Phytoncide was obtained by extracting essential oil from the leaves of C. obtusa and P. densiflora growing in South Korea.

The essential oil of plants is produced by various methods such as steam distillation, critical fluid extraction, expression, and solvent extraction, etc. Among them, steam distillation is known as the most widely used method of essential oil extraction (Božović et al., 2017; Masango, 2005).

In this study, C. Obtusa and P. densiflora essential oils were extracted using steam distillation method. Each essential oil was extracted through the following processes. After the C. Obtusa or P. densiflora needles being mixed with the distilled water and throwing in the steam distillation extraction device, it was heated at 100°C for 2 hours and the steam distillation was enforced. Then, the extracted essential oils were dehydrated over anhydrous sodium sulfate.

Mixed phytoncide was used by blending C. obtusa and P. densifola essential oils extracted by steam distillation at the same ratio.

In order to analyze the phytoncide components of the extracted essential oils, a professional analysis agency (P CAM Korea, Daejeon, Korea) was requested. Table 1 shows the phytoncide components of C. Obtusa essential oil and P. densiflora essential oil. A total of 19 components, including Sabinene, Limonene, and Bornyl acetate, were detected in C. Obtusa essential oil, and a total of 18 components, including alpha-Terpineol, alpha-Terpinolene, and 1,8-Cineole, were detected in P. densiflora essential oil (Table 1).

The bacteria used to determine the antibacterial activities of different phytoncide extracts were as follows: Gram-positive: Staphylococcus aureus and S. taphylococcus intermedius; Gram-negative: Escherichia coli, Proteus mirabilis, and Pseudomonas aeruginosa. All bacterial strains were obtained from the culture collection of the Veterinary Medicine and Microbiology Department (Chungnam National University, Daejeon, Korea). Lysogeny broth (LB) agar plates were prepared by mixing 8 g of nutrient broth and 15 g of agar with 1 L of water, and the medium was autoclaved at 120°C for 30 min. All strains were grown on LB plates at 28°C and cultured at 37°C in LB medium.

The antibacterial activities of P. densiflora and C. obtusa extracts were evaluated using a standard disc diffusion method. The phytoncide extract was incorporated into 6-mm-diameter sterile filter paper discs and dried. Three discs were placed on LB agar plates seeded with bacteria and incubated overnight at 37°C.

All dogs (2–6 years of age) were obtained from the animal reproduction and physiology laboratory of the animal science and biotechnology department (Chung-Nam National University, Daejeon, Korea). This study used ten beagle dogs (2–6 years of age). Beagles were randomly divided into two groups (n = 5 per group). The skin of the beagles in the phytoncide group was sprayed twice a day for 8 weeks. The control group was treated with sterilized distilled water. After 8 weeks, blood was collected from the common carotid artery, centrifuged at 12,000 rpm for 15 min at 4°C, and serum was separated. The serum was separated and stored at −70°C until analysis. Serum was used to assess immune responses and cortisol levels. Body weight was recorded at weeks 0 and 8. All dogs were housed in an approved breeding facility provided by the Chung-Nam National University and were housed in an environment with temperature (22–23°C), humidity (50–60%), and light (12 h light) control. The dogs were provided with a continuous supply of water and fed twice a day.

Blood was collected from the common carotid artery of beagles and centrifuged at 12,000 rpm for 15 minutes at 4°C. The separated serum was commissioned by DKKorea Inc. (Seoul, Korea) and evaluated for white blood cells (WBCs), neutrophils, lymphocytes, and cortisol using an XN-1000 analyzer (Sysmex, Kobe, Japan).

The concentrations of immunoglobulin G (IgG), tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukin (IL)-2 were assessed using an enzyme-linked immunosorbent assay (ELISA) kits following the manufacturer’s instructions (Abcam, Cambridge, UK). Absorbance readings were taken using a BioTek absorbance microplate reader (BioTek, Santa Clara, CA, USA).

All statistical analyses were performed using Sigma Plot software (version 12.0, Systat Software Inc., Chicago, IL, USA). Comparisons between groups were analyzed using the t-test, and the results are expressed as mean ± standard deviation (SD). Graphical representations of the results were made using GraphPad Prism software (version 5.0; GraphPad Software, Inc., San Diego, CA, USA). A probability p-value of less than 0.05 was considered statistically significant.

