Absence of Entourage : Terpenoids Commonly Found in Cannabis sativa Do Not Modulate the Functional Activity of Δ9-THC at Human CB1 and CB2 Receptors, Marina Santiago et al., 2019

Absence of Entourage : Terpenoids Commonly Found in Cannabis sativa Do Not Modulate the Functional Activity of Δ9-THC at Human CB1 and CB2 Receptors

doi : 10.1089/can.2019.0016


Introduction: Compounds present in Cannabis sativa such as phytocannabinoids and terpenoids may act in concert to elicit therapeutic effects. Cannabinoids such as Δ9-tetrahydrocannabinol (Δ9-THC) directly activate cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2); however, it is not known if terpenoids present in Cannabis also affect cannabinoid receptor signaling. Therefore, we examined six common terpenoids alone, and in combination with cannabinoid receptor agonists, on CB1 and CB2 signaling in vitro.

Materials and Methods: Potassium channel activity in AtT20 FlpIn cells transfected with human CB1 or CB2 receptors was measured in real time using FLIPR® membrane potential dye in a FlexStation 3 plate reader. Terpenoids were tested individually and in combination for periods up to 30 min. Endogenous somatostatin receptors served as a control for direct effects of drugs on potassium channels.

Results: α-Pinene, β-pinene, β-caryophyllene, linalool, limonene, and β-myrcene (up to 30–100 μM) did not change membrane potential in AtT20 cells expressing CB1 or CB2, or affect the response to a maximally effective concentration of the synthetic cannabinoid CP55,940. The presence of individual or a combination of terpenoids did not affect the hyperpolarization produced by Δ9-THC (10 μM): (CB1: control, 59%±7%; with terpenoids (10 μM each) 55%±4%; CB2: Δ9-THC 16%±5%, with terpenoids (10 μM each) 17%±4%). To investigate possible effect on desensitization of CB1 responses, all six terpenoids were added together with Δ9-THC and signaling measured continuously over 30 min. Terpenoids did not affect desensitization, after 30 min the control hyperpolarization recovered by 63%±6% in the presence of the terpenoids recovery was 61%±5%.

Discussion: None of the six of the most common terpenoids in Cannabis directly activated CB1 or CB2, or modulated the signaling of the phytocannabinoid agonist Δ9-THC. These results suggest that if a phytocannabinoid–terpenoid entourage effect exists, it is not at the CB1 or CB2 receptor level. It remains possible that terpenoids activate CB1 and CB2 signaling pathways that do not involve potassium channels; however, it seems more likely that they may act at different molecular target(s) in the neuronal circuits important for the behavioral effect of Cannabis.



An enduring notion in the medicinal Cannabis and cannabinoid field is that of entourage: the idea that use of the whole plant may exert substantially greater effects than the sum of its individual parts.1 Entourage is usually construed as a positive attribute, with the assumption that superior therapeutic actions, or a more favorable “high,” will be obtained from consuming the whole Cannabis plant rather than individual components such as Δ9-tetrahydrocannabinol (Δ9-THC). Somewhat surprisingly, the evidence for this widely cited notion is relatively sparse.

Cannabis contains ∼150 phytocannabinoids, the most common of which are Δ9-THC and cannabidiol (CBD), together with their acid precursors THCA and CBDA.2Cannabis also contains a large number of monoterpene and sesquiterpene compounds (together called terpenoids), the most common of which include α-pinene, β-pinene, linalool, limonene and β-myrcene (monoterpenes) and β-caryophyllene and caryophyllene oxide (sesquiterpenes).3 Terpenoids are volatile compounds that are synthesized alongside phytocannabinoids mainly in the trichomes of the cannabis plant, and provide cannabis with its distinctive aroma and flavor.4 Terpenoids are often lost if the extraction process involves heating.5

The entourage concept applied to cannabis can encompass the potential for both cannabinoid–cannabinoid and cannabinoid–terpenoid interactions. With regard to the former, Δ9-THC-CBD synergy in producing analgesia was reported in an animal model of neuropathic pain6 while in humans, CBD has been proposed to ameliorate some of the adverse psychotomimetic and anxiogenic effects of Δ9-THC.7,8 This claim is controversial, however, with a number of contrary findings.9,10 CBD may modulate Δ9-THC effects at the receptor level acting as a CB1 negative allosteric modulator,11 providing some biological plausibility to a modulatory interaction.

Scientific evidence for cannabinoid–terpenoid interactions is essentially absent, and mostly comes from websites and dispensaries extolling the virtues of proprietary Cannabis chemical varieties, or chemovars.12,13 However, some terpenoids do have intrinsic psychoactive and physiological effects, and modulatory effects on Δ9-THC actions are not farfetched.1,14 For example, in studies with laboratory animals, limonene displayed anxiolytic effects, pinene increased gastrointestinal motility, linalool was sedative, anticonvulsant, and anxiolytic, while myrcene produced sedation, analgesia, and muscle relaxant effects (summarized in Russo and Marcu14). Lewis et al.13 reported that in a low terpenoids variety (1.1% terpenoids) myrcene concentration is 0.45%, while in a high variety (4.8% total) myrcene concentration is as high as 3.44%. Compelling evidence for cannabinoid–terpenoid interactions or synergy does not yet exist. A report on perceived efficacy of Cannabis for childhood epilepsy identified the presence of three predominant terpenoids (β-caryophyllene, β-myrcene, and α-pinene); however, when extracts perceived as “effective” were compared with “ineffective” extracts, differences in terpenoid profile/content were not significant.15

With so many bioactive components present in cannabis, the systematic, granular elucidation of possible entourage effects poses a substantial combinatorial puzzle and scientific challenge. As a preliminary approach to addressing this challenge, this study examined whether the effects of Δ9-THC on its cognate cannabinoid receptors (CB1 and CB2) would be modified in the presence of terpenoids that are commonly found in cannabis, either alone or in combination. The demonstration of such a receptor-level entourage effect might lead to predictions regarding functional cannabinoid–terpenoid interaction effects that could be tested in vivo.