*4.3. GC*/*MS Analysis*

GC/MS analysis of ECB was performed in a GC-MS (GC: 6890 A, Agilent Technologies, Santa Clara, CA, USA; MS: 5973, Agilent Technologies) using a DB-5MS column (15 m, 0.25 mm i.d., 0.25 μm film thickness). ECB (0.1 μL) was injected on split mode at a 1:30 ratio. The oven temperature was set at 50 ◦C for 1 min, followed by a temperature gradient of 5 ◦C/min. When the temperature reached 160 ◦C, it was kept steady for 20 min. Then, a step of 5.0 ◦C/min was applied until oven temperature was 250 ◦C, where it was kept for 15 min. Helium was used as carrier gas with a flow rate of 1 mL/min. Injector and transfer line temperatures were both set at 280 ◦C. The mass spectrometer operated in the electron impact mode with the electron energy at 70 eV. Identification of volatile components was performed by matching their retention tmes and mass spectra with those of authentic standards.

#### *4.4. Cell Culture*

AsPC-1 human pancreatic adenocarcinoma, HT-29 human colon adenocarcinoma, A549 and NCI-H358 human lung adenocarcinoma, and human L132 normal lung epithelial cells were obtained from the Korean cell line bank (Seoul, Korea) and cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 units/mL), and streptomycin sulfate (100 μg/mL). Cells were maintained at 37 ◦C in a humidified atmosphere of 5% CO2.

#### *4.5. MTT Assay*

To examine cell viability, the MTT assay was conducted using the previous modified method as described by Lee et al. [32]. Cells were harvested during the logarithmic growth phase and seeded in 96-well plates at a density of 2 <sup>×</sup> 104/mL in a final volume of 190 <sup>μ</sup>L/well. After 24 h incubation, 10 <sup>μ</sup><sup>L</sup> ECB, or β-caryophyllene full-range concentration was added to 96-well plates. After 48 h, 50 μL of MTT (5 mg/mL stock solution in PBS) was added to each well for 4 h. Subsequently, the supernatant was removed, and MTT crystals were solubilized with 100 μL anhydrous DMSO each well. The optical density was measured at 540 nm.

#### *4.6. PI Staining Analysis*

After treatment with different concentrations of ECB or β-caryophyllene for various times, the cells were collected with trypsin and rinsed with ice-cold PBS twice. The pellets were resuspended and fixed in 70% EtOH at 4 ◦C overnight. Before detected by flow cytometry, the cells were washed twice with PBS and resuspended in a PI solution containing PI (1 μg/mL) and RNase A (10 μg/mL) for 30 min. The cells were evaluated by fluorescence-activated cell sorting (FACS) cytometer (Beckman Coulter, CA, USA).

#### *4.7. PI and Annexin V Double Staining*

Cells were harvested and washed twice with ice-cold PBS. Cells were resuspended with 1 × binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2), and then the cell suspension (100 μL) was incubated in dark place with 5 μL of annexin V-FITC and 10 μL of PI (50 μg/mL) for 15 min. Cells were analyzed for PI-annexin V double staining using FACS flow cytometry (Becton Dickinson Co, Heidelberg, Germany).

#### *4.8. Determination of Mitochondria Membrane Potential (MMP)*

Cells were collected and washed twice with ice-cold PBS. After treatment of 30 μg/mL ECB for 48 h, the cells were stained with DiOC6 at a final concentration of 30 nM for 30 min at 37 ◦C in the dark. The fluorescence intensity was analyzed with FACS cytometer (Beckman Coulter, CA, USA)

#### *4.9. Western Blot Analysis*

After the treatment of various concentrations of ECB or β-caryophyllene for different times, the cells were lyzed in protein lysis buffer (Intron, Seoul, Korea) for 30 min in ice. Cell debris was removed by microcentrifuge (4 ◦C, 15,000 rpm, 30 min) and then the protein concentration of supernatants was ascertained by bio-rad protein assay reagent, following the manufacturer's instruction. Measures of 20–30 μg of cell extracts were fractionated by 8–15 % SDS PAGE, and transferred to a PVDF. After incubating for 1 h with 5% skim milk in Tween 20/Tris-buffered saline (TBST) at 20 ◦C, the membranes were incubated with primary antibody at 4 ◦C for overnight. Membranes were washed three times with TBST and incubated with secondary antibody for 2 h at 25 ◦C, rewashed three times with TBST. Blots were developed using enhanced chemiluminescence detection agents (Amersham, Buckinghamshire, England).

#### *4.10. Statistical Analysis*

Data are expressed as mean ± S.D. Statistically significant values were compared using one-way ANOVA and Dunnett's post hoc test with the GraphPad Prism 5 statistical software, and *p*-values of less than 0.05 was considered statistically significant.

**Author Contributions:** K.-S.C., J.Y.H., and K.-T.L. were involved in the design of the study and analysis of data. J.-H.L. and H.-J.L. were involved in the technical support and conducted the experiments. K.-S.C., J.Y.H., J.Y.P., J.-H.C., J.H., and K.-T.L. were involved in the writing, reviewing, and revision of the manuscript. H.-J.P. and J.H. were involved in the material support. All authors have read and approved the final submitted manuscript.

**Funding:** This research was supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Science and ICT (NRF-2017R1A5A2014768).

**Conflicts of Interest:** The authors declare no conflict of interest.
