The plant extraction was performed as previously described by Jung et al (15) with a few modifications. CO was obtained from the Dongguk University Oriental Hospital (Korea). The roots of CO (50 g) were blended, and the crude powder was precipitated with 3,000 ml of methanol (80%) at 37°C for 3 days. The methanol extracts were concentrated using a rotary evaporator at 60°C under vacuum conditions. The extract was dissolved in 50 ml of sterile deionized water. The aqueous solution was then lyophilized by freeze-drying at −60°C. The cell culture materials were purchased from Thermo Fisher Scientific (Boston, MA, USA). Antibodies and other laboratory reagents were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively.

Human hepatoma cells (HepG2) were purchased at the Korean Cell Line Bank (Seoul, Korea). The Chang liver cells were obtained from Seoul National University (Seoul, Korea). HepG2 cells and the Chang liver cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cells were maintained under an atmosphere of 95% air/5% CO₂ at 37°C. These cells were trypsinized and maintained every 1–2 days.

The effects of different concentrations of CO (100–1,000 µg/ml) on viability of the Chang liver cells and HepG2 cells were investigated by the 2,3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) assay (EZ-Cytox cell viability assay kit; Daeil Lab Service, Seoul, Korea), respectively. Briefly, 5×10⁴ cells were seeded in a 96-well microplate and incubated in DMEM under 5% CO₂ at 37°C for 24 h. Then the cells were treated with different concentrations of CO (dissolved in DMEM) for 24 h. To measure cell viability, 10 µL EZ-Cytox was added to each well and the absorbance was measured at 460 nm using a microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). The viability of CO-treated cells was expressed as a percentage of the control cell viability.

CO extract-induced apoptosis in HepG2 cells was assessed by Hoechst 33258 staining. The cells were seeded at a density of 5×10⁴ cells/well in a 96-well culture plate and were incubated in DMEM at 37°C in 5% CO₂ for 12 h. Then the cells were pretreated with CO extract (0, 100, 250, 500, 750 and 1,000 µg/ml) and incubated again for 24 h. Following treatment, the media were removed and stained with 1 µM Hoechst 33258 staining at room temperature for 60 min. After which, all the solutions were removed and then washed twice with 100 µl PBS, and observed by the Cytation 3 Cell Imaging Multi-Mode reader (BioTek Instruments, Inc., Winooski, VT, USA).

Flow cytometry analysis was used to measure the proportion of HepG2 cells in the different stages of the cell cycle. HepG2 cells were seeded in 6-well plates at 1×10⁶ cells/ml and incubated for 24 h. The cells were incubated with CO extract (0–1,000 µg/ml) for 24 h, and were then harvested and washed twice with phosphate-buffered saline (PBS). Each sample was fixed in 1 ml of 70% ethanol for 2 h at −20°C and the samples were centrifuged. The collected cells were resuspended in PBS containing 50 µg/ml propidium iodide (PI) and 100 µg/ml RNAse A and incubated in the dark for 30 min at room temperature. Cell cycle was analyzed using the BD FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The data were analyzed using the BD CellQuest Pro software.

Total RNA was isolated from HepG2 cells pre-treated with varying concentrations of the extract using TRI-reagent (MRC, Cincinnati, OH, USA) according to the manufacturer's instructions. Briefly, cDNA was synthesized using 0.5 µg of total RNA and the Improm II reverse transcription system with oligo-deoxythymidine (oligo-dT) primers (Promega, Madison, WI, USA). The following semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) conditions were used for amplification; initial denaturation at 95°C for 10 min and 32 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min and extension at 72°C for 1 min. The following primers were used for RT-PCR; caspase-3, sense primer, 5′-TTTTTCAGAGGGGATCGTTG-3′, and antisense primer, 5′-CGGTTAACCCGGGTAAGAAT-3′; Bcl-2, sense primer, 5′-CTGCGAAGAACCTTGTGTGA-3′, and antisense primer, 5′-TGTCCCTACCAACCAGAAGG-3′; p53, sense primer, 5′-GCTCTGACTGTACCACCATCC-3′, and antisense primer, 5′-CTCTCGGAACATCTCGAAGCG-3′; CDK4, sense primer, 5′-ATGGCTGCCACTCGATATGAACCC-3′, and antisense primer, 5′-GTACCAGAGCGTAACCACCACAGG-3′; cyclin D, sense primer, 5′-AGACCTGCGCGCCCTCGGTG-3′, and antisense primer, 5′-GTAGTAGGACAGGAAGTTGTTC-3′; and GAPDH, sense primer, 5′-CGAGATCCCTCCAAAATCAA-3′, and antisense primer, 5′-AGGTCCACCACTGACACGTT-3′. Transcripts were quantified using electrophoresis on 2% agarose gels. All assays were performed in triplicate.

