In Defense of the “Entourage Effect”: Terpenes Found in Cannabis sativa Activate the Cannabinoid Receptor 1 In Vivo

Abstract

Cannabis sativa, or marijuana, is the most commonly used illicit drug in the United States. Recreational and medical use has increased over the past decade due to loosening laws and legalization in 11 states. Anecdotal evidence from users suggests the therapeutic value of C. sativa is greater when the whole plant is consumed versus individual constituents such as D9‐tetrahydrocannabinol (D9‐THC). This observation has been termed the “entourage effect” and is hypothesized to derive from the combinatorial effect of phytocannabinoids and terpenes; the sum of the constituents found in the whole plant. C. sativa varieties contain ~150 phytocannabinoids and over 100 terpenoids. Prevalent monoterpenes include a‐pinene, linalool, limonene, and b‐myrcene, while prevalent sesquiterpenes include b‐caryophyllene (a known cannabinoid receptor 2 (CB2) agonist) and a‐humulene. These terpenoid compounds dictate the specific aromas and flavors of cannabis. Although terpenes can be lost during heat extractions, cold extraction using CO2 can increase terpene content upwards of 10‐fold. With increased terpene concentrations, a stronger case for the “entourage effect” can be made. Recent evidence has been published against the “entourage effect”, however, terpenes in these studies were used at very low doses or were studied in a single in vitro model. We sought to test the question: is the “entourage effect” a possible physiological phenomenon? We thus tested individual terpenes of C. sativa for cannabinoid receptor 1 (CB1R) activity in comparison with the synthetic cannabinoid WIN55,212. Four terpenes, varying in amounts in C. sativa, were chosen for screening: linalool and a‐humulene (med‐high), b‐pinene (low‐med), and geraniol (low). Preliminary in vitro results demonstrated that each of these terpenes were agonists at the CB1R. In mice, activation of CB1Rs by CB1 agonists induces four distinct physiological responses known as the CB1R tetrad: antinociception, hypolocomotion, catalepsy, and hypothermia. Each terpene was subsequently tested for activity in the tetrad. Intraperitoneal injection (£ 200 mg/kg) of a‐humulene, b‐pinene, and geraniol resulted in a dose‐dependent increase in tail flick thermal latency, an indication of antinociception; linalool had no effect. Interestingly, at 200 mg/kg a‐humulene, b‐pinene, and geraniol were able to induce an effect greater than or equivalent to 5.6 mg/kg WIN55,212. These effects were at least partially mediated by the CB1R, as 3 mg/kg rimonabant (CB1R antagonist) caused partial reversal. At 200 mg/kg, each terpene caused a significant decrease in hypolocomotion, an increase in catalepsy, and decrease in core temperature. These preliminary results suggest these terpenes may induce CB1R‐dependent behaviors in mice, thus promoting the possibly of an “entourage effect”. Ongoing studies are examining the interaction between known CB1R ligands and these terpenes to identify whether their interactions at the CB1R are competitive or allosteric in nature, and whether these ligands interact behav...

Recent advances in Cannabis sativa genomics research

“…Cannabis is a prolific producer of terpenes and these compounds are thought to contribute to the therapeutic efficacy of cannabis preparations via the ‘entourage effect’, by acting in combination with cannabinoids (Ben‐Shabat et al ., 1998; LaVigne et al ., 2020). However, evidence for the existence of the entourage effect is largely anecdotal and lacks mechanistic explanation, for example whether the effect is additive or multiplicative.…”

Terpenes and Cannabinoids Yields and Profile from Direct-Seeded and Transplanted CBD-Cannabis sativa

“…However, the OFCT production system resulted in EO with lower percentages of 1,8-cineole, (E)-caryophyllene, γ-elemene, αhumulene, α-selinene, selina-4(15),7(11)-diene, selina-3,7(11)-diene, and germacrene B. The EO profile plays a significant role in the biological activity of cannabinoids, because both synergistic (entourage effect purported in humans) or antagonistic interactions 29,30 have been reported. In this study, the length of the steam distillation altered the EO profile.…”

Cannabinoids in Medicine: A Multifaceted Exploration of Types, Therapeutic Applications, and Emerging Opportunities in Neurodegenerative Diseases and Cancer Therapy

“…For instance, evidence has emerged that a holistic botanical preparation of cannabis exhibits greater potency in both in vitro and in vivo models for breast cancer treatment compared to isolated 9-THC [ 198 ]. Furthermore, in vivo experiments yielded compelling results, showing additive effects when cannabis terpenes such as α-humulene and β-pinene were combined with WIN55,212-2 in mouse models [ 199 ]. These findings suggest that the inclusion of terpenes enhances the activity of isolated cannabinoids, likely through a synergistic mechanism.…”