The Impact of Argan Oil and Polyhexanide on Healing Wounds with Tissue Loss Infected by Staphylococcus aureus in Mice

The wound is defined as the deterioration of soft tissue integrity (Alkan, 1987). One of the most important complications of the wound is infection of the tissue, due to the colonization of pathogenic bacteria in cases of normal skin flora and skin integrity are disturbed. Microorganisms such as Streptococcus and Staphylococcus are responsible for most bacterial soft tissue infections (Citil et al., 2015). Bacterial infection of the wound is a complication that may delay wound healing and result in irreversible tissue loss (Gardner et al., 2001). In addition, wound infection increases the cost of health care seriously. Antibiotic therapy is used as a standard treatment for wound infection control. However, the usage of empirical antibiotics also causes drug resistance development over time (Gardner et al., 2001; Dealey et al., 2004). The reliability of antiseptics in wound treatment should be discussed because of their toxic effects (Asada et al., 2012).

This study aimed to use herbal compositions and antiseptics that reduce these toxic effects in infected wounds. Modern medicine also benefits from plants that have been used in traditional treatments for centuries. The usage of plants has been a guide in the development of many drugs. The chemical structure of their active compounds giving the medical properties of many plants has been determined, isolated, and synthetically produced and started to be used (Yildiz et al., 2015; Mohan and Kumar, 2018; Yavuz et al., 2022). It was reported that argan [(Argonia spinosa (L.) Skeels1911] oil grows naturally in Morocco (Marfil et al., 2011). Virgin argan oil is obtained by a cold-pressed technique (Charrouf and Guillaume, 2009). Interestingly, this oil’s unsaponifiable fraction is notably abundant in antioxidant compounds like tocopherols, which are found in greater quantities than in olive oil (Morton and Voss, 1987; Khallouki et al., 2003). In addition, the therapeutic properties of the argan plant are based on anti-inflammatory and analgesic effects, and it also stimulates granulation and is used in wound healing since it supports tissue regeneration (El Babili et al., 2010; Avsar et al., 2016). One of these antiseptics polyhexanide (polyaminopropyl biguanide, polyhexamethylene biguanide, polyhexamethylene guanide), a modern wound antiseptic, is widely used to prevent bacterial infections and the treatment of mucous membranes and wounds (Horrocks, 2006; Valenzuela and Perucho, 2008; Hubner and Kramer, 2010). It is especially preferred in current treatments of chronic wounds, burn injuries, and methicillin-resistant Staphylococcus aureus (S. aureus) (MRSA) infections (Kramer et al., 2004; Rietkotter et al., 2007). Even at very low concentrations (10 mg/L) it has bactericidal and fungicidal properties. It causes bacterial death by adhering to the cell wall of bacteria disrupting the membrane and reducing the permeability. It also adheres to the DNA of bacteria and creates deadly DNA damage (Allen et al., 2006). It has very low toxicity on human cells and has a broad spectrum against both gram-positive and gram-negative bacteria (Rosin et al., 2002). In a study that was conducted on the model of palate defects in rats; it was reported that the highest rate (87.61%) of decrease in defect size in the group that has been used polyhexanide solution compared to the control (Sahin et al., 2009). Valenzuela and Perucho reported that treatment of chronic skin wounds with 0.1% polyhexanide gel has made a significant reduction in wound size, an increase in granulation tissue, more decrease in bacterial biofilm level compared to the group of physiological saline  (Valenzuela and Perucho, 2008).

In this research, our goal was to explore the effects of argan oil, a herbal drug commonly used in the treatment of various diseases, and polyhexanide which is used as an antiseptic agent in modern medicine, in wounds infected with S. aureus in mice, which causes wound infections in hospitals that are difficult to treat.

Chemicals and ethical approval
 
Argan oil and polyhexanide were purchased from commercial suppliers. Argan oil originates from the south of Morocco. Argan oil is derived from the hard kernel of the fruit by a conventional hand-pressing method and has been used without any prior processing. It is kept in a brown bottle at room temperature to protect it from light. Commercial preparation of polyhexanide was bought and used (Actolind W solution Acto CmbH). Our research was conducted at Çukurova University Health Sciences Experimental Application and Research Center between May and August 2018, after receiving the approval of the Çukurova University Experimental Animals Local Ethics Committee (approval number: 2018-01/05). In our study, 45 albino male BALB/c mice aged 3-4 weeks, weighing 25±5 g, were used. All animals were housed in room conditions to provide standard environmental conditions, and fed with standard food and tap water.

