*4.9. Valencene*

The sesquiterpene valencene produces an aroma that is often associated with citrus fruits such as Valencia oranges [141]. In cannabis, valencene has been reported in several different cultivars, but only at low concentrations [70]. Aside from its appealing scent, valencene's bioactivity has been explored through various essential oil profiles, speculating anti-inflammatory [142], neuroprotective [143], anti-allergic [144], and antimicrobial [145] properties.

While minor terpenes may not be the most abundant in cannabis, they have the potential to aid in the biological activities of cannabis. These terpenes demonstrate overlapping activity with each other, often targeting the same biological function (even if mechanism of action remains unknown) (Figure 3). Likewise, an overlap of therapeutic benefit between cannabinoids and these secondary terpenes may be inferred based on current cannabinoid research [60], suggesting a potential to increase the efficacy of these cannabinoids in an additive or synergistic manner.

**Figure 3.** Bioactivities of the nine secondary terpenes covered in this review, along with cannabis's principal cannabinoids (CBD, cannabidiol; THC, tetrahydrocannabinol; CBG, cannabigerol; CBN, cannabinol; CBC, cannabichromene). Bioactivity circles are color-coded to match the legend in the middle; the presence of a particular shading in the circle is indicative that the terpene or cannabinoid has been reported to possess that bioactivity. The figure demonstrates that not only do terpenes have multiple potential bioactivities, but different compounds possess overlapping activities, suggesting their potential to exert combination effects.

#### **5. Mechanism of Action for Terpenes—Pharmacologic Receptor Targets (TRPs)**

Several studies have investigated the pharmacodynamics of the receptors for the major terpenoids found in cannabis (e.g., β-caryophyllene, β-myrcene, β-pinene, α-humulene, linalool). For instance, β-caryophyllene has been found to be an agonist at the cannabinoid receptor 2 (CB2), peroxisome proliferator-activated receptor gamma (PPARγ), and the toll-like receptor 4 (TLR4)/CD14/MD2 complex, while β-myrcene is an agonist at α2- adrenergic receptors and transient receptor potential cation channel subfamily V member 1 (TRPV1) [13,146–149]. The information on the receptors modulated by the minor terpenes found in cannabis is much more variable and will be the focus of the discussion below.

Borneol is an agonist of TRPM8. This activation of TRPM8 by borneol has been found to be temperature sensitive and dose-dependent across a range of concentrations, from 10 μM to 2 mM; however, no EC50 was reported because the study failed to reach a maximal response [150,151]. The activation of TRPM8 receptors by borneol has been found to activate glutamatergic and GABAergic transmission in the spinal cord, leading to anti-nociceptive activity [152,153]. The activation of TRPM8 by borneol has also been shown to enhance the chemosensitivity of lung cancer cell lines to doxycycline [151]. Borneol is also an agonist of TRPV3 (EC50 = 3.45 mM) channels [154]. Furthermore, borneol is an antagonist of the TRPA1 channel, with an IC50 of 0.2–0.3 mM in cell-based assays [155,156]. The activation of TRPV3 and inhibition of TRPA1 also likely contribute to the antinociceptive properties of borneol. Of note, the antagonist/agonist profile of borneol at these receptors matches that of several cannabinoids, including CBD, CBG, and THC; however, these effects occur at relatively high levels.

Camphor is a major terpenoid constituent of cannabis but is best known as an isolate from the camphor laurel (*Cinnamomum camphora*). This compound is FDA-approved as an additive to soothing creams and ointments and as a component of over-the-counter respiratory treatments. Camphor has been found to act as an agonist at TRPM8 and TRPV3 and an antagonist at TRPA1, which is perhaps not surprising considering the structural similarity of camphor to borneol. Despite this structural similarity, camphor is less potent at both TRPV3 (EC50 = 6.03 mM) and TRPA1 (IC50 = 1.26 mM) compared to borneol [154,156]. At TRPM8, camphor has an EC50 of approximately 4.5 mM [157]. Additionally, camphor has been shown to be a partial agonist at TRPV1, with similar potency as at TRPV3 and TRPA1 (EC50 > 3 mM) [158,159]. The action of camphor at these receptors likely accounts for its analgesic activities. In addition, activation of the TRPV family of receptors has been linked to the ability of camphor to relax the trachea in rats, which may help explain its anti-congestive activities [160].

