All the chemicals and reagents used in this experiment were purchased from Sigma (St. Louis, MO, USA) unless otherwise specified. Lupenone (purity, 98.0%), lupeol (purity, 98.0%), and taraxerol (purity, 98.0%) (Figure 1) were isolated, purified and identified by analytical chemists in the Laboratory of Natural Product Science, Division of Bioscience, Dongguk University (Gyeongju, Korea). Briefly, roots of Adenophora triphylla var. japonica (cultivated in Jeongsun-gun, Gangwon-do, Korea) were provided and authenticated by Professor Je-Hyun Lee (College of Oriental Medicine, Dongguk University, Korea). Air-dried and chopped roots (300 g) were extracted with each of distilled water and 70% ethanol twice at 95℃ for 3 hours. The combined extracts were filtered and concentrated under reduced pressure to give 31.1 g (10.7%) and 38.4 g (12.8%) of crude extracts, respectively. In order to isolate its active constituents the ethanol extract was suspended in distilled water, then consecutively partitioned with organic solvents to give the corresponding hexane (3.6 g), dichloromethane (0.23 g), ethylacetate (0.09 g), and n-butanol (1.4 g) fractions. The main hexane fraction was subjected to column chromatographic separation on a silica gel column using a stepwise elution with dichloromethane, dichloromethane-MeOH 50:1, and dichloromethane-MeOH 1:1 to yield fractions F1-F12. Fraction F3 was recrystallized with dichloromethane-EtOH (two-phase system) 1:1 to give taraxerol (10.8 mg, 0.3%). Fraction F7 was further chromatographed with dichloromethane to afford five subfractions SF1-SF5. Among the subfractions, SF5 was separated by silica gel column chromatography using dichloromethane and MeOH (10:1 to 1:1 gradient) to furnish lupenone (136.8 mg, 3.8%) and lupeol (28.8 mg, 0.8%). The isolated compounds were identified by comparison of their spectral data with literature values141516.

NCI-H292 cells, a human pulmonary mucoepidermoid carcinoma cell line, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) in the presence of penicillin (100 units/mL), streptomycin (100 µg/mL), and HEPES (25 mM) at 37℃ in a humidified, 5% CO₂/95% air, water-jacketed incubator. For serum deprivation, confluent cells were washed twice with phosphate-buffered saline (PBS) and recultured in RPMI 1640 with 0.2% FBS for 24 hours.

After 24 hours of serum deprivation, cells were pretreated with varying concentrations of lupenone, lupeol, or taraxerol for 30 minutes and treated with TNF-α (0.2 nM), epidermal growth factor (EGF; 25 ng/mL), or phorbol 12-myristate 13-acetate (PMA; 10 ng/mL) for 24 hours in serum-free RPMI 1640. Lupenone, lupeol, and taraxerol were dissolved in dimethylsulfoxide and treated in culture medium (final concentrations of dimethylsulfoxide were 0.5%). The final pH values of these solutions were between 7.0 and 7.4. Culture medium and 0.5% dimethylsulfoxide did not affect mucin gene expression and production from NCI-H292 cells. After 24 hours, cells were lysed with buffer solution containing 20 mM Tris, 0.5% NP-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, and protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA) and collected to measure the production of MUC5AC protein (in 24-well culture plate). The total RNA was extracted for measuring the expression of MUC5AC gene (in 6-well culture plate) by using reverse transcription-polymerase chain reaction (RT-PCR).

Total RNA was isolated by using Easy-BLUE Extraction Kit (Intron Biotechnology, Inc., Seongnam, Korea) and reverse transcribed by using AccuPower RT Premix (Bioneer Co., Daejeon, Korea) according to the manufacturer's instructions. Two micrograms of total RNA was primed with 1 µg of oligo(dT) in a final volume of 50 µL (RT reaction). Two microliters of RT reaction product was polymerase chain reaction (PCR) amplified in a 25 µL by using Thermorprime Plus DNA Polymerase (ABgene, Rochester, NY, USA). Primers for MUC5AC were (forward) 5'-TGA TCA TCC AGC AGG GCT-3' and (reverse) 5'-CCG AGC TCA GAG GAC ATA TGG G-3'. As quantitative controls, primers for Rig/S15 rRNA, which encodes a small ribosomal subunit protein, a housekeeping gene that was constitutively expressed, were used. Primers for Rig/S15 were (forward) 5'-TTC CGC AAG TTC ACC TAC C-3' and (reverse) 5'-CGG GCC GGC CAT GCT TTA CG-3'. The PCR mixture was denatured at 94℃ for 2 minutes followed by 40 cycles at 94℃ for 30 seconds, 60℃ for 30 seconds, and 72℃ for 45 seconds. After PCR, 5 µL of PCR products were subjected to 1% agarose gel electrophoresis and visualized with ethidium bromide under a transilluminator.