The lysates from HepG2 cells were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to previously described protocols (16). Supernatant protein concentrations were determined using the Bio-Rad DC protein assay reagents (Bio-Rad, Hercules, CA, USA). The proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK), which were blocked overnight at 4°C in Tris-buffered saline containing 0.1% Tween-20 and 5% bovine serum albumin (BSA). The membranes were then incubated overnight at 4°C with specific antibodies diluted at 1:1,000. Immune complexes were further incubated with their respective peroxidase-conjugated secondary antibodies diluted at 1:1,000 for 1 h. The membranes were analyzed using chemiluminescent reactions (ECL Plus kit; Amersham Pharmacia Biotech), after which protein expressions were visualized and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Determination of standard components by high-performance liquid chromatography (HPLC). The methanol extract of CO was analyzed using an HPLC system (Agilent 1100 series; Agilent Technologies, Santa Clara, CA, USA) equipped with a Phenomenex column (C18, 4.06_250 mm). An isocratic mixture of CH₃CN:MeOH:H₂O (60:30:10) was used as the mobile phase at a flow rate of 1.0 ml/min. Absorbance was measured at 210 nm. Standard stock solutions of cnidilide, ligustilide, neocnidilide, butylphalide, senkyunolide, tetramethylpyrazine, caffeic acid, ferulic acid, and perlolyrine were prepared by dissolving 1 mg/ml of analytical standard in methanol and stored at 4°C prior to use.

The results were expressed as mean ± standard deviation (SD) of three independent experiments. Statistical differences among means were calculated by using ANOVA (one-way) followed by Duncan's multiple range test. The results of cell cycle analysis were tested using Pearson's Chi-square test. Differences with P<0.05 were considered significant. The statistical software package SPSS 10.0 (SPSS Institute, Chicago, IL, USA) was used for these analyses.

The composition of CO extract was analyzed by determining the presence of nine standard compounds, including cnidilide, ligustilide, neocnidilide, butylphalide, senkyunolide, tetramethylpyrazine, caffeic acid, ferulic acid, and perlolyrine by HPLC. As shown in Fig. 1, major four compounds were identified as including ferulic acid, cnidilide, senkyunolide, and ligustilide.

The effect of CO on the Chang liver cell viability was determined by XTT assay. The results indicated that CO was not cytotoxic at concentrations between 100–1,000 µg/ml (Fig. 2A). The viability of HepG2 cells treated for 24 h with increasing concentrations of CO extract was assessed. As shown in Fig. 2B, HepG2 cell viability significantly decreased after treatment with CO extract at 100, 250, 500, 750, and 1,000 µg/ml.

In order to investigate whether CO has an apoptotic effects, HepG2 cells treated with CO extract were stained by Hoechst 33258 staining and identified cells undergoing apoptosis by cell imaging reader. As shown in Fig. 3, the marked cells (yellow color) indicate the apoptotic cells. Apoptotic cells in the group treated with CO extract were increased compared with the untreated group in a dose-dependent manner.

To further examine the apoptotic characteristics in HepG2 cells treated with CO extract, the cell cycle phases were analyzed. As shown in Fig. 4, the percentage of S phase cells reduced to 3.5, 4.4, 5.9, 7.3, and 9.9% at 24 h following treatment with 100, 250, 500, 750, and 1,000 µg/ml CO extract, respectively but did not show any significant difference.

As shown in Fig. 5, mRNA levels of caspase-3 increased to 11.4, 34.5, 49.4, 80.0, and 84.7% and those of p53 to 17.1, 39.4, 51.0, 63.5, and 70.7%, respectively, after treatment with 100, 250, 500, 750, and 1,000 µg/ml CO. Conversely, the same concentrations of CO decreased the levels of Bcl-2 by 9.2, 20.0, 47.6, 62.0, and 64.7%, respectively. In addition, CDK4 expression decreased after treatment with the same concentrations of the CO extract to 14.1, 23.9, 66.0, 87.1, and 86.4%, respectively, and those of cyclin D decreased by 13.6, 24.2, 64.2, 92.8, and 92.0%, respectively.

We analyzed the expression of cell cycle- and apoptosis-related proteins to investigate the molecular mechanism by which CO extract triggered cell cycle regulation and apoptosis in HepG2 cells. The protein levels of caspase-3 and p53 increased after CO treatment in a dose-dependent manner. In contrast, those of Bcl-2 and cell cycle enzymes decreased in a dose-dependent manner after treatment with the same concentrations of CO extract (Fig. 6).

The expression levels of caspase-3 in HepG2 cells were determined following treatment with CO extract using western blot analysis. As shown in Fig. 7, the percentage of cleaved caspase-3 increased to 32.4, 64.3, 93.5, 127.0, and 133.5% following a 24-h treatment with CO extract at concentrations of 100, 250, 500, 750, and 1,000 µg/ml, respectively.