Cedrene has been identified as a potent agonist of the olfactory receptor 10J5 (OR10J5), a GPCR that is also found in liver and muscle tissue [161]. In human hepatocytes, cedrane has been shown to lower lipid levels through OR10J5. Furthermore, cedrane has been shown to reduce muscle atrophy induced by a high fat diet in mice, this action is mediated through the mouse ortholog of OR10J5, MOR23 [162]. This study also found that cedrane increased muscle mass and strength, possibly through increasing expression of IGF1.

Isopulegol has been identified as an agonist of the most abundant GABAAR in the brain, α1β2γ2, with an EC50 of approximately 3.25 μM. Activation of the GABAR produces sedative effects, and these receptors are targets for both analgesics and anticonvulsant medications [163]. Isopulegol is also an agonist at TRPM8 and may also antagonize the TRPV1 receptor [164,165]. Either of these actions may account for the anti-nociceptive properties of isopulegol that have been described in mice [165].

Phytol and its metabolites can act as natural ligands for a variety of transcription factor receptors. This list includes the peroxisome proliferator-activated receptor (PPAR) α and γ; however, an EC50 was not reported because the assay did not reach a plateau at 100 μM, the highest concentration tested [166,167]. Additionally, phytol has been shown to be an agonist of retinoid X receptors (RXR), with EC50 estimates ranging from 41.9 to 67.2 μM, depending upon the isotype [168]. Through activation of these receptors, phytol has been shown to reduce cancer cell viability in a number of cancer cell lines. Indeed, it has been found to have a lower IC50 in the lung adenocarcinoma cell line, A549, than the chemotherapeutic agen<sup>t</sup> methotrexate [169]. Phytol induced apoptosis in this system through the activation of the TNF receptor, TRAIL, and FAS. Additionally, the authors used molecular docking to sugges<sup>t</sup> that phytol may bind to glucose-6-phosphate dehydrogenase to inhibit tumor progression. In vitro, phytol has also been shown to increase the release of CA2+ reserves via activation of GPR40, a G-protein-coupled receptor that normally binds to free fatty acids, with an EC50 of 34.5 μM [170]. The activation of PPARs, RXRs, and GPR40 by phytol may also be of potential therapeutic benefit for the treatment of diabetes, and because of the ability to activate RXR receptors, phytol is also being pursued by the cosmeceutical industry as an anti-aging treatment in lieu of retinol (which is not well tolerated by all individuals due to its activation of TRPV1) [171,172].

Using a recently developed in vitro receptor binding assay, pulegone was shown to be the component in *Ziziphora clinopodioides* that binds and potentially activates β1- adrenoceptors [173]. Pulegone has been found to be an agonist of avian TRPM8 at low concentrations; however, it antagonizes this receptor at higher concentrations [174]. This study also found that pulegone is an antagonist of TRPA1 at both low and high concentrations. Taken together, these data sugges<sup>t</sup> that pulegone may have anti-nociceptive and analgesic utility.

Computer-based molecular docking research predicted that sabinene may be a potent interactor with L-asparginase from the bacterial pathogen, *Salmonella typhimurium* [175]. This study found that sabinene had a higher docking score than the antibiotic ciprofloxacin, suggesting that sabinene may have antibacterial properties and may be a good candidate for antibiotic development. Additionally, sabinene has been found to reduce levels of the inflammatory marker nitric oxide in cells exposed to lipopolysaccharide [129]. Another molecular docking study suggested that sabinene may interact with the spike protein on the SARS-CoV2 (COVID-19) virus along with three cell membrane proteins (transmembrane serine protease 2, cathepsin B, and cathepsin L) that play a role in mediating viral entry into cells [176]. Additional studies will be needed to determine if any of these interactions occur in vivo, as well as which receptors might mediate the decrease in nitric oxide production caused by sabinene. An in silico study suggested that thujene may have a modest binding affinity for the SARS-CoV2 main protease and papain-like protease, but further work will be needed to confirm these findings [177]. Additionally, no studies could be located that identified potential human receptors for this terpene.