Application of experimental protocol

General anaesthesia was induced by intramuscular administration of a mixture of 50 mg/kg ketamine (Ketalar®) and 10 mg/kg xylazine hydrochloride (Alfazyne®, 2%) in mice. The operation areas of animals were shaved under deep anaesthesia. After the cleaning and disinfection of the wound area, the model described by Park et al. (2012) was applied as an experimental wound model. The dorsal skin of the mouse was picked up at the midline and punched through two layers of skin (10 mm in diameter and 1-2 mm deep). After surgery, mice received carprofen (Adamson et al., 2010) 5 mg/kg dose subcutaneously every day for 14 days for analgesia during all of the experimental procedures.

Preparation of infection strains
 
The type strain ATCC 25923 of S. aureus was cultured on trypticase soy agar plates containing 5% sheep blood by swap. Cultur plates were incubated for 48 h at 37°C under aerobic conditions. After the isolation of pure cultures, identiûcation of S. aureus was performed based on enzymatic activity by catalase and plasma coagulase tests after microscopic examination. A loopful of bacteria was collected on agar plates transferred to 10 mL of nutrient broth and incubated at 37°C for 15-18 h to replicate the organisms. After this incubation period, to be used to create experimental wound infection, bacterial cell concentration was adjusted to Mc Farland 0.5 turbidity equal to 1.5×108 cfu/ml at 590 nm using a UV/vis spectrophotometer. The prepared bacteria suspension was transferred to the surface of the wounds received with a swap to create an infection.
 
SmaI-PFGE
 
All S. aureus (ATCC-25923) strains cultured on TSA and identified by biochemical properties were used for PFGE. The chromosomal DNA had been extracted similarly to the standard protocol described by Mulvey et al. (2001). Electrophoresis was done with a contour-clamped homogeneous electric field (CHEFDRII; Bio-Rad Laboratories, Nazareth, Belgium). PFGE-generated DNA fingerprints were digitized and analyzed with the GelCompar II software system (version 5.0; Applied Maths, Sint-Martens Latem, Belgium) only 3 clusters were classified by using 95% similarity as a cutoff. This result has shown us that the strains isolated from wounds were the same strain as the test strain used to create an infection.
 
The planning of the experimental groups
 
After waiting for a day, to reduce the mortality, a suspension of S. aureus (ATCC) isolated in the microbiology laboratory of the Faculty of Medicine, University of Çukurova was inoculated to wounds on the back of the mice. Inoculation as defined by Calame et al. (1991); a suspension bacteria 2×108 cfu/ml was administered subcutaneously as 100 μl. The wound was moistened with 0.9% saline and the operation terminated later and the mice were left to awaken from the anaesthesia. After 48 hours, wound dressings were opened and a touching sample of the wound for culturing was taken from each rat, then it was platted in the EMB agar in the microbiology laboratory of the Faculty of Medicine, University of Çukurova. This process was repeated every 48 hours until culturing of the S. aureus was achieved. Treatment of mice was started by our drug treatment every 24 hours when S. aureus was detected in culture. Mice were separated from each group before the treatment was started. Mice were sacrificed by applying a high-dose anaesthetic agent at the end of the experiment. The wound was also examined to reveal histopathologic findings of the infection in the wound area of mice.

Control group (n=14): An open ulcerative wound on the back of the mice was created. No drug was administered to mice in this group and during the experimental period of 14 days, the natural healing process in the wound area was observed.

Argan oil group (n=14): Open ulcerative wounds on the back of the mice were created and after 24 hours, a suspension of S. aureus was inoculated. After the infection was established, the treatment was started. 1 mL of argan oil (2 mg/kg dose) was applied daily via a syringe on the wound area. During the experimental period of 14 days, the process in the wound area was monitored.

Polyhexanide group (n=14): Open ulcerative wounds on the back of the mice were created and after 24 hours, a suspension of S. aureus was inoculated. 1 mL of polyhexanide (2 mg/kg) was applied daily via a syringe on the wound area. During 14 days of the experimental period, the process in the wound area was monitored.

It continued to receive wound swab cultures every 48 hours from all mice in the groups. In the same way, the wound diameters were measured and photographed. The progress of infection with cultures, wound diameter measurements, and changes in wound healing were observed.

Wound size measurement

The change in wound size was measured and the wound was evaluated as macroscopically. Wound size was measured as 10 mm on the 1^st day of wound creation. S. aureus (ATCC) was inoculated twice with intervals of 24 and 48 hours on the wound area after 5 days from wound formation. The wound size was measured on the very first day of the infection.

