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)-β-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 π–π 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. Cannabis

CB2 cannabinoid receptor

foodstuff

inflammation

natural product

Plant essential oils are typically composed of volatile aromatic terpenes and phenylpropanoids. These lipophilic volatiles freely cross cellular membranes and serve various ecological roles, like plant-insect interactions (1, 2). The sesquiterpene (E)-β-caryophyllene [(E)-BCP] (Fig. 1) is a major plant volatile found in large amounts in the essential oils of many different spice and food plants, such as oregano (Origanum vulgare L.), cinnamon (Cinnamomum spp.) and black pepper (Piper nigrum L.) (3–5). In nature, (E)-BCP is usually found together with small quantities of its isomers (Z)-β-caryophyllene [(Z)-BCP or isocaryophyllene] and α-humulene (formerly α-caryophyllene) or in a mixture with its oxidation product, BCP oxide (Fig. 1). Because of its weak aromatic taste, (E)-BCP is commercially used as a food additive and in cosmetics (6). (E)-BCP is also a major component (up to 35%) in the essential oil of Cannabis sativa L (7). Although Cannabis contains >400 different secondary metabolites, including >65 cannabinoid-like natural products, only Δ9-tetrahydrocannabinol (THC), Δ8-tetrahydrocannabinol, and cannabinol have been reported to activate cannabinoid receptor types 1 (CB 1 ) and 2 (CB 2 ) (8). Here, we show that the essential oil component (E)-BCP selectively binds to the CP55,940 binding site (i.e., THC binding site) in the CB 2 receptor, leading to cellular activation and antiinflammatory effects.

CB 1 and CB 2 cannabinoid receptors are GTP-binding protein (G protein) coupled receptors that were first cloned in the early 1990s (9, 10). Although the CB 1 receptor is expressed in the central nervous system and in the periphery, the CB 2 receptor is primarily found in peripheral tissues (11). In vivo, CB receptors are activated by arachidonic acid-derived endocannabinoids, such as 2-arachidonoyl ethanolamine (anandamide or AEA) and 2-arachidonoylglycerol (2-AG) (12, 13). In addition to a wide range of primarily CB 1 receptor-mediated physiological effects on the central nervous system, different cannabinoid ligands have been reported to modulate immune responses (14). In particular, CB 2 receptor ligands have been shown to inhibit inflammation and edema formation (15), exhibit analgesic effects (16), and play a protective role in hepatic ischemia-reperfusion injury (17). In the gastrointestinal tract, CB 2 receptor agonists have been shown to prevent experimental colitis by reducing inflammation (18). Moreover, the CB 2 receptor has been described as a potential target for the treatment of atherosclerosis (19) and osteoporosis (20). Consequently, CB 2 receptor-selective agonists that are devoid of the psychoactive side effects typically associated with CB 1 receptor activation are potential drug candidates for the treatment of a range of different diseases.