Valencene has been reported to be cardioprotective following myocardial infarction in rats, and this protection is mediated through the inhibition of the NF-κB pathway, oxidative stress, and cardiac hypertrophy; however, the receptors that mediate this inhibition were not examined [178]. Valencene has been found to be an antagonist of the calcium ion channel TRPV1 and the slow release calcium release-activated calcium channel protein 1 (ORAI1), which inhibited the melanin content in UVB exposed melanoma cells, and may therefore be useful for treating photo-aging of the skin [179]. This inhibition, may also mediate the ability of valencene to potentially treat atopic dermatitis [180].

#### **6. Synergy and the Entourage Effect: Beyond Cannabinoids**

Natural product discovery efforts are traditionally reductionist in nature, devoted to condensing a complex botanical extract down to a single bioactive agen<sup>t</sup> for drug development purposes. This is true for cannabis research and development, where the single molecule approach remains the dominant approach [181]. However, botanical medicines, including cannabis, are in fact complex diverse concoctions of phytochemicals that have the potential of exerting differing and potentially complementary biological effects. Indeed, it is often observed that these mixtures work in concert to achieve a specific physiological effect [182]. Compounds can work in a synergistic manner, in which each active compound potentiates the other to achieve a greater than expected benefit when combined (i.e., 1 + 1 > 2). If one compound, having no activity of its own, impacts the efficacy of an active molecule to increase activity (i.e., 1 + 0 > 1), this is known as an entourage effect [26]. As cannabis research has evolved, there has been a growing body of evidence that cannabinoids beyond THC demonstrate efficacy in humans [183,184] and that synergy/entourage could potentially play a large role in the bioactivity of cannabis extracts and products [22,185].

Botanical synergy and entourage have been demonstrated in cannabis, first in the combination of THC with other, "minor", cannabinoids. Johnson et al. (2010) tested a cannabis-based extract for patients with intractable pain and found that, while the THC dominant extract did not improve patient outcome versus the placebo (the mean pain Numerical Rating Scale (NRS) was a nonsignificant change of −1.01 vs. −0.69), a whole plant extract (the only difference being the presence of CBD) demonstrated a significant improvement in pain outcome (mean NRS of −1.37 vs. −0.69) compared to the placebo [186]. Animal studies focusing on analgesia also evidenced greater response from a full-spectrum cannabis extract as compared to pure CBD dosing [187]. Recently, experiments with a seizure mouse model looked at the effects of different strains of cannabis that all contained an equivalent CBD concentration. While all were effective, there were noticeable differences between the strains, and profiling 94 phytocannabinoids across 36 of the most commonly used *Cannabis* plants prescribed to patients in Israel led to the conclusion that these other cannabinoids have an impact on the overall efficacy of cannabis plant extracts [188]. In one in vitro study, one study of breast cancer cell lines revealed that the extract of the whole cannabis was more effective than a preparation featuring THC by itself; the boost in activity was attributed to the presence of "minor" cannabinoids cannabigerol (CBG) and tetrahydrocannabinolic acid (THCA) [189]. Complex fractions from cannabis extracts demonstrated synergistic interactions on colorectal cancer cell lines [190].

Cannabinoids have been widely studied for the treatment of epilepsy [191,192]; complex extracts containing multiple cannabinoids were found to treat severe epilepsy, such as Dravet and Lennox-Gastaut syndromes, at lower doses than trials using purer preparations (e.g., Epidiolex, which contains 97% CBD) [66,193]. A 2018 meta-analysis by Pamplona et al. of 11 studies demonstrated that the response rate at 50% improvement of seizure frequency was similar between the two groups, but the average daily doses were significantly different: 27.1 mg/kg/d for purified CBD as opposed to 6.1 mg/kg/d. for cannabis extracts [194]. Moreover, the incidence of adverse events was discernably higher in the CBD versus complex extract treatments (*p* < 0.0001), a result that the authors attributed to the lower dose utilized, which was achieved in their opinion by the synergistic contributions of other entourage compounds.

