Inflammatory diseases are associated with cytokine and adhesion molecule expression levels[1–2]. Asthma and chronic obstructive pulmonary disease are obstructive airway diseases involving chronic inflammation of the respiratory tract, but having two distinct modes of action with different patterns of inflammatory cell and mediator involvement being observed. Allergic asthma is characterized by airway hyper-responsiveness to a variety of specific and nonspecific stimuli including chronic pulmonary eosinophilia, elevated serum immunoglobulin E (IgE), and excessive airway mucus production. IgE is an important mediator of allergic reactions including allergic asthma and plays a central role in asthma-related symptoms, airway inflammation, and possibly airway remodeling. The pathophysiology of asthma is thought to be mediated by CD4+T lymphocytes producing a type 2 cytokine profile. Binding of IgE molecules to the surface of an immune cell sensitizes the cell to the specific allergen. The sensitized immune cell immediately expresses an inflammatory response, including the release of histamine, which induces the early phase of an allergic reaction. After IgE release, the immune cells synthesize other inflammatory molecules such as interleukins (ILs)[7–8].
Immunosuppressive drugs are currently being used to control undesired immune responses, such as autoimmune diseases, allergies, and allograft rejection. FK506, cyclophosphamide (CTX) and prednisone are typical immunosuppressive drugs that are being used in the clinical treatment for many years.
Pinus koraiensis (P. koraiensis) also known as the Korean pine, is a species of Pinus in the Pinaceae family. Most Pinus species grow in the northern hemisphere and some have been used in folk medicine for a long time. Previous studies have reported the anti-oxidant and anti-inflammatory activities of the pine pollen and the anti-nociception and anti-inflammatory effects of the pine bark and its essential oil. Larix kaempferi (L. kaempferi) belongs to the family Pinaceae in the Pinopsida class. It is a medium-sized to large deciduous coniferous tree reaching 20-40 meters in height, with a trunk up to 1 meter in diameter. The extract oil from L. kaempferi has been used in folk remedies to reduce allergic reactions. Several previous studies have examined the alleviating effect of P. koraiensis on allergic dermatitis in a mouse model[12,14–15]. In addition, L. kaempferi effectively suppresses the levels of serum IgE and proinflammatory cytokines such as ILs, and affects mast cell appearance[4,16–17]. L. kaempferi, regarded as an effective medicinal plant, contains active terpene compounds with effective pharmacological molecules.
Volatile organic compounds (VOCs) are reported to be associated with asthma and immune responses. However, to the best of our knowledge, there are no reports indicating that exposure to VOCs of P. koraiensis or L. kaempferi reduces inflammatory symptoms and, in particular, affects specific IgE and cytokine release. In addition, the mechanisms underlying the alleviative effects of VOCs have not been elucidated. The aim of the current study was to determine whether exposure to VOCs improves the inflammation in a mouse LPS-induced inflammatory model and is a suitable candidate for use as a pharmaceutical and functional material.
To investigate the anti-inflammatory effects of VOCs of P. koraiensis in the inflammatory BALB/c mouse model, each mouse was administered LPS via the intraperitoneal route. Compared to the VE group, marked induction of serum IgE and PGE2 was observed in the LPS-treated group. The elevated serum IgE levels were recovered in the DEX group (Fig. 1). Treatment with VOCs of P. koraiensis also resulted in a decrease in serum IgE levels. In addition, treatment with VOCs of P. koraiensis and L. kaempferi reduced the PGE2 levels as compared to those in the LPS-treated group. These anti-inflammatory effects on serum IgE and PGE2 levels indicated that exposure to VOCs of P. koraiensis and L. kaempferi relieved the systemic inflammatory condition, suggesting that VOCs of both P. koraiensis and L. kaempferi can be used for their continuous inflammation-relieving effect.
We investigated whether the VOCs of P. koraiensis and L. kaempferi inhibited the expression of inflammatory cytokines in PBMCs of LPS-treated mice. Expression levels of COX-2, TNF-α, IL-1β, and IL-13 mRNA in PBMCs were examined by performing real-time PCR (Fig. 2). Exposure to VOCs of both P. koraiensis and L. kaempferi recovered the COX-2, TNF-α, IL-1β, and IL-13 mRNA expression levels as compared to the LPS-treated group. Based on the observed effects on serum cytokines (Fig. 1) and the changes in expression of inflammatory cytokines in PBMCs, the VOCs of both P. koraiensis and L. kaempferi showed anti-inflammatory properties.
