Cannabis terpenoids: neglected entourage compounds?

Terpenoids are EO components, previously conceived as the quintessential fifth element, ‘life force’ or spirit & form the largest group of plant chemicals

Terpenoids are EO components, previously conceived as the quintessential fifth element, ‘life force’ or spirit (Schmidt, 2010), and form the largest group of plant chemicals, with 15–20 000 fully characterized (Langenheim, 1994). Terpenoids, not cannabinoids, are responsible for the aroma of cannabis. Over 200 have been reported in the plant (Hendriks et al., 1975; 1977; Malingre et al., 1975; Davalos et al., 1977; Ross and ElSohly, 1996; Mediavilla and Steinemann, 1997; Rothschild et al., 2005; Brenneisen, 2007), but only a few studies have concentrated on their pharmacology (McPartland and Pruitt, 1999; McPartland and Mediavilla, 2001a; McPartland and Russo, 2001b). Their yield is less than 1% in most cannabis assays, but they may represent 10% of trichome content (Potter, 2009). Monoterpenes usually predominate (limonene, myrcene, pinene), but these headspace volatiles (Hood et al., 1973), while only lost at a rate of about 5% before processing (Gershenzon, 1994), do suffer diminished yields with drying and storage (Turner et al., 1980; Ross and ElSohly, 1996), resulting in a higher relative proportion of sesquiterpenoids (especially caryophyllene), as also often occurs in extracts. A ‘phytochemical polymorphism’ seems operative in the plant (Franz and Novak, 2010), as production favours agents such as limonene and pinene in flowers that are repellent to insects (Nerio et al., 2010), while lower fan leaves express higher concentrations of bitter sesquiterpenoids that act as anti-feedants for grazing animals (Potter, 2009). Evolutionarily, terpenoids seem to occur in complex and variable mixtures with marked structural diversity to serve various ecological roles. Terpenoid composition is under genetic control (Langenheim, 1994), and some enzymes produce multiple products, again supporting Mechoulam’s ‘Law of Stinginess’. The particular mixture of mono- and sesquiterpenoids will determine viscosity, and in cannabis, this certainly is leveraged to practical advantage as the notable stickiness of cannabis exudations traps insects (McPartland et al., 2000), and thus, combined with the insecticidal phytocannabinoid acids (Sirikantaramas et al., 2005), provides a synergistic mechano-chemical defensive strategy versus predators.

As observed for cannabinoids, terpenoid production increases with light exposure, but decreases with soil fertility (Langenheim, 1994), and this is supported by the glasshouse experience that demonstrates higher yields if plants experience relative nitrogen lack just prior to harvest (Potter, 2004), favouring floral over foliar growth.

EO composition is much more genetically than environmentally determined, however (Franz and Novak, 2010), and while cannabis is allogamous and normally requires repeat selective breeding for maintenance of quality, this problem may be practically circumvented by vegetative propagation of high-performance plants under controlled environmental conditions (light, heat and humidity) (Potter, 2009), and such techniques have proven to provide notable consistency to tight tolerances as Good Manufacturing Practice for any pharmaceutical would require (Fischedick et al., 2010).

The European Pharmacopoeia, Sixth Edition (2007), lists 28 EOs (Pauli and Schilcher, 2010). Terpenoids are pharmacologically versatile: they are lipophilic, interact with cell membranes, neuronal and muscle ion channels, neurotransmitter receptors, G-protein coupled (odorant) receptors, second messenger systems and enzymes (Bowles, 2003; Buchbauer, 2010). All the terpenoids discussed herein are Generally Recognized as Safe, as attested by the US Food and Drug Admin-istration as food additives, or by the Food and Extract Manufacturers Association and other world regulatory bodies. Germane is the observation (Adams and Taylor, 2010) (p. 193), ‘With a high degree of confidence one may presume that EOs derived from food are likely to be safe’. Additionally, all the current entries are non-sensitizing to skin when fresh (Tisserand and Balacs, 1995; Adams and Taylor, 2010), but may cause allergic reactions at very low rates when oxidized (Matura et al., 2005). For additional pharmacological data on other common cannabis terpenoids not discussed herein (1,8-cineole, also known as eucalyptol, pulegone, a-terpineol, terpineol-4-ol, r-cymene, borneol and D-3-carene), please see McPartland and Russo (2001b).

