Allergy is a disorder of the immune system often also referred to as atopy. Allergic reactions occur to normally harmless environmental substances known as allergens; these reactions are acquired, predictable, and rapid. Strictly, allergy is one of four forms of hypersensitivity and is called type I hypersensitivity. It is characterized by excessive activation of mast cells and basophils by a type of antibody known as IgE, resulting in an extreme inflammatory response [16]. The cytotoxic effect of PE was evaluated in BMMC by MTS assay (see Section 3). Figure 1 shows that treatment with PE at concentrations of up to 1000 μg/mL did not affect the viability of BMMC.

IL-6 is released in a coordinated network and plays an important role in chronic disease. As such, the pattern of cytokine expression largely determines the nature and persistence of the inflammatory response [17]. IL-6 is a potent mediator of inflammatory processes, and a pleiotropic inflammatory cytokine produced by T cells, macrophages, monocytes, and synovial fibroblasts. To evaluate the effect of PE on the production of IL-6, we pretreated cells with PE (100 or 1000 μg/mL) before stimulation with PMA (50 nM) and A23187 (1 μM) for 6 h, followed by analysis using ELISA. As shown in Figure 2, the level of IL-6 considerably increased after stimulation with PMA plus A23187 in BMMC. Pretreatment of cells with PE (100 or 1000 μg/mL) inhibited these increases in a concentration-dependent and statistically significant manner.

BMMC exhibit biphasic PGD₂ biosynthetic responses over time, in addition to COX-1-dependent immediate and COX-2-dependent delayed responses [18]. The immediate PGD₂ generation occurring within 2 h is associated with the coupling of COX-,1 and the delayed PGD₂ generation, which occurs after several hours of culturing (between 2–10 h), is associated with the de novo induction and function of COX-2 after stimulation with particular stimuli [18]. This cell model also appears to be suitable for assessing the effect of 5-LOX inhibitors, since the immediate LTC₄ generation elicited by the IgE-dependent or cytokine-initiated stimulus occurs in BMMC through 5-LOX [19]. Therefore, the BMMC system is useful for screening selective COX-1/COX-2 or 5-LOX and COX-2/5-LOX dual inhibitors from various sources [20,21]. After stimulation with PMA + A23187, cell-cultured mediums were harvested to measure the level of PGD₂ production. PGD₂ production was clearly detected 2 h after stimulation with PMA + A23187. PGD₂ production also peaked in PMA (50 nM) + A23187 (1 μM). Next, an investigation of the inhibitory effect of PE on the PMA + A23187 induced PGD₂ production in BMMC was performed. Figure 3 shows PE significantly inhibited PMA plus A23187-induced PGD₂ production in dose-dependent manner. These results show that PE can inhibit PMA plus A23187 induced PGD₂ production in BMMC.

Arachidonic acid can also be converted to leukotrienes (LTs) by the action 5-lipoxygenase (LOX) in BMMC. The inhibition of 5-LOX is believed to be the ideal treatment for allergic diseases and asthma [22]. Therefore, the inhibitory activity of PE on the generation of LTC₄ in the BMMC was examined. Figure 4 shows that the BMMC stimulated with PMA + A23187 for 15 min produced approximately 2.3 ng/mL LTC₄, and preincubation of the BMMC with PE resulted in the dose-dependent suppression of this LTC₄ biosynthesis. After stimulation with PMA plus A23187, cell-cultured mediums were harvested to measure the level of LTC₄ production. LTC₄ production was clearly detected 15 min after stimulation with PMA + A23187. LTC₄ production also peaked in PMA (50 nM) plus A23187 (1 μM). Next, an investigation of the inhibitory effect of PE on the PMA and A23187 induced LTC₄ production in BMMC was performed. Figure 4 shows PE significantly inhibited PMA plus A23187-induced LTC₄ production in dose-dependent manner. These results show that PE can inhibit PMA + A23187 induced LTC₄ production in BMMC.

COX-2 is strongly induced in various activated cells. It has been reported that PGD₂, which is the COX-2 metabolite released from activated mast cells, is also essential for the pathogenesis of eosinophilic airway [23]. The inhibitory effect of the PE on PGD₂ production was examined to determine if it is a direct effect of the COX-2 protein or if this inhibition is mediated by some other mechanism. Western blot analysis was performed to determine if the inhibitory effect of PE on PGs were related to modulation of the COX-2 protein expression. As shown in Figure 5, the COX-2 protein was not detected in unstimulated BMMC. However, in response to PMA plus A23187, COX-2 protein was strongly expressed, and COX-2 protein expression was inhibited in a dose-dependent manner by PE.