Most synergy studies have focused primarily on the interactions between cannabinoid structures, despite the fact that the original definition of the entourage effect arose from the interaction of 2-acyl-glycerol esters with cannabinoids [26]. As terpenes are a large and diverse family of phytochemicals found in cannabis, they have the potential to serve as potentiating agents working in concert with cannabinoids. Terpenes, broadly speaking, have been found to be broadly synergistic, helping modulate the activity of a number of other botanicals, pharmaceuticals, and compounds. The terpenes highlighted in this review also have a strong history of synergistic activity with other compounds. Borneol was shown to synergize with curcumin to induce apoptosis in human melanoma cells [195], potentiate the activity of berberine and baicalein in inhibiting in vitro and in vivo fungal growth [196], and function as a potentiating agen<sup>t</sup> to sensitize cancer cells to doxorubicin treatment [151]. Sabinene [197] and pulegone [198] each indicated the potential for synergistic interactions with prescription antibiotics in treating bacterial infections, and β-caryophyllene and phytol demonstrated combination effects inducing apoptosis in skin epidermoid cancer cells [199]. Terpenes have also demonstrated efficacy in the treatment of mood and anxiety disorders, suggesting the possibility of combination effects with cannabinoids for more effective treatments [200]. Thus, while the potential of cannabis terpenes to possess additive or synergistic properties was originally posited as hypothetical based upon similar bioactivities [23], more recent studies have explored this possibility in earnest.

A 2021 study by LaVigne et al. found that α-humulene, geraniol, linalool, and β-pinene were cannabimimetic at the CB1 receptor and produced cannabinoid-like behaviors in a mouse model. Furthermore, the terpenes potentiated the effects of a cannabinoid agonist, suggesting synergistic activity [201]. Di Giacomo et al. treated triple negative breast cancer cells (MDA-MB-468) with hemp inflorescences and pure compounds of CBD, caryophyllene and cannabichromene. The presence of these other compounds induced the potentiating effects of CBD, likely mediated through CB2 activation [202]. However, separate studies observed that none of the terpenes α-pinene, β-pinene, β-caryophyllene, linalool, limonene, and β-myrcene were found to alter potassium channel signaling in AtT20 cells expressing CB1 and CB2 receptors, and did not interact with THC at the receptor [203], nor did they affect changes in intracellular calcium at the human transient receptor potential ankyrin 1 (hTRPA1) or human transient receptor potential vanilloid 1 (hTRPV1) channels [204]. Using a radioligand ([3H]-CP55,940) to measure binding at the CB1 and CB2 receptors, none of the tested terpenes (myrcene, α-pinene, β-pinene, β-caryophyllene, and limonene) had interactions with receptors, nor did they modulate the binding of THC or CBD [205]. Similarly, no synergy was detected between myrcene and CBD in modulating inflammation and analgesic properties in a rat adjuvant monoarthritis model [206]. Research on colorectal cancer cells did not detect any enhancement of activity when terpenes were included as part of a complex CBD oil compared to the effect of CBD alone [207].