Histopathological evaluation

Seven mice from each group on the 7^th day of drug treatment were sacrificed. Afterward, on the 14^th day of drug application, the remaining mice in the groups were sacrificed with a high-dose of anaesthetic agent. The wound areas of mice were sampled. Macroscopically the tissues were sectioned in 2 mm thickness. Formaline fixed paraffin-embedded blocks were serially sectioned at 5 µm. Routine hematoxylin-eosin (H and E) stained slides were examined for ulceration, inflammation, reepithelization, collagen accumulation, tissue granulation, and angiogenesis in the wound area by light microscopy.
 
Statistical analysis
 
One-way ANOVA was used for statistical analysis, followed by Tukey’s HSD for multiple comparisons. A p-value of less than 0.05 was considered statistically significant. The data are reported as mean ± standard error of the mean (SEM).
 

Wound size measurement and examination

Changes in wound size in all groups are shown in Fig 1. The change in wound size was measured and the wound was evaluated macroscopically. Wound size was measured as 10 mm on the 1^st day of wound creation. S. aureus (ATCC) was inoculated twice with 24 and 48-hour intervals on the wound area after 1 day of wound formation. The wound size was measured on the first day of the infection. No statistically significant difference was observed in wound size among the groups on the first day of the infection (5 days of wound creation) (P>0.05). Treatment was started immediately after the microorganism was determined in microbiological culture (after two infections). The wound healing was more effective in the treatment groups compared to the control group at the 7^th and 14^th days of treatment (P<0.001). The wound healing rate was found to be faster in the argan oil group compared to the polyhexanide group when the two treatment groups were compared (P<0.05), (Fig 1).



Histopathological results

The parameters of the histopathological lesions and the evaluation of the healing are shown in Table 1. It was inoculated with a suspension of S. aureus (ATCC) on the wound area of all mice before leaving the groups. After two times of inoculations, the infection and strain isolated from the wound were the same as shown with PFGE in collected pretreatment samples. In all mice, intense inflammatory cells were observed in histopathological examination (Fig 2). On the 7^th day of drug administration, the formation of re-epithelization was higher in the treatment groups compared to the control group (Fig 3). On the 7^th day of the application of argan oil, intense collagen formation in the dermis is observed and re-epithelization is completed in the histopathological examinations of sections. Granulation tissue and angiogenesis levels were higher than the polyhexanide group (Fig 3A, B). Histopathologic examinations of the polyhexanide group revealed that the surface epithelium was completed and granulation tissue and angiogenesis levels were higher than the control group (Fig 3C, D). On the 7^th day of the drug application, in the histopathologic examination of the sections of the control group appeared that the surface epithelium was not complete. There was no difference in collagen accumulation in the histopathological sections of the three groups (Fig 3E, F). On the 14^th day of drug application, it was revealed that surface epithelialization was completed and collagen accumulation was high in both groups in the histopathologic examination of sections of argan oil and polyhexanide groups. The granulation texture and angiogenesis formation (Fig 4) were higher in the group treated with argan oil according to the Polyhexanide group (Fig 5). On the 14^th day of drug application, epithelization was not completed in the histopathological examination of the wound sections of the control group. Collagen accumulation was at the same level as the treatment groups (Fig 6).













Wound is defined as disruption of tissue integrity, which includes trauma, insect bites, burns, surgery, vaccinations, skin piercing, acne, and infections (Gauglitz et al., 2011). Wound healing is a dynamic and complex process that can be complicated by microorganisms and inflammatory components. It consists from three phases: inflammation, re-epithelization-granulation, matrix formation-tissue formation (Sahin et al., 2009). One of the most important complications that delay wound healing in the inflammatory phase is wound infection (Gardner et al., 2001). Antibiotic therapy is used as a standard control of wound infection. However, by time excessive use of antibiotics leads to the development of drug resistance. Reliability should be discussed because of the toxic effects of existing antiseptics (Asada et al., 2012; Shukla et al., 2022). We also aimed to use herbal compositions and antiseptics with less toxic effects in infected wounds. In wounds with tissue loss, the wound area is regenerated by primary or secondary wound healing. If the gap between the two wound lips is too wide to be approached, the healing of the wounds happens with secondary healing. First, a clot forms after the bleeding in the wound area, and the ulcer area is filled with granulation tissue. In this stage, acute inflammatory reaction, reepithelialization, and angiogenesis occur in the wound site. On the surface of the wound, while epithelial mitosis and migration occur, the granulation tissue underneath proliferates.  This granulation tissue until it reaches the surface, the epithelial cells at the wound edges can not completely cover the wound surface. As a result of the contraction of the fibroblasts formed within granulation tissue, wound contraction is shaped and closure of the ulcer is provided (Sahin et al., 2009).

