Dulbecco's modification of Eagle's medium (DMEM) with 4.5 g/l glucose, L-glutamine and sodium pyruvate and antibiotics [penicillin and streptomycin (P/S)] were purchased from Corning (Mediatech, Inc., Manassas, VA, USA). Fetal bovine serum (FBS) was from HyClone (Logan, UT, USA). β-caryophyllene, baicalin, (+)-catechin, curcumin, and (+)-aromatic turmerone were obtained from Cayman Chemical (Ann Arbor, MI, USA). TNF-α was obtained from R&D Systems (Minneapolis, MN, USA). Sodium butyrate, roscovitine, sulforaphane, PD98059, wortmannin, dibucaine, and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified. The caspase-3 inhibitor (Caspase 3/CPP32 inhibitor W-1, biotin conjugate; Wako Pure Chemical Industries, Ltd., Osaka, Japan) was diluted in phosphate-buffered saline (PBS) and the other reagents were dissolved in 100% ethanol or dimethyl sulfoxide for use in the experiments.

Mouse RAW267.4 cells (murine macrophages) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) (9,10).

The RAW267.4 cells (1×10⁵/ml/well) cells were cultured using a 24-well plate in DMEM containing 10% FBS and 1% P/S for 1, 2, 3 or 7 days in a water-saturated atmosphere containing 5% CO₂ and 95% air at 37°C, as previously described (11). The RAW267.4 cells were cultured in DMEM containing 10% FBS and 1% P/S in the presence or absence of absence of either the vehicle (0.1% ethanol as a final concentration), β-caryophyllene [1 (5 µM), 10, 50, 100 or 200 µg/ml of medium], baicalin [1 (2.24 µM), 10, 50, 100 or 200 µg/ml], (+)-catechin [1 (3.45 µM), 10, 50, 100 or 200 µg/ml], curcumin [1 (2.72 µM), 10, 50, 100 or 200 µg/ml], or (+)-aromatic turmerone [1 (4.62 µM), 5, 10, 25 or 50 µg/ml] for 3 days. In separate experiments, the RAW267.4 cells were cultured in DMEM containing 10% FBS and 1% P/S with either the vehicle (0.1% ethanol as a final concentration) or mixture of β-caryophyllene (10 µg/ml of medium), baicalin (10 µg/ml) and (+)-catechin (10 µg/ml) in the presence or absence of either sodium butyrate (10 and 100 µM), roscovitine (10 and 100 nM), sulphoraphane (1 and 10 nM), dibucaine (0.1 or 1 µM), PD98059 (1 or 10 µM), or wortmannin (0.1 or 1 µM) for 3 days. Following culture, the cells were detached from the culture dishes and counted using the same method as described below in 'Cell counting'.

The RAW267.4 cells (1×10⁵/ml/well) were cultured using a 24-well plate in DMEM containing 10% FBS and 1% P/S for 3 days until they reached confluence, as previously described (12), and the cells were then cultured for an additional 2 days in the presence or absence of absence of either the vehicle (0.1% ethanol as a final concentration), β-caryophyllene (1 or 10 µg/ml of medium) and/or baicalin or (+)-catechin (1 or 10 µg/ml of medium) with or without caspase-3 inhibitor (10 µM). Following culture, the cells were detached from the culture dishes.

Following trypsinization of each culture dish using 0.2% trpysin plus 0.02% EDTA in Ca²⁺/Mg²⁺-free PBS for 2 min at 37°C, the detached cells from the dish were collected following centrifugation at 150 × g for 5 min at a room temperature, as previously described (12,13). The cells were resuspended in PBS solution and stained with eosin. Cell numbers were counted under a microscope (Olympus MTV-3; Olympus, Tokyo, Japan) using a hemocytometer plate (12,13). For each dish, we took the average of two countings. The cell number was calculated as the number of cells/well of each plate.