Phytoncides are known to exhibit antibacterial activity, hence we investigated the antibacterial activity of two phytoncide extracts against a representative bacteria known to cause skin diseases in dogs. It has been shown that phytoncides have an antibacterial effect on oral pathogenic bacteria (Kang et al., 2007). The phytoncide significantly enhanced the antibacterial activity of ampicillin against S. mutans and S. sobrinus. Similarly, phytoncides have an antibacterial effect on Porphyromonas gingivalis, one of the most important causative agents of periodontitis and halitosis (Kim et al., 2007). In evaluating the anti-bacterial activity of P. densiflora and C. obtusa phytoncides, we surprisingly found that anti-microbial activity was the highest in mixed phytoncide extracts, as compared to single-species extracts. The antibacterial activity of phytoncides was confirmed by measuring the diameter of the inhibition zone formed using the paper disc diffusion method when five types of bacteria that can cause skin infections in dogs were exposed to extracts (Fig. 1). The diameter of the inhibition zone was measured more than three times. Korean red pine phytoncide had high anti-bacterial activity in P. seudomonas aeruginosa (11.43 mm) than other phytoncides but no anti-bacterial activity in P. roteus mirabilis alone. Hinoki cypress phytoncide showed antibacterial activity in all bacteria and had high anti-bacterial activity in S. taphylococcus aureus (16.13 mm). However, mixed phytoncide from Korean red pine and Hinoki cypress showed evenly distributed antibacterial activity across all bacteria and high anti-bacterial activity in S. taphylococcus intermedius, P. roteus mirabilis, and E. scherichia coli (13.50 mm, 29.60 mm, and 20.20 mm, respectively). Based on these results, we conclude that the mixed Based on these results, we conclude that the mixed phytoncide extracts of Korean red pine and Hinoki cypress have a strong antibacterial effect against bacteria capable of causing skin infections in dogs. Additionally, these extracts seem to be particularly effective against S. taphylococcus intermedius, P. roteus mirabilis, and E. scherichia coli. These findings suggest that the anti-bacterial action of phytoncides extract from P. densiflora and C. obtusa can be effective in preventing and treating bacterial skin infections by inhibiting bacteria.

Phytoncides affect immune response (Li et al., 2012). Previous studies have shown that phytoncides increase the number of natural killer (NK) cells, a type of cytotoxic lymphocyte, and improve immune responses (Kim et al., 2018; Li, 2010). Since inhalation of phytoncides has been shown to increase the number of NK cells in the body’s circulatory system and help reduce human stress, it has also been shown to be beneficial to humans (Li et al., 2006). Thus, we aimed to evaluate the effects of phytoncides on the immune system of dogs. To investigate the effect of phytoncides on the immune response of dogs, levels of WBC, IgG, IFN-γ, TNF-α, and IL-2 were determined using the serum.

The level of IgG was significantly heightened (approximately 2-fold) in the phytoncide group compared to that in the control group (Fig. 2A). The levels of TNF-α and IL-2 slightly increased in the phytoncide group compared to those in the control group, but the difference was not significant (Figs. 2C, 2D). However, IFN-γ levels were significantly increased (3- to 5-fold) in the phytoncide group compared to the control group (Fig. 2B). Treatment with mixed phytoncides of P. densiflora and C. obtusa was shown to be associated with increased the levels of WBCs, lymphocytes, IgG, and IFN-γ in dogs at 8 weeks compared to levels in the control group.

However, the body weight of dogs has remained constant (Table 2). We also demonstrated that phytoncide treatment significantly increased the proportion of lymphocytes and mononucleotides in WBC and decreased the proportion of granulocytes (Table 3).

WBCs prevent the establishment of an infection and therefore play an important role in the body (Honda et al., 2016). IgG modulates macrophage function and promotes immune response and IFN-γ, TNF-α, and IL-2 are cytokines responsible for immune responses and are produced by activated macrophages or lymphocytes (Anderson et al., 2002; Van der Meide and Schellekens, 1996). These act as important markers of the immune system. Our study indicated that phytoncide exposure may contribute to an improved immune response in dogs.

In humans, phytoncides are well-known for their stress-reducing qualities (Woo and Lee, 2020). A forest environment enriched with phytoncides suppresses sympathetic nervous activity, reduces blood pressure, reduces heart rate, and decreases the level of stress hormones such as cortisol (Antonelli et al., 2019; Farrow and Washburn, 2019; Ideno et al., 2017). Additionally, forest bathing in the presence of phytoncides promotes brain functioning by reducing mental stress (Wen et al., 2019). Previous studies have reported that the inhalation of phytoncide extract from pine trees and cypress trees is partially effective in alleviating stress in college students (Nam and Uhm, 2008). Cortisol is commonly known as a stress hormone and is recognized as a major indicator of stress responses (Payne and Nadel, 2004). Stress impairs immune responses and may promote the initiation and progression of disease (Cohen et al., 2007; Padgett and Glaser, 2003). During stress, cortisol secretion is increased through the increased activation of the hypothalamus-pituitary-adrenal system and sympathetic nervous system (Kudielka and Wüst, 2010). Cortisol plays a crucial role in body and brain function as it regulates numerous underlying processes, such as blood pressure, glucose metabolism, inflammation, and immune responses. Overall, cortisol allows organisms to flexibly adapt to various environmental challenges (Dedovic et al., 2009; Denson et al., 2009; Fraser et al., 1999; Khani and Tayek, 2001; Nijm and Jonasson, 2009). We investigated the effects of phytoncides on stress hormones in dogs. In the present study, we measured the concentration of cortisol in serum after exposure to phytoncides. We found that cortisol levels were significantly lowered (approximately 2-fold) in the phytoncide group compared to levels in the control group at 8 weeks (Fig. 3). These results suggest that phytoncide inhalation can help relieve stress in dogs.