MATERIALS AND METHODS

Plant materials: An ethnobotanical survey was conducted in the Halla Mountain of Jeju Island from April to December 2012. Voucher specimens were identified by Dr. C.G. Hyun and deposited in Department of Chemistry. The plant was identified immediately after collection and air-dried at room temperature for later analysis. In the case of Prunus yedoensis, only the flower part was used as the material in this experiment. The leaves and branches were used together for the plants Saururus chinensis and Zanthoxylum piperitum.

Supercritical fluid extraction: Each plant material (2,000 g) was milled and sieved. The materials with an average particle diameter of 0.4 mm (collected between sieves of 0.2 and 0.6 mm) were used for the experiments. Extractions with supercritical carbon dioxide (SC CO₂) were performed on laboratory scale equipment. The apparatus was manufactured by Supercritical Laboratory (Model No. SFE0350R1) located in South Korea. The pressure and temperature conditions for the extraction of the first fraction were 10 Mpa and 40°C, respectively while the SC CO₂ flow rate was 0.67 kg h^-1 (sample name: EO-CO₂). After the plant material was exhausted, the pressure was raised to 35 Mpa and the extraction of the second fraction followed. The SC CO₂ flow rate was 0.32 kg h^-1 (sample name: AO-CO₂). Commercial carbon dioxide (99% purity) supplied by Tehnogas (Messer-Tehnogas, Serbia) was used for SC CO₂. Dichloromethane and methanol (GC purity, Sigma-Aldrich, Germany) was used for dissolution of supercritical extracts prior to GC-FID-MS analyses.

Gas chromatography-mass spectrometry: Gas chromatographic analyses were performed on a Hewlett-Packard 5890 gas chromatograph equipped with a polar Supelcowax column (30 mx0.25 mx0.25 μm), an apolar DB-1HT column (30 mx0.25 mm) and a split-splitless injection port (split mode). The temperature was set at 40°C for 5 min, ramped to 210°C at rate of 10°C min^-1 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 mx0.32 mmx0.1 μm). The retention indices and mass spectra of each compound were compared with those in the literature.

Cell culture and viability: Murine RAW 264.7 macrophages were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 U mL^-1 penicillin and 100 μg mL^-1 streptomycin, all from GIBCO (Grand Island, NY, USA), in an incubator at 37°C in a humidified atmosphere of 95% air and 5% CO₂. Cell viability was determined by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. RAW 264.7 cells were cultured in 96-well plates for 18 h, followed by treatment with various concentrations of the PYE, SCE and ZPE. After 24 h incubation, MTT was added to the medium for 4 h. Finally, the supernatant was removed and formazan crystals were dissolved in DMSO. Absorbance was measured at 540 nm. The percentage of cells showing cytotoxicity was determined relative to the control group.

Determination of nitric oxide (NO) products: After pre-incubation of RAW 264.7 cells (2.0x10⁵ cells mL^-1) with LPS (1 μg mL^-1) for 24 h, the quantity of nitrite in the culture medium was measured as an indicator of NO production. Briefly, 100 μL of cell culture medium was mixed with 100 μL of Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride in 2.5% phosphoric acid), the mixture with different concentration (12.5, 25.0 and 50.0 μg mL^-1) of oils was incubated at room temperature for 10 min and the absorbance at 540 nm was measured in a microplate reader. Fresh culture medium was used as a blank in every experiment. The quantity of nitrite was determined from a sodium nitrite standard curve.

Measurement of pro-inflammatory cytokine (TNF-α, IL-1β and IL-6) production: The inhibitory effects of PYE, SCE and ZPE on pro-inflammatory cytokine (IL-1β, IL-6 and TNF-α) production in LPs-treated RAW 264.7 cells were determined by ELISA as described in the manufacturer's instructions (R & D Systems, Minneapolis, MN).

Statistical analysis: The data are expressed as the Mean±Standard Error (SE). A statistical comparison was performed via., a one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (Duncan 1955). p-values of less than 0.05 were considered to be significant.

RESULTS AND DISCUSSION

As part of our ongoing alternative medicine program, we have directed our attention to explore essential oils possessing biological effects including anti-inflammatory, anti-aging and anti-cancer activities (Kim et al., 2009a, 2011; Yang et al., 2010; Yoon et al., 2009, 2010). Herein, we report the chemical compositions and anti-inflammatory activities of the plants, PYE, SCE and ZPE. The essential oils were obtained by supercritical extraction from the blossoms of P. yedoensis, the leaves and branches of S. chinensis and Z. piperitum. The identified compounds and quantitative analytical results from Gas Chromatography equipped with Mass detector (GC/MS) are shown in Table 1-3 according to their elution orders on a DB1-HT column. The GC/MS retention indices were calculated using a homologous series of n-alkanes, C₆-C₃₁.

Analysis of GC/MS for the PYE led to the identification of 10 different components, most of which are volatile monoterpenoids, representing 100% of the total oil (Table 1). The major components detected in the oil were α-pinene (22.38%), 2-β-pinene (20.09%), camphor (13.07%), sylvestrene (7.45%), caryophyllene oxide (9.39%), trans-caryophyllene (8.76%), junipene (7.73%), germacrene-d (4.18%), δ-cadinene (3.60%) and camphene (3.35%).

Table 1:	Chemical composition (%) of Prunus yedoensis essential oils

The general chemical profiles of the SCE including the content percentage and the retention indices are summarized in Table 2. Selecting only components with MS data matches over 80%, a total of 13 components were characterized through a typical library search which represented about 97% of SCE oil. The major components detected in the oil were bicyclic sequiterpenes, δ-cadinol (22.51%) and δ-cadinene (19.74%). On the other hand, C₆-C₉ saturated linear-chain alcohols and their acids such as octanoic acid (13.37%) and n-heptanol (9.82%) were identified as the major components in ZYE (Table 3).