Materials and Methods Drugs and Antibodies. See SI Materials and Methods . Data Analysis. Results are expressed as mean values ± SD or ± SEM for each examined group. Statistical significance of differences between groups was determined by the Student's t test (paired t test) with GraphPad Prism4 software. Outliners in a series of identical experiments were determined by Grubb's test (ESD method) with alpha set to 0.05. Statistical differences between treated and vehicle control groups were determined by Student's t test for dependent samples. For animal experiments, statistical differences between treated and vehicle control groups were determined by repeated measurements (ANOVA) and post hoc least square difference tests. Differences between the analyzed samples were considered as significant at P ≤ 0.05. Nonlinear regression analysis (curve fitting) was performed with GraphPad Prism4 software. Cell Cultures. See SI Materials and Methods . FACS Analysis of CB 2 Expression. Cellular surface expression of the CB 2 receptor was quantified by immunofluorescence as described in ref. 29 (for additional details, see SI Materials and Methods ). Radioligand Displacement Assays on CB 1 and CB 2 Receptors. [3H]CP-55,940 binding and displacement experiments were performed as described in ref. 29. (For additional details, see SI Materials and Methods .) Data were fitted in a sigmoidal curve and graphically linearized by projecting Hill plots, which for both cases allowed the calculation of IC 50 values. Derived from the dissociation constant (K D ) of [3H]CP-55,940 (0.39 nM) and the concentration-dependent displacement (IC 50 value), inhibition constants (K i ) of competitor compounds were calculated by using the Cheng–Prusoff equation [K i = IC 50 /(1 + L/K D )] (40). Molecular Modeling. The CB 2 receptor homology model used for molecular modeling was described in ref. 23. Docking of (E)-BCP, (Z)-BCP and α-humulene and CB 2 protein-ligand complex MD/MM studies were performed on the basis of published docking protocols, using Tripos molecular modeling packages Sybyl7.3.3 and Tripos force field (23, 29). (For additional details, see SI Materials and Methods .) cAMP Assay. Human CB 2 -receptorexpressing CHO-K1 cells were plated in 96-well plates at a density of 3 × 105 cells per ml and incubated overnight. After aspirating the media, the cells were chilled for 10 min at room temperature in RPMI medium 1640 (w/o supplements) containing 500 μM 3-isobutyl-1-methylxanthine. Cells were then treated with different concentrations of test-compounds and incubated for 30 min at 37°C in a total volume of 100 μl. After another 30 min of incubation at 37°C with 20 μM forskolin, intracellular cAMP levels were detected by HitHunter for adherent cells EFC chemiluminescent detection assay (Amersham; catalog no. 90000302) according to the manufacturer's instructions and measured on a Microlumat Plus Microplate Luminometer LB 96V (EG&G Berthold). The high-affinity CB receptor ligand WIN55,212–2 was used as positive control. Measurement of [Ca2+] i . Intracellular Ca2+ was quantified in HL60 cell lines by FACS measurements as described in ref. 29. (For additional details, see SI Materials and Methods .) CBA Quantification of Cytokines in Human Blood Plasma. Human peripheral whole blood cultures were obtained as described in ref. 29. Cytokine production in human peripheral whole blood was analyzed in blood plasma of whole blood cultured for 18 h at 37°C, 5% CO 2 , using Cytometric Bead Arrays (BD Biosciences; human inflammation CBA kit 551811) as described in ref. 29. (For additional details, see SI Materials and Methods .) Determination of p38, Erk1/2, and JNK1/2 Activation. Phsophorylation of p38 and Erk1/2 was analyzed in HL60 CB 2 -positive cells and CD14+ peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from human buffy coats by density gradient centrifugation as reported in ref. 29. Phosphoproteins were quantified with CBA Phospho p38 MAPK Flex Set 560010 (T180/T182), Phospho Erk1/2 Flex Set 560012 (T202/Y204), and the Phospho JNK1/2 Flex Set (T183/Y185) from BD Biosciences according to the manufacturer's instructions. Western Blot Analysis. Western blots were carried out by standard methods (see SI Materials and Methods ). Animals. Male CB 2 knockout mice (Cnr2−/−) on a C57BL6/J congenic background (32) and their C57BL6/J (Cnr2+/+) wild-type controls ≈3 months of age were used. Animals were housed in groups of 3–5 and had access to water and food ad libitum. The housing conditions were maintained at 21 ± 1°C and 55 ± 10% relative humidity in a controlled light–dark cycle (light on between 7:00 a.m. and 7:00 p.m.). All experimental procedures and animal husbandry were conducted according to standard ethical guidelines. Animal Treatment. (E)-BCP was dissolved in olive oil (Fluka) and gavage-fed to the animals (with the help of feeding needle) 30 min (50 mg/kg) and 60 min (5 and 10 mg/kg) before carrageenan treatment. Olive oil without (E)-BCP was gavage-fed to the animals as vehicle control. Carrageenan–Paw Edema. The experiments were performed as described in ref. 41. Briefly, Carrageenan (2%, 20 mg/ml suspended in saline; Sigma) was injected intraplantar in a volume of 30 μl into the hind right paw, using a 27-gauge needle. The left paw received the same amount of saline and it was used as control. Edema was measured by using a Volume meter (TSE) at a several time points after carrageenan injection. Edema was expressed in milliliters as the difference between the right and left paw. GC Measurements. Gas chromatography measurements were carried out as described in the European Pharmacopoeia 5.5 (Pinus silvestris oil) with a Thermo Electron Focus GC instrument (Thermo Fisher Scientific) fitted with a BGB wax column (60 m, 0.25-mm diameter, 0.25-μm film; serial no. 13651937). (For additional details, see SI Materials and Methods ).

Acknowledgments We thank Dr. Irmgard Werner and Alex Hermann for their help and technical assistance for the GC measurements, Andreas Nievergelt-Meier for his help with the CBA analyses, and Dr. Michael Detheux (Euroscreen S.A., Brussels, Belgium) for the CB 2 -transfected CHO-K1 cell line. This work was supported by the Deutsche Forschungsgemeinschaft Grants FOR926 and GRK804.