New Research Casts Doubt on the Entourage Effect

Many cannabis users believe in the entourage effect, the idea that cannabinoids and other cannabis compounds have a greater effect combined than consumed alone. But new research has cast some doubt over the entourage effect’s existence. 

Published in pre-print in bioRxiv, the study from Macquarie University and the University of Sydney, Australia, investigated the possibility of a cannabinoid-terpenoid entourage effect by studying the responses of cells transfected with human CB1 or CB2 cannabinoid receptors in the presence of common terpenoids and THC. 

But the researchers found that the receptors weren’t altered by any of these six major terpenoids, either when used individually or when mixed.

Scientific evidence for the entourage effect

Given that the cannabis plant is made up of over 400 distinct chemical entities, the notion that some of these compounds might interact may not be so surprising. 

There are two main classes of entourage effect that are theorized: cannabinoid-cannabinoid and cannabinoid-terpenoid interactions. Cannabinoids include the likes of tetrahydrocannabinol (THC) and cannabidiol (CBD). The terpenoids, a collective name given to the large number of monoterpene and sesquiterpene compounds found in the essential oils of plants, are highly volatile compounds that give plants like cannabis their distinctive aroma and flavor. 

Cannabinoid-cannabinoid interactions have been recorded before in the medical literature, with interactions between THC and CBD reportedly producing analgesia for neuropathic pain in animal tests. In human trials, CBD has also been seen to mitigate some of the adverse effects caused by THC, implying some level of synergy between the compounds — though this finding remains controversial with some studies producing contradictory results.

Compared to the research of cannabinoid-cannabinoid reactions, there is very little in the way of scientific evidence for cannabinoid-terpenoid synergy, though this has not stopped the idea from taking hold in product marketing. On a base level, the idea appears to make sense. Like CBD and THC, many terpenoids also produce beneficial medical effects when taken at sufficient dosages. For example, linalool is known to work as a sedative, myrcene shows promise as an analgesic and muscle relaxant, and limonene displays some anti-anxiety effects.

Experiments reveal no significant evidence for cannabinoid-terpene synergy

In the new study, the researchers conducted assays of mouse wild-type At20 FlpIn cells that were transfected with human CB1 or CB2 receptors. Changes in membrane potential (and thus activation of cannabinoid receptors) in the cells were measured by fluorescence using diluted membrane potential dye. 

As well as investigating the immediate effects on CB1 and CB2 cannabinoid signaling, the researchers also tested whether the terpenoids might accelerate cannabinoid receptor desensitization, as previous research by the same team had uncovered evidence of negative allosteric modulators for the CB1 receptor which did the same. 

For this, the researchers exposed the cells to THC for a prolonged time period. This produced a hyperpolarization that reversed substantially over the course of the following 30 minutes. To test whether the terpenoids altered the desensitization time-course, this test was repeated in the presence of the six terpenoids used in the study, and the amount of signal desensitization again recorded after 30 minutes. No significant difference was observed between the two readings, indicating that terpenoids also do not interfere with desensitization of cannabinoid signaling. 

Where a previous study concluded that β-caryophyllene was a CB2 agonist, this new work was unable to verify these results. 

So does this mean there is no entourage effect?

The study authors do note that this work has limitations, the largest being that the study design means that only one CB1 and CB2 signaling pathway was examined. “Cannabinoid receptors couple to multiple G proteins as well as signaling through other pathways,” write the study authors. “It is possible that entourage effects of terpenoids are mediated through modulation of a subset of the cannabinoid receptor signaling repertoire.”

If there is some kind of synergy or an entourage effect between the cannabinoids and the terpenoids, it seems clear from this research that it is related to the function of the cannabinoid receptors in the body. But as the study authors suggest, this does not mean that the terpenoids can’t be influencing the outcomes of cannabis administration through interacting with other targets. THC is known to have some influence over a number of non-cannabinoid receptor targets, and so perhaps it could be that the terpenoids are having an effect through these pathways.

As the authors note, "the quest for entourage does not end here; in many ways it has only just begun.”