The RAW267.4 cells (1×10⁵ cells/well in 2 ml of medium) were cultured in DMEM containing 10% FBS and 1% P/S in the presence or absence of either the vehicle (0.1% ethanol as a final concentration), β-caryophyllene (1 µg/ml of medium), baicalin (1 µg/ml), and/or (+)-catechin (1 µg/ml) for 3 days. In a separate experiment, the cells were cultured in the presence or absence of a combination of β-caryophyllene (1 µg/ml), baicalin (1 µg/ml) and (+)-catechin (1 µg/ml) for 3 days, and then were they cultured for an additional 24 h with or without the addition of TNF-α (10 ng/ml). Following culture, the cells were washed twice with ice-cold PBS and removed from the dish by scraping in cell lysis buffer containing protein inhibitors. The lysed cells were homogenized by sonication in 0.4 ml of ice-cold cell lyses buffer containing protein inhibitors. The homogenate was centrifuged for 5 min at 1,500 × g, and the protein concentration of the supernatant was determined for western blotting using bovine serum albumin as a standard. Samples of supernatant protein (30 µg/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nylon membranes for western blotting using specific rabbit antibodies against Akt, mitogen-activated protein kinase (MAPK), caspase-3, cyclooxygenase (COX)-1, -2, p65, and β-actin (all form Cell Signaling Technology, Danvers, MA, USA). β-actin was used as a loading control. Donkey anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as the secondary antibody for all the above antibodies. A minimum of 2 blots from independent experiments was scanned on an Epson Perfection 1660 Photo scanner, and bands quantified using ImageJ. Data from independent experiments were normalized to a percentage of control before averaging.

Statistical significance was determined using GraphPad InStat version 3 for Windows XP (GraphPad Software Inc., La Jolla, CA, USA). Multiple comparisons were performed by one-way analysis of variance (ANOVA) with Tukey-Kramer multiple comparisons post test for parametric data as indicated. A value of p<0.05 was considered to indicate a statistically significant difference.

The effects of various botanical molecules [β-caryophyllene, baicalin, (+)-catechin, curcumin and (+)-aromatic turmerone], which exert anti-inflammatory effects on RAW267.4 cells in vitro have not been investigated thus far. Thus, to determine these effects, the cells were cultured for 3 days in the presence or absence of each compound. The proliferation of the RAW267.4 cells was suppressed in the presence of β-caryophyllene (50, 100 and 200 µg/ml of medium) (Fig. 1A) or curcumin (100 and 200 µg/ml) (Fig. 1B). Culture with baicalin (1, 10, 50, 100 and 200 µg/ml) (Fig. 1C) or (+)-catechin (1, 10, 50, 100 and 200 µg/ml) (Fig. 1D) did not exert a significant effect on RAW267.4 cell proliferation. Culture with (+)-aromatic turmerone (25 and 50 µg/ml) significantly increased the proliferation of RAW267.4 cells (data not shown).

Subsequently, the effects of the combination with various botanical molecules on the proliferation of RAW267.4 cells in vitro were determined. Cell proliferation was not suppressed by the combination of β-caryophyllene (1 or 10 µg/ml) with either baicalin (1 or 10 µg/ml) (Fig. 2A), (+)-catechin (1 or 10 µg/ml) (Fig. 2B), curcumin (1 or 10 µg/ml) (Fig. 2C) or (+)-aromatic turmerone (1 and 10 µg/ml) (Fig. 2D).

Notably, the combination of baicalin (80 or 160 µg/ml) and (+)-catechin (20 or 40 µg/ml) at comparatively higher concentrations exerted a significant suppressive effect on cell proliferation (Fig. 3A). Moreover, the combination of β-caryophyllene, baicalin and (+)-catechin at a lower concentration of each (1 or 10 µg/ml), which did not independently exert a significant effect on cell proliferation, was found to exert a potent synergistic suppressive effect on cell proliferation (Fig. 3B). Such an effect was also observed with a higher concentration (100 µg/ml) of β-caryophyllene, baicalin and (+)-catechin (Fig. 3B). In addition, the synergistic effects of β-caryophyllene, baicalin and (+)-catechin on cell proliferation were also observed with the combination of β-caryophyllene (1 and 10 µg/ml), baicalin (0.8 or 8 µg/ml) and (+)-catechin (0.2 or 2 µg/ml) (Fig. 3C). The synergistic effects observed with the combination of β-caryophyllene, baicalin and (+)-catechin on cell proliferation were not enhanced in the presence of curcumin (10 µg/ml) (Fig. 3D). Thus, the combination of β-caryophyllene, baicalin and (+)-catechin was shown to exert a potent synergistic suppressive effect on RAW267.4 cell proliferation in vitro.

