The psychoactive cannabinoids from Cannabis sativa L. and the arachidonic acid-derived endocannabinoids are nonselective natural ligands for cannabinoid receptor type 1 (CB1) and CB2 receptors. Although the CB1 receptor is responsible for the psychomodulatory effects, activation of the CB2 receptor is a potential therapeutic strategy for the treatment of inflammation, pain, atherosclerosis, and osteoporosis. Here, we report that the widespread plant volatile (E) Cannabis ͉ CB2 cannabinoid receptor ͉ foodstuff ͉ inflammation ͉ natural product P lant 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)-␤-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)-␤-caryophyllene [(Z)-BCP or isocaryophyllene] and ␣-humulene (formerly ␣-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 Ͼ400 different secondary metabolites, including Ͼ65 cannabinoid-like natural products, only ⌬ 9 -tetrahydrocannabinol (THC), ⌬ 8 -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 ...

Alkylamides (alkamides) from Echinacea modulate tumor necrosis factor ␣ mRNA expression in human monocytes/macrophages via the cannabinoid type 2 (CB 2 ) receptor (Gertsch, J., Schoop, R., Kuenzle, U., and Suter, A. (2004) FEBS Lett. 577, 563-569). Here we show that the alkylamides dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (A1) and dodeca-2E,4E-dienoic acid isobutylamide (A2) bind to the CB 2 receptor more strongly than the endogenous cannabinoids. The K i values of A1 and A2 (CB 2 ϳ60 nM; CB 1 >1500 nM) were determined by displacement of the synthetic high affinity cannabinoid ligand [ 3 H]CP-55,940. Molecular modeling suggests that alkylamides bind in the solvent-accessible cavity in CB 2 , directed by H-bonding and -interactions. In a screen with 49 other pharmacologically relevant receptors, it could be shown that A1 and A2 specifically bind to CB 2 and CB 1 . A1 and A2 elevated total intracellular Ca 2؉ in CB 2 -positive but not in CB 2 -negative promyelocytic HL60 cells, an effect that was inhibited by the CB 2 antagonist SR144528. At 50 nM, A1, A2, and the endogenous cannabinoid anandamide (CB 2 K i >200 nM) up-regulated constitutive interleukin (IL)-6 expression in human whole blood in a seemingly CB 2 -dependent manner. A1, A2, anandamide, the CB 2 antagonist SR144528 (K i <10 nM), and also the non-CB 2 -binding alkylamide undeca-2E-ene,8,10-diynoic acid isobutylamide all significantly inhibited lipopolysaccharide-induced tumor necrosis factor ␣, IL-1␤, and IL-12p70 expression (5-500 nM) in a CB 2 -independent manner. Alkylamides and anandamide also showed weak differential effects on anti-CD3-versus anti-CD28-stimulated cytokine expression in human whole blood. Overall, alkylamides, anandamide, and SR144528 potently inhibited lipopolysaccharide-induced inflammation in human whole blood and exerted modulatory effects on cytokine expression, but these effects are not exclusively related to CB 2 binding.

The identification of targets whose interaction is likely to result in the successful treatment of a disease is of growing interest for natural product scientists. In the current study we performed an exemplary application of a virtual parallel screening approach to identify potential targets for 16 secondary metabolites isolated and identified from the aerial parts of the medicinal plant Ruta graveolens L. Low energy conformers of the isolated constituents were simultaneously screened against a set of 2208 pharmacophore models generated in-house for the in silico prediction of putative biological targets, i. e., target fishing. Based on the predicted ligand-target interactions, we focused on three biological targets, namely acetylcholinesterase (AChE), the human rhinovirus (HRV) coat protein and the cannabinoid receptor type-2 (CB 2 ). For a critical evaluation of the applied parallel screening approach, virtual hits and non-hits were assayed on the respective targets. For AChE the highest scoring virtual hit, arborinine, showed the best inhibitory in vitro activity on AChE (IC 50 34.7 μM). Determination of the anti-HRV-2 effect revealed 6,7,8-trimethoxycoumarin and arborinine to be the most active antiviral constituents with IC 50 values of 11.98 μM and 3.19 μM, respectively. Of these, arborinine was predicted virtually. Of all the molecules subjected to parallel screening, one virtual CB 2 ligand was obtained, i.e., rutamarin. Interestingly, in experimental studies only this compound showed a selective activity to the CB 2 receptor (Ki of 7.4 μM) by using a radioligand displacement assay. The applied parallel screening paradigm with constituents of R. graveolens on three different proteins has shown promise as an in silico tool for rational target fishing and pharmacological profiling of extracts and single chemical entities in natural product research.

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The cannabinoid CB(2) receptor is known to modulate osteoclast function by poorly understood mechanisms. Here, we report that the natural biphenyl neolignan 4'-O-methylhonokiol (MH) is a CB(2) receptor-selective antiosteoclastogenic lead structure (K(i) < 50 nM). Intriguingly, MH triggers a simultaneous G(i) inverse agonist response and a strong CB(2) receptor-dependent increase in intracellular calcium. The most active inverse agonists from a library of MH derivatives inhibited osteoclastogenesis in RANK ligand-stimulated RAW264.7 cells and primary human macrophages. Moreover, these ligands potently inhibited the osteoclastogenic action of endocannabinoids. Our data show that CB(2) receptor-mediated cAMP formation, but not intracellular calcium, is crucially involved in the regulation of osteoclastogenesis, primarily by inhibiting macrophage chemotaxis and TNF-α expression. MH is an easily accessible CB(2) receptor-selective scaffold that exhibits a novel type of functional heterogeneity.