Introduction

Propionibacterium acnes is a skin-colonizing, gram-positive bacterium associated with sebum-excreting pilosebaceous follicles. P. acnes is involved in the development of inflamed lesions during the course of acne, in which an induced inflammatory response is presumably directed against propionibacterial antigens [1]. In contrast, Staphylococcus epidermidis, an aerobic organism, is usually involved in superficial infections within the sebaceous unit [2].

Mild to moderate inflammatory acne sometimes responds well to topical treatment with anti-bacterial agents, usually erythromycin or clindamycin and less often tetracycline [3, 4]. Over the past 2½ decades, the incidence of resistance to erythromycin, clindamycin and/or tetracycline has increased among propionibacterial populations associated with inflammatory acne; this resistance presents a worldwide problem for the treatment of this condition [3, 5]. The development of antibiotic resistance is multifactorial, involving the specific nature of the relationship of bacteria to antibiotics, how the anti-bacterial is used, host characteristics and environmental factors. To overcome the problem of antibiotic resistance, essential oils have been extensively studied as alternative treatments for disease [2, 6]. Therapeutic agents for acne are also employed to inhibit inflammation. However, these remedies have been known to induce side effects. Benzoyl peroxide and retinoid cause xerosis cutis and skin irritation if they are used excessively [7], and several reports also suggest that prolonged use of antibiotics can lead to organ damage and immunohypersensitivity [8–10]. In addition, triclosan is converted into an environmental hormone when exposed to light, inducing severe environmental pollution [10]. Therefore, many researchers have sought to develop therapeutic agents for acne that have high antibacterial and anti-inflammatory activities with no side effects [11–16].

As part of our ongoing alternative medicine programs, we have directed our attention toward the identification of essential oils that combine a relatively narrow spectrum of activity against erythromycin- and clindamycin-resistant strains with good anti-inflammatory activity for potential topical applications in patients with mild to moderate inflammatory acne. Here we report on the anti-bacterial and anti-inflammatory activities of Abies koreana essential oil (AKE) against drug-resistant P. acnes and S. epidermidis.

Experimental Procedures

An ethnobotanical survey was conducted on Jeju Island, South Korea, in October 2007. Voucher specimens were deposited at the herbarium of Jeju Biodiversity Research Institute (Jeju, Korea). The AKE was extracted by hydrodistillation as described by Baik et al. [17]. Briefly, approximately 1 kg of fresh A. koreana leaves was immersed in 3.5 l of distilled water in a 5-l three-neck flask. The sample was steam distilled for 12 h at atmospheric pressure. Gas chromatographic analyses were performed on a Hewlett-Packard 5890 gas chromatograph equipped with a polar Supelcowax column (30 m × 0.25 mm × 0.25 μm), an apolar DB-1HT column (30 m × 0.25 mm) and a split-splitless injection port (split mode). The temperature was set at 40°C for 5 min, ramped to 210°C at 10°C/min and held at 250°C for 28 min. Compounds were identified by their retention indices on both columns and by GC-MS using a Hewlett-Packard MSD 5972 mass spectrometer at 70 eV coupled to an HP 5890GC equipped with a DB-1HT column (30 m × 0.32 mm × 0.1 μm).

Nitric oxide production was assayed in the culture medium of cells stimulated with LPS (1 μg/ml) for 24 h in the presence of AKE. Cytotoxicity was determined using the LDH method. Supernatants were collected and PGE2 concentration in the supernatants was determined by ELISA. iNOS and COX-2 mRNA expression and protein levels were determined by RT-PCR and immunoblotting, respectively. The primers used in this study were: β-actin (forward primer 5′-GTGGGCCGCCCTAGGCACCAG-3′ and reverse primer 5′-GGAGGAAGAGGATGCGGCAGT-3′), iNOS (forward primer 5′-CCCTTCCGAAGTTTCTGGCAGCAGC-3′ and reverse primer 5′-GGCTGTCAGAGCCTCGTGGCTTTGG-3′), COX-2 (forward primer 5′-CACTACATCCTGACCCACTT-3′ and reverse primer 5′-ATGCTCCTGCTTGAGTATGT-3′). PCR products were electrophoresed in 1.5% agarose gels and stained with ethidium bromide. The β-actin, iNOS and COX-2 primers produced the expected amplified products of 603, 496 and 696 bp, respectively. The Student’s t-test and one-way ANOVA were used to determine statistically significant differences between the values for the various experimental and control groups. Data are expressed as means ± standard errors (SEM) and the results are taken from at least three independent experiments performed in triplicate. Values are the mean ± SEM of triplicate experiments. *P < 0.05; **P < 0.01