The Nitric Oxide (NO) is produced by activated human macrophages and play critical roles in inflammatory diseases like sepsis and arthritis (Murakami and Ohigashi, 2007). Hence, the inhibition of NO by down-regulation of its synthetic enzyme iNOS in inflammatory cells offers a new therapeutic strategy for the treatment of inflammation.

Table 2:	Chemical composition (%) of Saururus chinensis essential oils

Table 3:	Chemical composition (%) of Zanthoxylum piperitum essential oils

In order to validate the potentials of the above essential oils as anti-inflammatory agents in traditional Korean medicine, we investigated the effects of PYE, SCE and ZPE on the production of NO in LPS-activated RAW 264.7 macrophages. RAW 264.7 cells were treated with various concentrations of samples for 24 h and cell viability was assessed using an MTT assay. The nitrite concentration was determined in the supernatant after treatment with LPS (1 μg mL^-1) alone or co-treated with PYE, SCE and ZPE (3.13-200 μg mL^-1) for 24 h using Griess reagent. As shown in Fig. 1a, the oils PYE, SCE and ZPE suppressed LPs-induced NO formation significantly in dose-dependent fashion. Among these, ZPE gave the most reliable result, where 62% NO inhibition was observed with 25 μg mL^-1 oil sample.

As shown in Fig. 1b, the numbers of viable activated macrophages were never affected by either of PYE, SCE or ZPE as determined by MTT assays, thus indicating that NO inhibition by these essential oils is not caused by cytotoxicity or cell death but is involved by other biological pathways.

It has been reported that cytokines such as TNF-α, IL-6 and IL-1β have proinflammatory effects both in vitro and in vivo. Moreover, TNF-α production is crucial to the synergistic induction of NO synthesis in IFN-γ and/or LPS-stimulated macrophages. TNF-α elicits a number of physiological effects such as septic shock, inflammation, cachexia and cytotoxicity while IL-6 is believed to be an endogenous mediator of LPS-induced fever (Feldmann, 2008; Ding et al., 2009; Kim et al., 2009b; Sugita, 2009; Yoon et al., 2010).

Fig. 1(a-b):	Effects of PYE, SCE and ZPE on the production of nitric oxide (A) and cyotoxicity (B) in RAW 264.7 cells. The production of nitric oxide was assayed from culture medium of cells stimulated with LPS (1 μg mL-1) in the presence of PYE, SCE and ZPE. NO production was determined by ELISA method. Parameters on x-axis denote as follows, LPS (-): without LPS-treatment; LPS (+): LPS-treated; (A): P. yedoensis (12.5 μg mL-1): P. yedoensis (12.5 μg mL-1) added to LPS-treated cell; (B): Control: without additives; P. yedoensis (12.5 μg mL-1): P. yedoensis (12.5 μg mL-1) added to control cell. Cytotoxicity was determined using MTT method. The data represent the Mean±SD of triplicate experiments

Thus, the inhibition of cytokine production or function is a key mechanism in the control of inflammation. In this experiment, levels of IL-1β, TNF-α and IL-6 in the culture supernatants were measured using ELISA kits. Lipopolysaccharide (1 μg mL^-1) stimulation for 24 h led to marked increases of TNF-α, IL-6 and IL-1β levels in the cell supernatants (Fig. 2). As shown in Fig. 2a, treatment of the oils PYE, SCE or ZPE inhibited the production of TNF-α in dose-dependent manner in RAW 264.7 cells. Among the tested samples, SCE exhibited the more prominent inhibition profile. In the examination of IL-6 production, PYE and SCE exhibited considerable inhibition activities but the effect of PYE was rarely appeared (Fig. 2b). Compared to TNF-α and IL-6, the decrease of IL-1β contents by the oils was observed only in a slight degree. The inhibitory effect of PYE was rarely observed in the same way (Fig. 2c).

Fig. 2(a-b):

Effects of PYE; SCE and ZPE on TNF-α; IL-6 and IL-1β production in LPS-stimulated RAW 264.7 cells. The cells were stimulated with 1 μg mL-1 of LPS alone or with a combination of LPS and various concentrations of PYE; SCE and ZPE for 24 h. (a) TNF-α (b) IL-6 and (c) IL-1β produced and released into the culture medium was assayed by the ELISA method. Parameters on x-axis denote as follows; LPS (-): without LPS-treatment; LPS (+): LPS-treated; (a) PYE (25): P. yedoensis oil (25 μg mL-1) added to LPS-treated cell; SCE (25): S. chinensis oil (25 μg mL-1) added to LPS-treated cell; ZPE (3.13): Z. piperitum oil (3.13 μg mL-1) added to LPS-treated cell. The data represent the Mean±SD of triplicate experiments

CONCLUSION

The chemical composition and anti-inflammatory activities of supercritical extracted essential oils from P. yedoensis (PYE), S. chinensis (SCE), Z. piperitum (ZPE) were investigated for the first time. We have demonstrated that treatment of RAW 264.7 cells with PYE, SCE and ZPE can decrease levels of pro-inflammatory mediators and cytokines in LPS stimulated cells. In fact, the oils PYE, SCE and ZPE considerably inhibited the release of NO, TNF-α, IL-6 and IL-1β in a concentration-dependent manner. Based on these findings, essential oils of P. yedoensis, S. chinensis and Z. piperitum appear to possess suitable properties as effective anti-inflammatory agents for the treatment of a variety of inflammatory diseases.

ACKNOWLEDGMENTS

This research was supported by a program of Business for Academic-industrial Cooperative establishment funded by Korea Small and Medium Business Administration in a year of 2011 (2011-00045876).