The so-called ‘entourage effect has become a well-known theory in recent years, referring to the potential synergistic relationship between cannabis compounds – in particular, cannabinoids and terpenes. This theory has gained significant traction and support in the cannabis sector, despite limited evidence.
A recent report aimed to investigate the effects of terpenes, both alone and with the cannabinoid agonist WIN55, 212 using in vivo and in vitro approaches.
What are Terpenes?
Terpenes are a type of phyto-compound – the basic constituent of essential oils – found in many plant species. These compounds have been found to have some therapeutic potential and have been used medicinally for thousands of years. Different terpenes may have different therapeutic potentials – for example, some evidence shows that this potential can vary from reducing inflammation to improving sleep, anxiety, and depression. They are also responsible for much of the flavour and colour of cannabis and other plants.
Even though both cannabinoids and terpenes are produced in the cannabis plant, the potential interaction between the compounds, when cannabis is consumed for both recreational and medicinal purposes, has barely been researched. 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
Researchers performed several experiments, designed to investigate whether select terpenes and terpenoids had activity at the CB1 receptor in contributing to reduced nociception, movement disorders, muscle tone, and hypothermia.
The terpenes and terpenoids used (α-Humulene, β-Pinene, Linalool, Geraniol, and β-Caryophyllene) were selected based on their presence in Cannabis Sativa and for their reported therapeutic potentials. Both in vitro and in vivo (in mouse models) experiments were carried out to identify a clearer picture of potential synergistic relationships between the compounds.
Results of the Study – In vivo
The researchers lay out several findings from the experiments carried out on the study, detailing how terpenes and cannabinoids were seen to work synergistically. These findings include:
Terpenes induce cannabinoid behaviours in mice
Terpenes were administered in several doses (50-200mg/kg) – much higher than those found during medical use of the plant – and effects were assessed in the ‘tail flick assay’ in male and female CD-1 mice. The tail flick assay is used as a test of nociception (pain detection) involving heat being applied to the tail of a mouse. The responsive tail flicks are then recorded to indicate pain or discomfort.
The 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 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 cannabinoid behaviours, the researchers used a CB1 selective antagonist/inverse agonist. It was first shown that the CB1 selective antagonist/inverse agonist could fully or partially reverse the behaviours induced by the positive control cannabinoid WIN55,212-2. Next, the rug was used in terpene tail flick anti-nociception – this showed that CB1 selective antagonist/inverse agonist 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 cannabinoid, 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, CB1 selective antagonist/inverse agonist 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 cannabinoid 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 CB1 selective antagonist/inverse agonist-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.
To date, few studies have aimed to identify the molecular targets and mechanisms for terpenes, either in vitro or in vivo. Furthermore, despite the fact that terpenes and cannabinoids have often been hypothesised to interact to produce an “entourage effect”, the few studies that have so far been carried have shown no interaction. Therefore, this study is the first to show that terpenes and cannabinoids can produce a synergistic effect when administered in combination.
The researchers do note, however the generally high concentrations of terpenes needed to see activation, which was especially apparent in the in vitro studies. Doses needed to see activation in vivo were not as extreme, producing full responses in most assays for most terpenes art 200mg/kg. These are concentrations much greater than that seen during medical use and therefore the clinical applicability of this is very much in doubt.