When mast cells are activated by various stimuli, the release of histamine bears a close parallel to that of β-Hex, which is one degranulation marker [24]. An investigation of the effect of PE on the PMA p+ A23187-induced β-Hex release was performed using ELISA. As shown in Figure 6, pretreatment of BMMC with PE at concentrations of 100 or 1000 μg/mL reduced β-Hex production.

HPLC analysis of saponins from Platycodon root was carried out on a Hitachi L-6200 instrument equipped with an evaporative light scattering detection (ELSD) system and SIL-9A auto injector (Shimadzu, Japan). A Zorbax SB-Aq C₁₈ column (150 × 4.6 mm, 5 μm particle size) from Agilent Technologies (Palo Alto, CA, USA) was used for all separations. HPLC conditions were as follows: eluent A, water; eluent B, acetonitrile; gradient, 0–6 min (10–15% B), 6–50 min (15–25% B), 50–60 min (25–47.5% B), and then equilibrated with 10% B for 8 min at a flow of 1 mL/min. The ELSD was set to a probe temperature of 70 °C, a gain of 7 and the nebulizer gas nitrogen adjusted to 2.5 bar. As shown in Figure 7, peaks were assigned by comparing their retention times with that of each reference compound eluted in parallel with a series of mobile phase and by spiking sample with reference compounds. The standard solutions containing 2–400 μg/mL corresponding to test ranges of 10 saponins were prepared in triplicate and 50 μL of each sample was injected into the HPLC column for the construction of calibration curves and linear ranges. The calibration curves were plotted by the peak area versus concentration of each analyte. The linearity was evaluated by linear regression analysis calculated by the least square regression method.

The samples used in the previous experiment was purchased at a local herbal market and originated from China. In addition, the sample was extracted with 100% methanol [25]. In this experiment, the roots were collected in Korea and were extracted with 70% ethanol. These differences between the samples might affect the variation of saponin composition (Table 1).

Platycodon roots were collected from JinAn, Korea, in February, 2009. They were identified by Dr. D.Y. Kwon. A voucher specimen was deposited in the Laboratory of Herbalogy, College of Pharmacy, Wonkwang University, Iksan, Korea. The dried aerial parts of the Platycodon roots (1 kg) were cut into small pieces and extracted repeatedly with 70% ethanol. The solution was filtered and evaporated in vacuo to yield a powered extract (177.4 g).

Male Balb/cJ mice (20–30 g) were purchased from DAMUL, Korea. All animals were kept in a temperature controlled room under a 12 h light/12 h dark cycle. The animals were provided with free access to commercial solid food (SCF Co., Ltd. Korea) and water ad libitum. Mice were acclimatized for at least one week prior to beginning the experiments.

RPMI 1640, penicillin and streptomycin and FBS (Fetal Bovine Serum) were obtained from Hyclone (Hyclone Labs Logan, UT). Bovine serum albumin (BSA), PMA and A23187 were purchased from Sigma (St. Louis, MO, USA). COX-2 and β-actin monoclonal antibodies and peroxidase conjugated secondary antibody were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

Bone marrow mast cells from male Balb/cJ mice were cultured for up to 10 weeks in 50% enriched medium (RPMI 1640 containing 2 mM L-glutamine, 0.1 mM nonessential amino acids, antibiotics, and 10% fetal bovine serum) and 50% WEHI-3 cell-conditioned medium as a source of IL-3. After 3 weeks, >98% of the cells were BMMC.

The cell viability was examined by a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Briefly, BMMC were seeded at a density of 5 × 10⁵ cells/mL in 96 well plates (Nunc, Denmark). Each batch of cells included a non-treated group as control. PE (100 or 1000 μg/mL) was then added to each well, after which the plates were incubated for 24 h at 37 °C under 5% CO₂. Next, MTS solutions (5 mg/mL) were added to each well. Next, the optical density was read at 490 nm. The cytotoxicity was then calculated by subtracting the ratio of the mean absorbance value for treated cells over the mean absorbance value for untreated cells from one.

Cells were seeded at a density of 1 × 10⁶ cells/mL in 24 well tissue culture plates and then pretreated with various concentrations of PE (100 or 1000 μg/mL) for 30 min prior to PMA (50 nM) plus A23187 (1 μM) stimulation. ELISA plates (Falcon, Becton Dickinson Labware) were then coated overnight at 4 °C with anti-mouse IL-6 antibody diluted in coating buffer (0.1 M carbonate, pH 9.5) and then washed three times with PBS containing 0.05% tween 20. Non-specific protein binding sites were then blocked by incubating the plates with assay diluent (PBS containing 10% FBS, pH 7.0) for at least 1 hour. Immediately following the incubation, each sample and IL-6 standard were added to the wells, after which the plates were incubated for 2 h. Next, working detector (biotinylated anti-mouse IL-6 monoclonal antibody and streptavidin-HRP reagent) was added to each well and the plates were incubated for an additional 1 h. Finally, substrate solution (tetramethylbenzidine: TMB) was added to the wells, after which the plates were then incubated in the dark for 30 min. Next, the reaction was stopped using stop solution (2NH₃PO₄) and the absorbance at 450 nm was then measured. All standards and samples were assayed in duplicate.