Are cannabis terpenoids actually relevant to the effects of cannabis?

Terpenoid components in concentrations above 0.05% are considered of pharmacological interest (Adams and Taylor, 2010). Animal studies are certainly supportive (Buchbauer et al., 1993). Mice exposed to terpenoid odours inhaled from ambient air for 1 h demonstrated profound effects on activity levels, suggesting a direct pharmacological effect on the brain, even at extremely low serum concentrations (examples: linalool with 73% reduction in motility at 4.22 ng·mL-1, pinene 13.77% increase at trace concentration, terpineol 45% reduction at 4.7 ng·mL 1). These levels are comparable to those of THC measured in humans receiving cannabis extracts yielding therapeutic effects in pain, or symptoms of multiple sclerosis in various randomized controlled trials (RCTs) (Russo, 2006; Huestis, 2007). Positive effects at undetectable serum concentrations with orange terpenes (primarily limonene, 35.25% increase in mouse activity), could be explainable on the basis of rapid redistribution and concentration in lipophilic cerebral structures. A similar rationale pertains to human studies (Komori et al., 1995), subsequently discussed. Limonene is highly bioavailable with 70% human pulmonary uptake (Falk-Filipsson et al., 1993), and a figure of 60% for pinene with rapid metabolism or redistribution (Falk et al., 1990). Ingestion and percutaneous absorption is also well documented in humans (Jäger et al., 1992): 1500 mg of lavender EO with 24.7% linalool (total 372 mg) was massaged into the skin of a 60 kg man for 10 min, resulting in a peak plasma concentration of 100 ng·mL-1 at 19 min, and a half-life of 13.76 min in serum (Jäger et al., 1992). EO mixtures (including limonene and pinene) also increase permeation of estradiol through mouse skin (Monti et al., 2002).

Government-approved cannabis supplied to patients in national programmes in the Netherlands and Canada is gamma-irradiated to sterilize coliform bacteria, but the safety of this technique for a smoked and inhaled product has never been specifically tested. Gamma-radiation significantly reduced linalool titres in fresh cilantro (Fan and Sokorai, 2002), and myrcene and linalool in orange juice (Fan and Gates, 2001).

D-limonene, common to the lemon and other citrus EOs (Table 2), is the second most widely distributed terpenoid in nature (Noma and Asakawa, 2010), and is the precursor to other monoterpenoids (Figure 2) through species-specific synthetic schemes. Unfortunately, these pathways have not yet been investigated in cannabis. The ubiquity of limonene serves, perhaps, as a demonstration of convergent evolution that supports an important ecological role for this monoterpene. Studies with varying methodology and dosing in citrus oils in mice suggest it to be a powerful anxiolytic agent (Carvalho-Freitas and Costa, 2002; Pultrini Ade et al., 2006), with one EO increasing serotonin in the prefrontal cortex, and dopamine (DA) in hippocampus mediated via 5-HT1A (Komiya et al., 2006). Compelling confirmatory evidence in humans was provided in a clinical study (Komori et al., 1995), in which hospitalized depressed patients were exposed to citrus fragrance in ambient air, with subsequent normalization of Hamilton Depression Scores, successful discontinuation of antidepressant medication in 9/12 patients and serum evidence of immune stimulation (CD4/8 ratio normalization). Limonene also produces apoptosis of breast cancer cells, and was employed at high doses in Phase II RCTs (Vigushin et al., 1998). Subsequent investigation in cancer treatment has centred on its immediate hepatic metabolite, perillic acid, which demonstrates anti-stress effects in rat brain (Fukumoto et al., 2008). A patent has been submitted, claiming that limonene effectively treats gastro-oesophageal reflux (Harris, 2010). Citrus EOs containing limonene proved effective against dermatophytes (Sanguinetti et al., 2007; Singh et al., 2010), and citrus EOs with terpenoid profiles resembling those in cannabis demonstrated strong radical scavenging properties (Choi et al., 2000). As noted above, limonene is highly bioavailable (Falk-Filipsson et al., 1993), and rapidly metabolized, but with indications of accumulation and retention in adipose tissues (e.g. brain). It is highly non-toxic (estimated human lethal dose 0.5–5 g·kg-1) and non-sensitizing (Von Burg, 1995)

