 12, and 21 after burning in a mouse model. A, NF; B, Negative control; and C, SSD at Day 1. D, NF; E, Negative control; and F, SSD at Day 12. G, NF; H, Negative control; I, SSD at day 21. NF: new formula, SSD: Silver sulfadiazine.

4.3. Histopathological and Immunohistochemical Findings

4.3.1. Day 4 After Burning

The burn areas in the negative control mice showed skin tissue characterized by extensive epidermal and dermal necrosis, with inflammatory cells in the central area. Some neutrophilic infiltration was seen in the peripheral tissues of the necrotic dermis. The burn areas of the mice treated with SSD showed extensive necrosis, along with dense transmural neutrophilic infiltration, especially in the subcutaneous fat. The burn areas in the NF treated group showed skin tissue with extensive necrosis and dense neutrophilic infiltration, with abscess formation in the subcutaneous fat.

4.3.2. Day 12 After Burning

The burn areas in mice treated with NF contained skin tissue showed focal re-epithelialization and marked fibroblastic proliferation. Granulation tissue formation and abscess formation in subcutaneous fat tissues were greater in the NF group than in the SSD group.

4.3.3. Day 21 After Burning

The NF-treated mice showed marked skin tissue re-epithelialization, granulation tissue formation, scattered inflammatory cells infiltration, and collagenization in the dermis and skin appendages. The collagenization and reepithelialization effects were significantly greater in the NF group than in the SSD group. The histopathological results (H and E and Masson’s trichrome staining) are shown in Table 4 and Figure 4. These findings were further confirmed by the immunohistological analysis with anti-pancytokeratin antibody (Figure 5).

Table 4.

Effect of NF Treatment on Burn Healing in Mice[a]

Groups	Day 4			Day 12			Day 21
	Untreated	SSD	NF	Untreated	SSD	NF	Untreated	SSD	NF
Inflammatory	++	+++	++	++	++	++	+	++	Trace
Angiogenesis	Trace	+	+	+	++	+++	++	++	+
Fibroblasts count	Trace	+	+	+	++	+++	++	++	+
Re-epithelialization	-	-	-	+	+	++	+++	++	++++
Necrosis	+++	+++	+++	+++	++	++	+	+	-

aAccording to the pathological findings, the grading of injuries was as follows: - (absent), Trace ~ weak, + (minimal), ++ (mild), +++ (moderate), and ++++ (marked).

Figure 4.

Effect of NF on Skin Repair in Mice

A, SSD; B, Negative control; and C, NF at Day 4 (H and E). D, SSD; E, Negative control; and F, NF at Day 21 (H and E). G, SSD; H, Negative control; I, NF at dat 21 (Trichrome staining). Skin repair after burning was evaluated using histological analysis with H and E (100×) and Masson’s trichrome staining (400×). The NF group showed marked tissue re-epithelialization, granulation tissue formation, scattered inflammatory cell infiltration, and collagenization in the dermis and skin appendages. NF, new formula; SSD, silver sulfadiazine (as positive control).

Figure 5.

Pancytokeratin antibody-labeled section of tissue 21 days after burning from the NF group, showing complete epithelialization, 100× and 400×. NF, new formula.

5. Discussion

This study demonstrated that NF increased the rate of healing of third degree burn wounds. We analyzed the progress in wound healing at different time points throughout the three weeks following treatment. Interestingly, a significantly greater wound healing effect was observed with NF than with SSD, a conventional therapy for burn injuries. Note that NF consists of a mixture of four structural components walnut, wild pistachio, hemp, and sesame oils that each play different roles in wound healing. The long history of the practice of traditional medicine has confirmed the greater effectiveness of a combination of several herbs in a single formula than a single constituent for the management of disease (31, 32).