The proliferation of the RAW267.4 cells was determined in the presence of various inhibitors that induce cell cycle arrest in vitro (Fig. 4). The cells were cultured for 3 days in the presence of butyrate (10 and 100 µM), roscovitine (10 and 100 nM), or sulforaphane (1 and 10 nM). The proliferation of RAW267.4 cells was suppressed in the presence of these inhibitors (Fig. 4A). Such effects were not altered in the cells cultured in the presence of the combination of β-caryophyllene, catechin and baicalin (Fig. 4B). Thus, the suppressive effects of the combined chemicals on the proliferation of RAW267.4 cells were not altered in the presence of butyrate, roscovitine or sulphoraphan, that induce cell cycle arrest. Roscovitine is a potent and selective inhibitor of the cyclin-dependent kinases, cdc2, cdk2m and cdk5 (13). Sulforaphane induces G2/M phase cell cycle arrest (14). Butyrate inhibits G1 progression (11). In this study, the combined use of the botanical molecules was found to induce G1 and G2/M phase cell cycle arrest in RAW267.4 cells.

Subsequently, to further determine the mechanistic characteristics responsible for the suppressive effects of the combined use of botanical molecules on cell proliferation, we examined whether the suppressive effects of the combined chemicals on the proliferation of RAW267.4 cells are modulated through various signaling factors that suppress cell proliferation. The proliferation of RAW267.4 cells was suppressed in the presence of wortmannin (0.1 or 1 µM), an inhibitor of phosphatidylinositol 3-kinase (PI3K) (15), PD98059 (1 or 10 µM), an inhibitor of extracellular signal-regulated kinase (ERK) and mitogen-activated protein kinase (MAPK) (16), or dibucaine (0.1 or 1 µM), an inhibitor of Ca²⁺/calmodulin-dependent protein kinase (11) (Fig. 5A). The suppressive effects of these inhibitors on cell proliferation were not observed when used in combination with β-caryophyllene, catechin and baicalin (Fig. 5B). The combined use of the botanical molecules was found to exert suppressive effects on cell proliferation by inhibiting various intracellular signaling pathways, such as the PI3K/Akt, ERK/MAPK and Ca²⁺ pathways in RAW267.4 cells.

To determine whether the combination of β-caryophyllene, baicalin and (+)-catechin induces apoptotic cell death in vitro, RAW267.4 cells were cultured for 3 days until they reached confluence, and the cells were then cultured for an additional 2 days in the presence or absence of either β-caryophyllene (10 µg/ml), baicalin (10 µg/ml), or (+)-catechin (10 µg/ml) with or without caspase-3 inhibitor (10 µM) (Fig. 6). The addition of β-caryophyllene, baicalin or (+)-catechin alone did not cause a significant alteration in the number of RAW267.4 cells (Fig. 6A). However, the combination of β-caryophyllene (10 µg/ml), baicalin (10 µg/ml) and (+)-catechin (10 µg/ml) caused a significant decrease in cell number, indicating that this treatment induces cell death. Culture with curcumin (10 µg/ml) did not exert an effect on cell death (Fig. 6A). Of note, the suppressive effects of combination of the botanical molecules on cell death were completely prevented in the presence of caspase-3 inhibitor (Fig. 6B). These results suggest that the combined use of the botanical molecules induce cell death related to caspase-3, that activates nuclear DNA fragmentation, which induces apoptotic cell death (17).

Alterations in the expression of key proteins related to cell proliferation, apoptotic cell death and inflammation were examined by western blot analysis (Fig. 7). The combination of β-caryophyllene (1 or 10 µg/ml), baicalin (1 or 10 µg/ml) and (+)-catechin (1 or 10 µg/ml) exerted a suppressive effect on Akt, p44/42 MAPK, p38 MAPK and COX-1 and -2 expression (Fig. 7A). Culture with this combination increased caspase-3 expression, but did not alter the levels of nuclear factor-κB (NF-κB) p65 (Fig. 7A). Moreover, culture with TNF-α (10 ng/ml) caused an increase in COX-2 and NF-κB p65 expression (Fig. 7B). This effect was suppressed by the combination of β-caryophyllene, baicalin and (+)-catechin at the concentration of 1 µg/ml (Fig. 7B).