We have tried to determine the effect of argan oil on the rate of healing of infected wounds with tissue loss in our study which we have experimentally formed.  Argan oil has been shown to provide powerful antioxidant, antithrombotic, and antidiabetic effects (Cherki et al., 2005; Samane et al., 2009; Mekhfi et al., 2012). Because it has antioxidant properties, it is protective against damage caused by free radicals (El Babili et al., 2010). Avsar and colleagues reported that in their studies evaluating the effect of argan oil in the treatment of second-degree burns, argan oil had an anti-inflammatory effect and epithelial regeneration was higher in the argan oil group compared to silver sulfadiazine (Avsar et al., 2016). In our study, the closure of the ulcer occurred in a shorter time in the group that applied argan oil according to the macroscopic findings. Histolophatological findings in the sections of this group supported macroscopic data. It was monitored that from the third day on granulation tissue started to occur and an increase in the following days continued. It is determined that declined over time of epithelial opening surrounding the wound surface. We aimed both to accelerate wound healing and to the treatment of infection by using together argan oil and polyhexanide.

One of these antiseptics, polyhexanide (polyhexamethylene biguanide), is a biguanide derivative that has a broad spectrum with bactericidal and fungicidal effects in wound healing. The spectrum of action includes microorganisms such as Staphylococcus Enterococcus, Pseudomonas aeruginosa and Escherichia coli. Polyhexanide does not adversely affect the formation of granulation tissue and therefore wound healing is not difficult. Since polyhexanide interacts with neutral phospholipids very little, it does not cause toxic effects on human cells (Rosin et al., 2002; Kramer et al., 2004; Allen et al., 2006; Horrocks, 2006; Rietkotter et al., 2007; Valenzuela and Perucho, 2008). In an experimental animal study conducted by Kramer et al., (2004), it was examined the healing effects of polyhexanide and octenedine on standardized skin wounds. Octenidine delayed the wound contraction significantly at the 9^th day of early healing, compared to polyhexanide and placebo when complete wound closure times were examined, it was reported to provide complete wound regeneration longer time than polyhexanide (Kramer et al., 2004). Schmit-Neuerburg et al., (2001) showed that polyhexanide is highly active and that it provides rapid wound healing and cell or tissue tolerability it is good, they studied the effect of gauze compresses soaked in 0.04% polyhexanide in comparison with Ringer solution in a study in 85 patients. The polyhexanide group showed improved wound healing with a significantly more rapid reduction of Gram-positive organisms and better tissue compatibility than the control group. In a study by Daeschlein et al., (2007), in treating second-degree burns, they compared the effects of polihexamethylene biguanide, povidone-iodine, and silver nitrate. It has been reported, that polihexamethylene biguanide has a clear superiority on epithelium formation in long-term burn treatment and the in-vitro tolerance is better. All these studies show the efficacy of polyhexanide in skin antisepsis. In our study similar to the findings of previous researchers (Schmit-Neuerburg et al., 2001; Kramer et al., 2004; Daeschlein et al., 2007). S. aureus (ATCC) could not be isolated during 3 days following wound healing in polyhexanide treated group and the wound healing rate was higher than the control group. This research has shown that polyhexanide can be used in the treatment of wound healing, as well as the antibacterial activity of polyhexanide in wounds with tissue loss infected by S. aureus (ATCC).

In conclusion, we believe that this antiseptic polyhexanide may be a good alternative in cases of widespread skin and wound colonization in wounds due to skin and soft tissue infections. At the same time, we consider that polyhexanide has beneficial effects on wound healing, both due to its antibacterial effect and high tissue compatibility, control of wound infection, and healing by accelerating the reduction of tissue defect diameters in wounds with tissue loss. In our research, it has been shown that argan oil seems to be a good choice, especially as a powerful remedy for the treatment of infected wounds.

We want to thank Çukurova University Health Sciences Experimental Application and Research Center and its staff, as well as Veterinary Health Technician/Operating Room Supervisor Faruk UĞUR, for allowing this study to be carried out.

Additional information 

This research received no grant from any funding agency/sector. The data supporting this study’s findings are available on request from the corresponding author (Selvinaz YAKAN). This study was presented as an oral presentation at the “1st International Conference on Life and Engineering Sciences (ICOLES), Kyrenia, Cyprus, 2018”.

Informed consent

The research was carried out at Çukurova University Health Sciences Experimental Application and Research Center between May and August 2018, after receiving the approval of the Çukurova University Experimental Animals Local Ethics Committee (approval number: 2018-01/05).

The authors declare that they have no conflict of interest. “I (We) declare that this study was done by the authors named in this article and all liabilities about claims relating to the content of this article will be borne by the authors”.