Results and Discussion

Abies koreana is a shrub or broadly pyramidal evergreen tree endemic to the high mountains in southern Korea, including Mt. Halla. Recent reports demonstrated that A. koreana has several medicinal functions, including anti-tumor [18, 19] and memory-enhancing effects [20]. In addition, Jeong et al. [21] elucidated 68 chemical components and demonstrated the anti-microbial activity of AKE. However, anti-inflammatory effects or anti-bacterial activities against drug-resistant skin pathogens by AKE have not been described. In our study, A. koreana leaves were subjected to steam distillation. Nineteen compounds were identified in A. koreana leaves by GC/MS, representing more than 85.05% of the volatile compounds (Table 1). Bornyl acetate (30.35%) and limonene (18.95%) were the major components. Other chemical components included α-pinene (8.10%), camphene (7.39%), α-elemene (3.51%), alloaromadendrene (2.45%), γ-selinene (2.21%) and borneol (1.96%). In contrast, the main compounds identified by Jeong were borneol (27.9%), α-pinene (23.2%), β-pinene (5.8%), terpinene-4-ol (3.8%), bornyl acetate (3.4%) and α-terpineol (3.1%). This difference in the main components may be due to the provenance of plant, harvest time or development stage, extraction technique, or the use of fresh or dried plant material. These are all factors that influence the chemical composition and biological activity.

Table 1 Chemical composition of the essential oils from A. Koreana
Full size table

The in vitro anti-bacterial activity of AKE against P. acnes and S. epidermidis was assessed by the presence or absence of inhibition zones and by MIC values. The MIC was recorded as the lowest concentration (highest dilution) of essential oils that inhibited visible growth (no turbidity). The MIC of AKE was determined using a two-fold serial dilution method. As shown in Table 2, AKE exhibited excellent anti-bacterial activity against drug-susceptible and -resistant P. acnes and S. epidermidis. We also determined the MIC of the major components of AKE (bornyl acetate, camphene, limonene, and α-pinene) and commercial essential oils (tea tree, lavender, and peppermint oils). In this experiment, the antibacterial activities of AKE were similar to those of the major components of AKE and the commercial essential oils. The MIC of AKE was far superior to those of the other compounds and oils against clindamycin-resistant P. acne SKA4.

Table 2 MIC values of AKE, its components, and commercial essential oils
Full size table

In many studies, anti-inflammatory compounds have been investigated for their potential inhibitory effects in vitro using lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Bacterial LPS is one of the best-characterized stimuli used to induce upregulation of pro-inflammatory proteins such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Inducible COX-2 is responsible for the high prostaglandin levels observed in many inflammatory pathologies. Similarly, iNOS produces large amounts of nitric oxide (NO) and is thought to play a central role in inflammatory disease. Numerous studies have reported that NO and prostaglandin E2 (PGE2) participate in inflammatory and nociceptive events.

Since acne vulgaris is the combined result of a bacterial infection and the inflammatory response to that infection, we next examined whether AKE possessed anti-inflammatory activity. To investigate the effect of AKE on NO production, we measured the accumulation of nitrite, a stable oxidized product of NO, in culture media [22, 23] of RAW 264.7 cells stimulated with LPS for 24 h in the presence or absence of AKE. Nitrite levels in LPS-stimulated cells increased significantly compared to that in control cells. As shown in Fig. 1a, AKE (12.5, 25 and 50 μg/ml) markedly and dose-dependently inhibited LPS-induced NO production by RAW 264.7 cells. COX-2 is induced by cytokines and other activators, such as LPS, in a variety of inflammatory cells, including macrophages, resulting in the release of large amounts of PGE2 at inflammatory sites. Therefore, we examined the effects of AKE on PGE2 production in LPS-stimulated RAW 264.7 macrophages. When macrophages were stimulated with LPS (1 μg/ml) for 24 h, the levels of PGE2 increased in the culture medium. As shown in Fig. 1b, AKE (12.5, 25 and 50 μg/ml) suppressed LPS-induced PGE2 production in a dose-dependent manner. As expected, the reference compounds, NS-398 (COX-2 inhibitor) and 2-amino-4-methyl pyridine (iNOS inhibitor) potently inhibited PGE2 and NO production at 20 μM, respectively.