We investigated whether the VOCs of P. koraiensis and L. kaempferi inhibited the expression of pulmonary inflammatory cytokines in PBMCs of nasal-LPS treated mice. We examined the expression levels of COX-2, TNF-α and NF-κB mRNA in lung cells by performing real-time PCR (Fig. 3). Exposure to VOCs of both P. koraiensis and L. kaempferi recovered the COX-2, TNF-α, and NF-κB mRNA expression levels compared to that in the LPS-treated group. These results indicated that VOCs of both P. koraiensis and L. kaempferi exerted an in vivo anti-inflammatory effect in the lungs.
For evaluation of lung damage, especially bronchus thickness and mucus secretion, lung (bronchial) tissues damaged by LPS treatment were stained with H&E. LPS induced an increase in bronchial wall thickness compared to that in the vehicle-treated mice, while the increased bronchial wall thickness was recovered by dexamethasone and VOCs of P. koraiensis and L. kaempferi (Fig. 4). Therefore, anti-inflammatory effects of the VOCs of P. koraiensis and L. kaempferi on nasal LPS induced lung inflammation provided a promising potential for inflammation recovery similar to the positive control, dexamethasone.
For one month, the terpene contents in VOCs were analyzed every other day by entrapping the gas. Quantitative analysis of the components of VOCs of P. koraiensis and L. kaempferi were shown in Table 1. The concentrations and amount of the components that diffused from L. kaempferi were higher than that from P. koraiensis. Both VOCs contained alpha-pinene, beta-pinene, carproaldehyde, limonene, terpinolene, alpha-terpineol, borneol, and camphor. In addition, P. koraiensis emited 3-carene, alpha-terpinene, 1, 8-cineole, isopulegol, pinocarveol, 4-terpineol, verbenone, caryophyllene, and alpha-cedrene, whereas L. kaempferi emited beta-farnesene. The concentrations of VOCs also differed in a closed system; P. koraiensis contained 564.37 ng/L VOC and L. kaempferi contained 80.91 ng/L VOC.
Retention time Compound Pinus koraiensis (ng/L) Larix kaempferi (ng/L) 26.3 N-carproaldehyde 131.24 50.57 29.1 1-hexanol 16.05 7.55 29.68 2-hexenal 0.42 0.17 31.4 Heptanal 5.29 4.00 32.31 α-pinene 74.19 85.64 33.51 Camphene 9.06 2.97 33.95 1-Heptanol 0.98 0.11 34.99 β-pinene 39.48 29.86 36.17 3-carene 54.38 - 36.43 α-terpinene 0.06 - 36.53 Benzaldehyde 9.88 0.96 36.6 2-ethyl-1-hexanol - 0.23 37.02 Limonene 60.11 13.64 37.02 Ocimene 57.96 trace 37.33 Meta-cymene 23.32 - 37.33 Para-cymene 26.09 trace 37.4 β-phellandrene 1.29 11.99 37.84 1, 8-cineole 3.05 - 39.93 Terpinolene 10.41 1.93 40.3 Nonanal 12.16 16.29 43.38 Isopulegol 0.50 - 43.56 Pinocarveol 1.27 - 44.65 Borneol 4.98 0.66 44.65 Camphor 5.08 0.04 44.65 4-Terpineol 0.53 - 45.1 α-terpineol 16.52 3.27 47.44 Verbenone 0.69 - 49.38 Bornyl acetate 1.95 0.10 49.55 Isobornyl acetate 2.47 0.45 51.98 α-Longipinene 9.76 0.18 52.76 Geranyl acetate 0.52 - 55.64 β-farnesene - 1.85 56.55 Caryophyllene 20.76 - 56.55 α-cedrene 4.42 - 66.33 Globulol - 0.19 Total 604.87 232.65
Table 1. Contents in VOCs in P. koraiensis and L. kaempferi panel