b-Myrcene is another common monoterpenoid in cannabis (Table 2) with myriad activities: diminishing inflammation via prostaglandin E-2 (PGE-2) (Lorenzetti et al., 1991), and blocking hepatic carcinogenesis by aflatoxin (De- Oliveira et al., 1997). Interestingly, myrcene is analgesic in mice, but this action can be blocked by naloxone, perhaps via the a-2 adrenoreceptor (Rao et al., 1990). It is nonmutagenic in the Ames test (Gomes-Carneiro et al., 2005). Myrcene is a recognized sedative as part of hops preparations (Humulus lupulus), employed to aid sleep in Germany (Bisset and Wichtl, 2004). Furthermore, myrcene acted as a muscle relaxant in mice, and potentiated barbiturate sleep time at high doses (do Vale et al., 2002). Together, these data would support the hypothesis that myrcene is a prominent sedative terpenoid in cannabis, and combined with THC, may produce the ‘couch-lock’ phenomenon of certain chemotypes that is alternatively decried or appreciated by recreational cannabis consumers.

a-Pinene is a bicyclic monoterpene (Table 2), and the most widely encountered terpenoid in nature (Noma and Asakawa, 2010). It appears in conifers and innumerable plant EOs, with an insect-repellent role. It is anti-inflammatory via PGE-1 (Gil et al., 1989), and is a bronchodilator in humans at low exposure levels (Falk et al., 1990). Pinene is a major component of Sideritis spp. (Kose et al., 2010) and Salvia spp. EOs (Ozek et al., 2010), both with prominent activity against MRSA (vide infra). Beyond this, it seems to be a broadspectrum antibiotic (Nissen et al., 2010). a-Pinene forms the biosynthetic base for CB2 ligands, such as HU-308 (Hanus et al., 1999). Perhaps most compelling, however, is its activity as an acetylcholinesterase inhibitor aiding memory (Perry et al., 2000), with an observed IC50 of 0.44 mM (Miyazawa and Yamafuji, 2005). This feature could counteract short-term memory deficits induced by THC intoxication (vide infra).

Representative plants containing each terpenoid are displayed as examples to promote recognition, but many species contain them in varying concentrations. 5-HT, 5-hydroxytryptamine (serotonin); AD, antidepressant; AI, anti-inflammatory; CB1/CB2, cannabinoid receptor 1 or 2; GABA, gamma aminobutyric acid; PGE-1/PGE-2, prostaglandin E-1/prostaglandin E-2; SSADH, succinic semialdehyde dehydrogenase.

Table 2 | Cannabis Terpenoid Activity Table

D-Linalool is a monoterpenoid alcohol (Table 2), common to lavender (Lavandula angustifolia), whose psychotropic anxiolytic activity has been reviewed in detail (Russo, 2001). Interestingly, linalyl acetate, the other primary terpenoid in lavender, hydrolyses to linalool in gastric secretions (Bickers et al., 2003). Linalool proved sedating to mouse activity on inhalation (Buchbauer et al., 1991; Jirovetz et al., 1992). In traditional aromatherapy, linalool is the likely suspect in the remarkable therapeutic capabilities of lavender EO to alleviate skin burns without scarring (Gattefosse, 1993). Pertinent to this, the local anaesthetic effects of linalool (Re et al., 2000) are equal to those of procaine and menthol (Ghelardini et al., 1999). Another explanation would be its ability to produce hot-plate analgesia in mice (P < 0.001) that was reduced by administration of an adenosine A2A antagonist (Peana et al., 2006). It is also anti-nociceptive at high doses in mice via ionotropic glutamate receptors (Batista et al., 2008). Linalool demonstrated anticonvulsant and antiglutamatergic activity (Elisabetsky et al., 1995), and reduced seizures as part of Ocimum basilicum EO after exposure to pentylenetetrazole, picrotoxin and strychnine (Ismail, 2006). Furthermore, linalool decreased K+-stimulated glutamate release and uptake in mouse synaptosomes (Silva Brum et al., 2001). These effects were summarized (Nunes et al., 2010, p. 303): ‘Overall, it seems reasonable to argue that the modulation of glutamate and GABA neurotransmitter systems are likely to be the critical mechanism responsible for the sedative, anxiolytic and anticonvulsant properties of linalool and EOs containing linalool in significant proportions’. Linalool also proved to be a powerful anti-leishmanial agent (do Socorro et al., 2003), and as a presumed lavender EO component, decreased morphine opioid usage after inhalation versus placebo (P = 0.04) in gastric banding in morbidly obese surgical patients (Kim et al., 2007).