Beneficial effects in wound healing have been reported for each of the components of NF. For example, sesame seeds contain two unique phenolic substances sesamin and sesamolin that have antioxidant properties and may prevent the spoilage of oils (17, 18). The use of antioxidants has been shown to promote wound healing (33) through the scavenging of free radicals from the site of injury. Free radicals are believed to impair the healing process by damaging cellular membranes, nucleotides, proteins, and lipids (34). Sesame oil also has antibacterial activities against common skin pathogens, such as Staphylococcus and Streptococcus ssp. and against common skin fungi such as athlete’s foot fungus. It also shows anti-viral and anti-inflammatory effects (15, 17, 24).

Wild pistachio is used in traditional medicine to treat swelling, inflammation, and ulcers (24). Studies on the chemical composition of oil from wild pistachio reported that the main fatty acids were oleic and linoleic acids. The oil also showed antioxidant properties due to its total phenol content (35). Pheophytin, which is the major pigment in wild pistachio oil, also shows potent anti-inflammatory activity (35-37). Djerrou and his colleagues (38) showed that Mastic (Pistacia lentiscus L.) fatty oil significantly promoted wound contraction and reduced epithelialization time in a rabbit model.

Walnut oil has long been recognized for its valuable effects on skin and is a common additive in cosmetics. Its moisturizing and free radical scavenging properties have led to its use as a component of moisturizing skin creams, anti-wrinkle treatments, and anticaking products (39). The benefits of walnut oil in cosmetic products may be partly due to the presence of essential fatty acids, including linoleic acid and linolenic acid (40, 41). These acids play a central role in the regulation of trans-epidermal water loss, which is one of the most important functions of the skin (42). The omega-3 and omega-6 fatty acids are also reported to have the potential to change the production rates of eicosanoids, especially prostaglandins, to improve and enhance wound healing (23).

Cannabis has been used in traditional medicine to treat swelling and inflammation (24). Hemp seed oil contains linoleic acid and linolenic acid and is valued primarily for its nutritional properties, as well as for the health benefits (43). Although ∆9-tetrahydrocannabinol (∆9-THC) is commonly accepted as the main factor responsible for the effects of cannabis, other components of this herb influence its pharmacological activity. One of these components is cannabidiol (CBD). Analgesic and anti-inflammatory effects have been reported in animal studies on the CBD present in hemp seed oil (16, 44, 45).

One of the contradictory results of this study was that SSD was less effective in burn wound healing when compared to the negative control group. A deleterious effect of SSD on burn wound healing has been reported in other studies (46). This effect may be related in part to the cytotoxic effects of SSD on skin keratinocytes (47).

Wound healing involves a cascade of events and is characterized by completion of specific biological processes in a certain order and within a certain time frame (48). It is a spontaneous and self-initiated process. However, numerous risk factors can delay the wound healing rate, including infection by organisms of endogenous and exogenous origin (49). For example, Pseudomonas aeruginosa is an important opportunistic pathogen that can seriously affect burn wounds, particularly in immunocompromised patients (50). Under these conditions, acceleration of the wound healing process is vital (51). The aim of burn therapy is to promote the wound healing time in order to reduce post-burn complications such as infection and scarring (52). In line with this fact, burns that heal in less than 21days typically exhibit minimal scar formation (53).

Many previous articles have reported side effects of silver sulfadiazine treatment, including retardation of wound healing, allergic reactions to its sulfadiazine moiety, and silver staining of the treated burn wound. Our NF avoids all these complications. It gave complete contraction and re-epithelialization times of less than 21 days after burning, without leaving any scarring or other post-healing disfigurement. The results of the present study support the introduction of NF as a new therapeutic remedy for the treatment of burn wounds and confirm the usefulness of traditional and folklore information. The limitations of our study include the lack of a scientific survey on a human model of burn wound healing, which could have different wound healing mechanisms when compared to mice. An additional limitation is the small sample size; future work involving a larger sample size is warranted. The advantages of our study include our identification of a traditional formula for burn wound healing with significant results that would be a cost-effective and clinically effective therapy. However, further clinical and molecular studies are needed to determine the exact mechanism(s) involved in the burn wound healing effects of NF.