The divergence of results involving potential synergy or entourage effects has led to doubt surrounding the entourage effect in cannabis and whether it really holds pharmaceutical potential. Cogan (2020) references several studies where individual cannabinoids did not improve the clinical performance of THC or CBD [208]. However, the intellectual leap to label cannabis potential synergistic interactions as "questionable" is perhaps premature. Botanicals or combinations exhibiting synergy or entourage does not necessitate that the effects take place at the same target to elicit a heightened response; compounds can exhibit "pharmacodynamic synergism" by acting at multiple cellular targets (seen in both antibiotic and cancer synergistic therapies) [209,210] and "pharmacokinetic synergism" by increasing the solubility or disposition (absorption, distribution, metabolism) of active constituents [211,212], and can limit side effects of the active constituent [213,214] or disrupt resistance mechanisms [215,216]. Indeed, the study Santiago et al. that purported the "absence of entourage" nevertheless suggested that synergy could still be taking place at a different molecular target than the CB receptors [203]. Therefore, as further studies are developed, it would be prudent to also employ phenotypic assays that encompass more than a single receptor/enzyme/target and can better deduce the combination effects at complementary sites and pathways to deliver heightened results. Thus, the heightened skepticism or dismissal of synergism in cannabis is perhaps unwarranted at this time, as there exists a growing body of evidence suggestive that not only do multiple cannabinoids work in concert to produce heightened effects (or potentially lower deleterious side effects), as seen in the prescription drugs that utilize multi-component cannabis extracts (Sativex® and Epidolex®) [186], but that terpenes/terpenoids can also potentially function as synergists with cannabinoids to deliver amplified results. Furthermore, studies from our own group, using an animal model of chemotherapeutic induced peripheral neuropathy, demonstrated an enhanced effect at reducing mechanical hypersensitivity by an extract containing equal parts CBG and CBD, in addition to other cannabinoids and terpenes, compared to pure CBG [217]. Additionally, in the same animal model we found that pure CBD was without an effect; however, when animals were treated with a complex hemp extract at the same CBD concentration, a reduction in mechanical sensitivity was observed [218]. These studies sugges<sup>t</sup> that there is a potential interaction between cannabinoids and terpenes that can enhance the effect of pure cannabinoids alone. Moreover, the lack of synergy in vitro may not hold true for results in intact organisms, and the entourage effect does not have to be present or absent in all systems or biomedical indications. The search for synergy and entourage effects within the diverse phytochemical landscape of cannabis remains in its infancy; to better understand these combination effects, further research on the potential

combination effects of cannabis's polypharmacy is essential to establish mechanisms of interaction, cellular targets of interest, and adverse events.

#### **7. Conclusions and Future Directions**

While commonly thought of as a psychoactive plant producing one of the most famous mind-altering chemicals discovered by humans, cannabis is a biosynthetic engine, producing hundreds of diverse phytochemicals that have the potential to impact a wide variety of human health conditions. In particular, cannabis produces 200 terpene structures that are of interest, both as independently bioactive molecules as well as by modulating or potentiating the effects of cannabinoids or other phytochemicals from cannabis. Terpenes are already widely implemented in traditional medicines and pharmaceuticals, as well as in industrial processes, perfumery, cosmetics, and food additives. They demonstrate generally low toxic profiles and high bioavailability and are highly selective to TRP channels, among other targets. There are known cannabimimetic activities of some terpenes, and they already have shown synergy amongs<sup>t</sup> each other in other in vitro and in vivo studies. Thus, there is a firm foundation for cannabis synergy and the involvement of terpenes in the flavor, aroma, and bioactivity of cannabis. Investigations into potential combination effects in cannabis is a growing field, one which requires rigorous experimental design and execution but has the possibility to evolve our understanding of cannabis's diverse pharmaceutical effects.

**Author Contributions:** Conceptualization, F.T.C., W.M.R.-K., K.E.V. and J.J.K.; writing—original draft preparation, F.T.C., K.E.V. and J.J.K.; writing—review and editing, F.T.C., W.M.R.-K., K.E.V. and J.J.K.; visualization, F.T.C.; supervision, J.J.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** J.J.K. is supported in part by the USDA National Institute of Food and Agriculture's Hatch Appropriations (PEN04772). K.E.V. (and the Penn State College of Medicine) is the recipient of research support from PA Options for Wellness (a state-approved medical marijuana clinical registrant). The funding sources were not involved in: study design; providing any experimental materials; data collection, analysis and interpretation; writing of the report; or the decision to submit the article for publication.

**Conflicts of Interest:** The authors declare no conflict of interest.