b-Caryophyllene (Table 2) is generally the most common sesquiterpenoid encountered in cannabis (Mediavilla and Steinemann, 1997), wherein its evolutionary function may be due to its ability to attract insect predatory green lacewings, while simultaneously inhibiting insect herbivory (Langenheim, 1994). It is frequently the predominant terpenoid overall in cannabis extracts, particularly if they have been processed under heat for decarboxylation (Guy and Stott, 2005). Caryophyllene is common to black pepper (Piper nigrum) and Copaiba balsam (Copaifera officinalis) (Lawless, 1995). It is anti-inflammatory via PGE-1, comparable in potency to the toxic phenylbutazone (Basile et al., 1988), and an EO containing it was on par with etodolac and indomethacin (Ozturk and Ozbek, 2005). In contrast to the latter agents, however, caryophyllene was a gastric cytoprotective (Tambe et al., 1996), much as had been claimed in the past in treating duodenal ulcers in the UK with cannabis extract (Douthwaite, 1947). Caryophyllene may have contributed to antimalarial effects as an EO component (Campbell et al., 1997). Perhaps the greatest revelation regarding caryophyllene has been its demonstration as a selective full agonist at CB2 (100 nM), the first proven phytocannabinoid beyond the cannabis genus (Gertsch et al., 2008). Subsequent work has demonstrated that this dietary component produced antiinflammatory analgesic activity at the lowest dose of 5 mg·kg-1 in wild-type, but not CB2 knockout mice (Gertsch, 2008). Given the lack of attributed psychoactivity of CB2 agonists, caryophyllene offers great promise as a therapeutic compound, whether systemically, or in dermatological applications such as contact dermatitis (Karsak et al., 2007). Sensitization reactions are quite rare, and probably due to oxidized product (Skold et al., 2006).

Nerolidol is a sesquiterpene alcohol with sedative properties (Binet et al., 1972), present as a low-level component in orange and other citrus peels (Table 2). It diminished experimentally induced formation of colon adenomas in rats (Wattenberg, 1991). It was an effective agent for enhancing skin penetration of 5-fluorouracil (Cornwell and Barry, 1994). This could be a helpful property in treating fungal growth, where it is also an inhibitor (Langenheim, 1994). It seems to have anti-protozoal parasite control benefits, as a potent antimalarial (Lopes et al., 1999; Rodrigues Goulart et al., 2004) and anti-leishmanial agent (Arruda et al., 2005). Nerolidol is nontoxic and non-sensitizing (Lapczynski et al., 2008).

Caryophyllene oxide (Table 2) is a sesquiterpenoid oxide common to lemon balm (Melissa officinalis), and to the eucalyptus, Melaleuca stypheloides, whose EO contains 43.8% (Farag et al., 2004). In the plant, it serves as an insecticidal/ anti-feedant (Bettarini et al., 1993) and as broad-spectrum antifungal in plant defence (Langenheim, 1994). Analogously, the latter properties may prove therapeutic, as caryophyllene oxide demonstrated antifungal efficacy in a model of clinical onychomycosis comparable to ciclopiroxalamine and sulconazole, with an 8% concentration affecting eradication in 15 days (Yang et al., 1999). Caryophyllene oxide is non-toxic and non-sensitizing (Opdyke, 1983). This agent also demonstrates anti-platelet aggregation properties in vitro (Lin et al., 2003). Caryophyllene oxide has the distinction of being the component responsible for cannabis identification by drug-sniffing dogs (Stahl and Kunde, 1973).

Phytol (Table 2) is a diterpene (McGinty et al., 2010), present in cannabis extracts, as a breakdown product of chlorophyll and tocopherol. Phytol prevented vitamin A-induced teratogenesis by inhibiting conversion of retinol to a harmful metabolite, all-trans-retinoic acid (Arnhold et al., 2002). Phytol increased GABA expression via inhibition of succinic semialdehyde dehydrogenase, one of its degradative enzymes (Bang et al., 2002). Thus, the presence of phytol could account for the alleged relaxing effect of wild lettuce (Lactuca sativa), or green tea (Camellia sinensis), despite the latter’s caffeine content.