The combination of β-caryophyllene, baicalin and catechin synergistically suppresses the proliferation and promotes the death of RAW267.4 macrophages in vitro

beta-caryophyllene, which is a constituent of many essential oils, has been known to be a selective agonist of the cannabinoid receptor type-2 and to exert cannabimimetic anti-inflammatory effects in animals. The effects of beta-caryophyllene on macrophages have not been investigated to date. This study was undertaken to determine whether beta-caryophyllene exerts suppressive effects on mouse RAW267.4 macrophages in vitro in comparison with other botanical molecules that exert antioxidant and anti-inflammatory effects. The proliferation of RAW267.4 cells was suppressed by culture with beta-caryophyllene (50; 100 and 200 mu g/ml of medium) or curcumin (100 and 200 mu g/ml). Culture with baicalin (1-200 mu g/ml) or (+)-catechin (1-200 mu g/ml) did not exert an effect on RAW267.4 cells. The combination of beta-caryophyllene (1 or 10 mu g/ml) with either curcumin (1 or 10 mu g/ml), baicalin (1 or 10 mu g/ml) or (+)-catechin (1 or 10 mu g/ml) did not exert any suppressive effects on cell proliferation. Of note, the combination of all three agents, beta-caryophyllene, baicalin and (+)-catechin at the concentration of either 1 or 10 mu g/ml was found to exert synergistic suppressive effects on cell proliferation. Moreover, the combination of beta-caryophyllene, baicalin and (+)-catechin synergistically promoted the death of RAW267.4 cells. Such an effect was blocked by culture with caspase-3 inhibitor. The combination of the three agents decreased the protein levels of Akt, mitogen-activated protein kinase (MAPK) and cyclooxygenase (COX)-1 or -2. On the whole, this study demonstrates that the combination of beta-caryophyllene, baicalin and (+)-catechin exerts synergistic suppressive effects on macrophages in vitro. This composition may be a useful as an anti-inflammatory treatment strategy.