MUC5AC airway mucin production was measured by enzyme-linked immunosorbent assay. Cell lysates were prepared with PBS at 1:10 dilution, and 100 µL of each sample was incubated at 42℃ in a 96-well plate, until dry. Plates were washed three times with PBS and blocked with 2% bovine serum albumin (fraction V) for 1 hour at room temperature. Plates were again washed three times with PBS and then incubated with 100 µL of 45M1, a mouse monoclonal MUC5AC antibody (1:200, NeoMarkers, Fremont, CA, USA), which was diluted with PBS containing 0.05% Tween 20 and dispensed into each well. After 1 hour, the wells were washed three times with PBS, and 100 µL of horseradish peroxidase-goat anti-mouse IgG conjugate (1:3,000) was dispensed into each well. After 1 hour, plates were washed three times with PBS. Color reaction was developed with 3,3',5,5'-tetramethylbenzidine peroxide solution and stopped with 1 N H₂SO₄. Absorbance was read at 450 nm.

Means of individual group were converted to percent control and expressed as mean±SEM. The difference between groups was assessed using one-way ANOVA and Holm-Sidak test as a post-hoc test. p<0.05 was considered as significantly different.

As can be seen in Figure 2, MUC5AC gene expression induced by TNF-α from NCI-H292 cells was inhibited by pretreatment with lupenone, lupeol, and taraxerol, respectively (Figure 2). Cytotoxicity was checked by lactate dehydrogenase assay and there was no remarkable cytotoxic effect of lupenone, lupeol, or taraxerol, at the treatment concentrations (data not shown).

Lupenone inhibited TNF-α-induced MUC5AC mucin production. The amounts of MUC5AC mucin in the cells of lupenone-treated cultures were 100±6%, 543±29%, 362±14%, 324±7%, and 114±7% for control, 0.2 nM of TNF-α alone, TNF-α plus lupenone 10^-6 M, TNF-α plus lupenone 10^-5 M, and TNF-α plus lupenone 10^-4 M, respectively (Figure 3A).

Lupeol also inhibited TNF-α-induced MUC5AC mucin production. The amounts of MUC5AC mucin in the cells of lupeol-treated cultures were 100±10%, 405±13%, 337±21%, 307±24%, and 110±11% for control, 0.2 nM of TNF-α alone, TNF-α plus lupeol 10^-6 M, TNF-α plus lupeol 10^-5 M, and TNF-α plus lupeol 10^-4 M, respectively (Figure 3B).

Taraxerol also inhibited TNF-α-induced MUC5AC mucin production. The amounts of MUC5AC mucin in the cells of taraxerol-treated cultures were 100±10%, 405±13%, 315±36%, 290±7%, and 77±7% for control, 0.2 nM of TNF-α alone, TNF-α plus taraxerol 10^-6 M, TNF-α plus taraxerol 10^-5 M, and TNF-α plus taraxerol 10^-4 M, respectively (Figure 3C).

Lupenone significantly inhibited EGF-induced MUC5AC production from NCI-H292 cells. The amounts of mucin in the cells of lupenone-treated cultures were 100±13%, 330±14%, 232±2%, 116±8%, and 75±4% for control, 25 ng/mL of EGF alone, EGF plus lupenone 10^-6 M, EGF plus lupenone 10^-5 M, and EGF plus lupenone 10^-4 M, respectively (Figure 4A).

Lupeol significantly inhibited EGF-induced MUC5AC production from NCI-H292 cells. The amounts of mucin in the cells of lupeol-treated cultures were 100±31%, 369±15%, 340±38%, 336±13%, and 79±18% for control, 25 ng/mL of EGF alone, EGF plus lupeol 10^-6 M, EGF plus lupeol 10^-5 M, and EGF plus lupeol 10^-4 M, respectively (Figure 4B).

Taraxerol significantly inhibited EGF-induced MUC5AC production from NCI-H292 cells. The amounts of mucin in the cells of taraxerol-treated cultures were 100±31%, 369±15%, 344±29%, 327±36%, and 100±11% for control, 25 ng/mL of EGF alone, EGF plus taraxerol 10^-6 M, EGF plus taraxerol 10^-5 M, and EGF plus taraxerol 10^-4 M, respectively (Figure 4C).

Lupenone significantly inhibited PMA-induced MUC5AC production from NCI-H292 cells. The amounts of mucin in the cells of lupenone-treated cultures were 100±14%, 247±2%, 261±1%, 147±8%, and 55±3% for control, 10 ng/mL of PMA alone, PMA plus lupenone 10^-6 M, PMA plus lupenone 10^-5 M, and PMA plus lupenone 10^-4 M, respectively (Figure 5A).

Lupeol significantly inhibited PMA-induced MUC5AC production from NCI-H292 cells. The amounts of mucin in the cells of lupeol-treated cultures were 100±6%, 273±6%, 262±14%, 205±7%, and 123±15% for control, 10 ng/mL of PMA alone, PMA plus lupeol 10^-6 M, PMA plus lupeol 10^-5 M, and PMA plus lupeol 10^-4 M, respectively (Figure 5B).

Taraxerol significantly inhibited PMA-induced MUC5AC production from NCI-H292 cells. The amounts of mucin in the cells of taraxerol-treated cultures were 100±5%, 378±24%, 426±48%, 308±14%, and 121±7% for control, 10 ng/mL of PMA alone, PMA plus taraxerol 10^-6 M, PMA plus taraxerol 10^-5 M, and PMA plus taraxerol 10^-4 M, respectively (Figure 5C).