Beta-caryophyllene is a dietary cannabinoid

Abstract

The psychoactive cannabinoids from Cannabis sativa L. and the arachidonic acid-derived endocannabinoids are nonselective natural ligands for cannabinoid receptor type 1 (CB(1)) and CB(2) receptors. Although the CB(1) receptor is responsible for the psychomodulatory effects, activation of the CB(2) receptor is a potential therapeutic strategy for the treatment of inflammation, pain, atherosclerosis, and osteoporosis. Here, we report that the widespread plant volatile (E)-beta-caryophyllene [(E)-BCP] selectively binds to the CB(2) receptor (K(i) = 155 +/- 4 nM) and that it is a functional CB(2) agonist. Intriguingly, (E)-BCP is a common constituent of the essential oils of numerous spice and food plants and a major component in Cannabis. Molecular docking simulations have identified a putative binding site of (E)-BCP in the CB(2) receptor, showing ligand pi-pi stacking interactions with residues F117 and W258. Upon binding to the CB(2) receptor, (E)-BCP inhibits adenylate cylcase, leads to intracellular calcium transients and weakly activates the mitogen-activated kinases Erk1/2 and p38 in primary human monocytes. (E)-BCP (500 nM) inhibits lipopolysaccharide (LPS)-induced proinflammatory cytokine expression in peripheral blood and attenuates LPS-stimulated Erk1/2 and JNK1/2 phosphorylation in monocytes. Furthermore, peroral (E)-BCP at 5 mg/kg strongly reduces the carrageenan-induced inflammatory response in wild-type mice but not in mice lacking CB(2) receptors, providing evidence that this natural product exerts cannabimimetic effects in vivo. These results identify (E)-BCP as a functional nonpsychoactive CB(2) receptor ligand in foodstuff and as a macrocyclic antiinflammatory cannabinoid in Cannabis.

Fig. 1.

Caryophyllane- and humulane-type sesquiterpenes found in C. sativa and numerous other plants. Shown are the chemical structures of the bicyclic sesquiterpenes (E)-β-caryophyllene, (Z)-β-caryophyllene, caryophyllene oxide, and the ring-opened isomer α-humulene (α-caryophyllene).

Fig. 2.

(E)-BCP and (Z)-BCP displace [³H]CP-55,940 from hCB₂ receptors expressed in HEK293 cells. (A) Sigmoidal displacement curves (R² = 0.93 for (E)-BCP and 0.98 for (Z)-BCP) show overall displacement (71% and 84%, respectively) with K_i values of 155 and 485 nM, respectively. Data show mean values of nine measurements ± SEM. (B) Hill plot showing linearized data and the corresponding K_i values. (C) Dixon plot of the competitive binding interaction of (E)-BCP with the CP-55,940 receptor binding site in the CB₂ receptor. Radioligand assays were performed by using 84 pM, 168 pM, and 252 pM [³H]CP-55,940.

Fig. 3.

Model of the putative interaction of (E)-BCP with the CB₂ receptor determined by Surflex–Dock and MD/MM calculations. (E)-BCP is located in the hydrophobic region of the amphipathic CB₂ receptor binding pocket where it closely interacts with hydrophobic residues F117, I198, W258, V113, and M265. In this model, significant π–π stacking interactions between the (E)-BCP double bonds and F117 and W258, respectively, facilitate binding.

Fig. 4.

G protein triggered effects of (E)-BCP upon CB₂ receptor binding. (A) The high-affinity CB ligand WIN55,212–2 and (E)-BCP dose-dependently inhibit forskolin-stimulated cAMP production in CB₂ receptor-transfected CHO-K1 cells. Data are mean values of three independent experiments measured in triplicates ± SEM in a nonlinear dose-response curve (R² > 0.97). (B) Like 2-AG, which was used as positive control, (E)-BCP dose-dependently triggers [Ca²⁺]_i transients in CB₂ expressing HL60 cells. The FACS histogram shows CB₂ immunofluorescence of HL60 cells. Data are mean values of three independent experiments ± SEM shown in a nonlinear dose-response curve (R² = 0.99). (C) No [Ca²⁺]_i transients were induced in HL60 cells devoid of CB₂ surface expression. The FACS histogram shows the lack of CB₂ Ab immunofluorescence in these HL60 cells. (D) Addition of the CB₂-selective antagonist SR144528 (1 μM) inhibited the [Ca²⁺]_i release triggered by (E)-BCP (10 μM) in CB₂-positive HL60 cells. Data are mean values from three independent experiments ± SEM (paired t test **, P < 0.01).

Fig. 5.

Anti-inflammatory effects of (E)-BCP in vitro and in vivo. (A) (E)-BCP (1 h incubation before stimulation) inhibits LPS-stimulated (313 ng/ml) IL-1β and TNF-α protein expression in human peripheral whole blood (determined after 18 h). Prior incubation with the CB₂ receptor antagonist AM630 (5 μM for 1 h) blocked the effect of (E)-BCP. Data show values from three independent experiments ± SEM (paired t test *, P < 0.05; **, P < 0.01; ***, P < 0.001). (B) Intraplantar carrageenan (30 μl)-induced edema formation is inhibited by orally administered (E)-BCP (5 and 10 mg/kg) in C57BL/6J wild-type (Cnr2^+/+) mice but not in CB₂ (Cnr2^−/−) knockout mice. Shown is the mean increase in paw volume over time of at least nine mice per group ± SEM (ANOVA, *, P < 0.05; **, P < 0.01; ***, P < 0.001). (C) Comparison of the effects of oral JWH133 (CB₂ selective agonist, K_i = 3.4 nM) and (E)-BCP on carrageenan (30 μl)-induced edema formation in C57BL/6J wild-type (Cnr2^+/+) mice after 240 min. Shown is the percentage of increase in paw volume (relative to control) of at least nine mice per group ± SEM (paired t test, *, P < 0.05; **, P < 0.01; ***, P < 0.001).