22nd July 2021
By Emily Ledger
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For those with experience in cannabis – whether that be recreationally or medically – the entourage effect will probably be a well-known phenomenon. The term refers to the different compounds found in cannabis working together to offer a synergistic effect – enhancing the effects of each compound alone.

The theory has gained significant traction and support in the cannabis sector, despite limited evidence. However, a recent report aimed to investigate the effects of terpenes, both alone and with a known cannabinoid agonist (called WIN55, 212) using both in vivo and in vitro approaches.

What are Terpenes?

Terpenes are a type of compound found in many plants and are the basic constituent of essential oils. In many cases, terpenes contribute to the appearance, smell, and taste of plants – however, they have also been found to have a number of potential therapeutic effects.

For example, evidence suggests that different terpenes may have the potential to reduce inflammation and improve sleep, anxiety, and stress.

Despite the entourage effect being a popular theory among both medical and recreational users, advocates, campaigners, and researchers, there is still little solid evidence on the subject. The existing literature on the matter – deductive reasoning arguments, some clinical suggestions, and pre-clinical studies – remain mixed, with some evidence for and against potential synergistic effects.

Report Design and Methods

The recent study, carried out by researchers at the University of Arizona, USA, was designed to “determine whether selected terpenes/terpenoids… had activity in the cannabinoid tetrad of behaviours mediated by the CB1 receptor: antinociception, hypo locomotion, catalepsy, and hypothermia.”

The terpenes/terpenoids used were α-Humulene, β-Pinene, Linalool, Geraniol, and β-Caryophyllene were selected based on their presence in Cannabis Sativa plants and for their reported therapeutic potentials. Both in vitro and in vivo (in mouse models) experiments were carried out.

Results of the Study

Terpenes induce cannabinoid tetrad behaviours in mice

Terpenes were administered in several doses (50-200mg/kg) and effects were assessed in the ‘tail-flick assay’ in male and female mice – a method used as a test of nociception (pain detection) involving heat being applied to the tail of a mouse. The responsive tail flicks are recorded to indicate pain or discomfort.

Terpenes were found to induce a range of efficacies in the tail-flick assay: Geraniol and α-Humulene exhibited moderate ~40–50% efficacy in a dose-dependent manner; β-Pinene showed low efficacy but not in a dose-dependent manner; Linalool demonstrated dose-dependent low efficacy, as did the β-Caryophyllene at the dose tested.

The positive control WIN55,212-2 demonstrated dose-dependent increases in thermal latency with greater efficacy than any of the tested compounds reaching near-threshold values at a 10 mg/kg dose.

Terpenes demonstrated cannabimimetic (similar to cannabis) effects in at least three out of the four classic cannabinoid tetrad behaviours each, while Vehicle and β-Caryophyllene controls did not.

Terpene tail-flick antinociception is CB1 mediated and is additive with cannabinoid

To determine the role of the CB1 receptor in the mediation of these tetrad behaviours, the researchers used the CB1 selective antagonist/inverse agonist rimonabant. It was first shown that rimonabant could fully or partially reverse the tetrad behaviours induced by the positive control cannabinoid WIN55,212-2.

Next, the rug was used in terpene tail-flick anti-nociception – this showed that “rimonabant pre-treatment fully blocked terpene response in this assay, suggesting that terpenes induce tail-flick anti-nociception via the CB1.”

Terpene hypothermia was also found to be additive with cannabinoids, however, experiments demonstrated that this was mostly not mediated by CB1. When researchers co-injected both terpenes and WIN55,212-2, hypothermia was increased over either treatment alone for all terpenes tested.

However, unlike for tail-flick, rimonabant was only partially able to reverse α-humulene hypothermia and had no effect on the other terpenes. This suggests that CB1 may only mediate α-humulene hypothermia and has no role for other terpenes.

Experiments also demonstrated that terpene catalepsy is partially additive with cannabinoids and mostly mediated by A2a (adenosine A2a receptor). Terpene hypo locomotion is also partially additive with cannabinoid and partially A2a mediated.

Terpenes Activate the CB1 Receptor In Vitro

As the results of the behavioural experiments suggested that the terpenes used potentially interact with the CB1 receptor (and likely others), the researchers sought to determine whether these selected terpenes acted as CB1 agonists in vitro. This was first done by assessing CB1-dependent ERK activation.

All of the terpenes tested were found to activate downstream ERK signalling in CB1-CHO cells. This activation was rimonabant-sensitive, supporting the findings of the in vivo studies.

In addition, the terpenes tested also caused ERK phosphorylation in CB2-expressing cells, suggesting that they may also interact with CB2, as previously demonstrated for β-Caryophyllene13.

Researcher Conclusions

The findings of this study suggest that terpenes could be used to enhance the analgesic properties of medical cannabis/cannabinoid therapy, without worsening the side effects of cannabinoids.

However, the authors conclude that this possibility must be confirmed “using relevant phytocannabinoids like THC instead of synthetic cannabinoid WIN55,212-2 used in this study.” Further research in this area – including identifying specific terpene:cannabinoid combinations with a maximized therapeutic index for a particular disease state – could provide a new means to improve treatment with these drugs.

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