To measure the inhibitory activity of PE on COX-2, cells were suspended in enriched medium at 1 × 10⁶ cells/mL and preincubated with aspirin (10 g/mL) for 2 h to irreversibly inactivate any pre-existing COX-1. After washing, the BMMC were activated with PMA (50 nM) + A23187 (1 μM) at 37 °C for 2 h in the presence or absence of PE dissolved in PBS. All reactions were quenched by centrifugation at 890 g at 4 °C for 5 min. The supernatant and cell pellet were frozen immediately in liquid N₂ and stored at −80 °C until needed for further analysis. Under these conditions, the COX-2-dependent phase of PGD₂ generation reached 1.3 ng/10⁶ cells. The data are reported as the arithmetic mean of triplicate determinations.

BMMC suspended in enriched medium at 1 × 10⁶ cells/mL were pretreated with PE for 30 min at 37 °C and stimulated with PMA (50 nM) + A23187 (1 μM). After 15 min of stimulation, the supernatants were isolated for further analysis by enzyme immunoassay (EIA). LTC₄ was determined using an EIA kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s instructions. Under the conditions employed, LTC₄ reached 2.3 ng/10⁶ cells. All data are the arithmetic means of triplicate determinations.

After activation with PMA (50 nM) + A23187 (1 μM), BMMC were washed once with 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl (PBS) and lysed in PBS containing 0.1% SDS and 10 mM β-mercaptoethanol at 1 × 10⁷ cells/mL. The lysate (1 × 10⁵ cells equivalent) was applied to 10% SDS-polyacrylamide gels. After running the gel, the protein bands were blotted onto nitrocellulose membranes (Schleicher and Schull, Dassel, Germany) using a semi-dry blotter (MilliBlot-SDE system, Millipore, Bedford, MA, USA) according to the manufacturer’s instructions. Membranes were then washed once with 10 mM Tris-buffered saline (TBS, pH 7.2) containing 0.1% tween-20 (TBS-T), and then blocked for 1 h in TBS-T containing 3% skim milk. After washing the membranes with TBS-T, an antibody directed against COX-2 was added at a dilution of 1:5000 in TBS-T. After incubation for 2 h followed by washing three times, membranes were treated for 1 h with horseradish peroxide-conjugated goat anti-rabbit IgG (Santa Cruz, CA, USA) (diluted to 1:7000) in TBS-T. The protein bands were visualized using an enhanced chemiluminesence (ECL) system (Amersham Corp., Newark, NJ, USA).

β-Hexosaminidase (β-Hex), a marker of mast cell degranulation, was quantitated by spectrophotometric analysis of p-nitrophenyl-2-acetamido-2-deoxy-β-D-glucopyranoside hydrolysis (Sigma, St. Louis, MO, USA). Briefly, after harvesting the supernatant, cells were lysed in the same volume of medium by three freeze/thaw cycles. Ten milliliters of the BMMC lysate or supernatant samples were mixed with 50 μL of β-Hex substrate solution (1.3 mg/mL p-nitrophenyl-2-acetamido-2-deoxy-β-D-glucopyranoside in 100 mM sodium citrate, pH 4.5) in the wells of 96-well plate and then incubated at 37 °C for 90 min. The reaction was stopped by adding 140 μL of 0.2 M glycine-NaOH (pH 10.7). Absorbance was measured at 410 nm in a microplate reader. The percentage of β-Hex released into the supernatant was calculated by the following formula: [S/(S + P)] × 100, where S and P are the β-Hex contents of the supernatant and cell pellet, respectively.

The chromatographic system consisted of a pump (Shimadzu LC-10AT VP) and a UV detector (Shimadzu LC-SPD-10A VP). A Shim-pack VP-ODS column (4.6 × 250 mm, Shimadzu) was used. An isocratic of CH₃CN:CH₃OH:H₂O (11:11:8) was used as the mobile phase at a flow rate of 1.0 mL/min. The HPLC was monitored at 254 nm.

The data from the experiments are presented as the mean ± S.E.M. The level of statistical significance was determined by analysis of variance (ANOVA) followed by Dunnett’s t-test for multiple comparisons. P values less than 0.05 were considered to be significant.