Fig. 1
figure 1

Inhibitory effects of AKE on iNOS and COX-2 expressions in macrophage RAW264.7 cells. Nitric oxide production was assayed in the culture medium of cells stimulated with LPS (1 μg/ml) for 24 h in the presence of AKE (a). Supernatants were collected and PGE2 concentration in the supernatants was determined by ELISA (b). iNOS and COX-2 mRNA expression (c) and protein levels (d) were determined by RT-PCR and immunoblotting, respectively. Cytotoxicity was determined using the LDH method (e). As positive controls, we used 2-amino-4-methyl pyridine (20 μΜ) and NS398 (20 μΜ), which inhibit NO and PGE2 production, respectively. Values are the mean ± SEM of triplicate experiments. *P < 0.05; **P < 0.01

Full size image

Western blot and RT-PCR analyses were performed to determine if the inhibitory effects of AKE on pro-inflammatory mediators (NO and PGE2) were related to the modulation of iNOS and COX-2 expressions. In unstimulated RAW 264.7 cells, iNOS and COX-2 protein and mRNA were not detected. But, LPS upregulated their protein levels, and pre-treatment with AKE inhibited this upregulation. To determine if the inhibition of LPS-stimulated NO and PGE2 production by AKE was mediated by iNOS and COX-2 gene regulation, RT-PCR analyses were performed. As shown in Fig. 1c, AKE reduced the expressions of iNOS and COX-2 mRNA in a dose-dependent manner. By comparison, AKE did not affect β-actin expression, a housekeeping gene. As shown in Fig. 1d, AKE also dose-dependently reduced the expressions of iNOS and COX-2 protein. In general, these results indicate that the inhibitory effects of AKE on LPS-induced NO and PGE2 production resulted from iNOS and COX-2 suppression. Furthermore, based on the RT-PCR analyses, the mRNA levels of iNOS and COX-2 correlated with their protein levels. The numbers of viable activated macrophages, as determined by LDH assays, were not altered by AKE, indicating that the inhibition of NO synthesis by AKE was not simply due to cytotoxic effects (Fig. 1e). Therefore, the inhibitory effect of AKE on iNOS and COX-2 gene expressions appears to be one of the mechanisms for the anti-inflammatory action of AKE.

We also investigated the in vitro anti-inflammatory effects of tea tree, lavender, and peppermint oils as commercially available essential oils in this study. As shown in Fig. 2, we found that low concentrations of commercial oils also inhibited the production of NO and PGE2 within LPS-stimulated murine macrophages. The inhibitory effect of AKE on NO and PGE2 production is greater than those of commercial oils in this study. Peppermint and tea tree oil exhibited cytotoxicities at 50 μg/ml. Also, many of the major components of AKE, including bornyl acetate, camphene, limonene, and α-pinene, had no inhibitory effect on NO and PGE2 production and caused severe cytotoxicities above 25 μg/ml. Only limonene had an inhibitory effect on PGE2 production (data not shown).

Fig. 2
figure 2

Inhibitory effects of oil extract of lavender (L), peppermint (P), and tea tree (T) oils on nitric oxide and PGE2 production in RAW 264.7 cells. Nitric oxide production was assayed in the culture medium of cells stimulated with LPS (1 μg/ml) for 24 h in the presence of commercial oils (12.5, 25 and 50 μg/ml), NS-398 (20 μM) and 2-amino-4-methyl pyridine (20 μM). Cytotoxicity was determined using the LDH method (b). (c), lavender oil; (d), peppermint oil; (e), tea tree oils. Values are the mean ± SEM of triplicate experiments. *P < 0.05; **P < 0.01

Full size image

IL-1β, IL-6 and TNF-α are produced primarily by activated monocytes or macrophages. Because AKE potently inhibited pro-inflammatory mediators, we investigated its effects on LPS-induced IL-1β, IL-6 and TNF-α release by enzyme immunoassay (EIA). After 18-h pre-incubation, mRNA expression of pro-inflammatory cytokines was determined from an 24-h culture stimulated with LPS (1 μg/ml) in the presence of AKE. After 24-h incubation with both LPS and AKE, TNF-α, IL-1β and IL-6 production were remarkably inhibited in RAW264.7 cells. However, NS-398 and 2-amino-4-methyl pyridine had no effect on inflammatory cytokine production (Fig. 3).

Fig. 3
figure 3

Inhibitory effects of AKE on TNF-α, IL-1β and IL-6 production by RAW 264.7 cells. Cells (1.5 × 105 cells/ml) were stimulated with LPS (1 μg/ml) for 24 h in the presence of AKE (12.5, 25, and 50 μg/ml). Supernatants were collected, and the pro-inflammatory cytokine concentrations in the supernatants were determined by ELISA. Values are the mean ± SEM of triplicate experiments. *P < 0.05; **P < 0.01

Full size image

In conclusion, we demonstrated that AKE has good anti-bacterial and anti-inflammatory effects. Therefore, we suggest that AKE should be explored further as a potential therapeutic agent to promote skin health.