Medicinal Properties of Terpenes Found in Cannabis Sativa and Humulus Lupulus

ACCEPTED MANUSCRIPT Medicinal properties of terpenes found in Cannabis sativa and Humulus lupulus Tarmo Nuutinen 1,2,* 1 Department of Environmental and Biological Sciences, Univerisity of Eastern Finland (UEF) 2 Department of Physics and Mathematics, UEF * Email: [email protected] Abstract Cannabaceae plants Cannabis sativa L. and Humulus lupulus L. are rich in terpenes – both are typically comprised of terpenes as up to 3-5% of the dry-mass of the female inflorescence. Terpenes of cannabis and hops are typically simple mono- and sesquiterpenes derived from two and three isoprene units, respectively. Some terpenes are relatively well known for their potential in biomedicine and have been used in traditional medicine for centuries, while others are yet to be studied in detail. The current, comprehensive review presents terpenes found in cannabis and hops. Terpenes’ medicinal properties are supported by numerous in vitro , animal and clinical trials and show anti-inflammatory, antioxidant, analgesic, anticonvulsive, antidepressant, anxiolytic, anticancer, antitumor, neuroprotective, anti-mutagenic, anti-allergic, antibiotic and anti-diabetic attributes, among others. Because of the very low toxicity, these terpenes are already widely used as food additives and in cosmetic products. Thus, they have been proven safe and well-tolerated. Keywords MANUSCRIPT Monoterpene; sesquiterpene; Cannabis sativa ; Humulus lupulus ; medicine; medicinal property Introduction Plants produce terpenes for interactions with other organisms [1]. Terpenes protect plants against pathogens like mold, fungus and bacteria, and can attract pollinating insects or repel herbivores. Thousands of terpenes have been found across the plantae , but only a small percentage of all terpenes have been identified [2]. Despite the diversity of the known terpenes, some are concentrated in certain phyla or families such as Cannabaceae . Terpenes found in Cannabis sativa (cannabis) and Humulus lupulus (hop), or more precisely, in their essential oils (EOs), are mainly mono- and sesquiterpenes: up to 99% of all terpenes found in the EO of hops [3] and up to 98% in cannabis EO [4]. Cannabis and hop produce and accumulate a terpene-rich resin in glandular trichomes, which are most abundant on the surface of female inflorescences. Some terpene synthases are specialized to produce strictly one terpene, while others are multi-substrate enzymes producing more than one terpene [5]. Recently, a transcriptome analysis of trichomes of the hemp variety “Finola” identifiedACCEPTED 33 complete terpene synthase (TPS) genes and an additional 18 putative TPSs. At the protein level, 40 enzymes involved in the synthesis of terpenes were identified in hop [6]. Cannabis and hops have been used in traditional medicine for millennia around the world. However, all of the active constituents and their mechanisms of action have not yet been explored. Naturally, the action of cannabis is mostly based on cannabinoids, but not all of its medicinal properties. Hops, which are devoid of cannabinoids, have been used as sedative means e.g. for the treatment of insomnia, depressive symptoms, irritation, nervous tension, delirium, anxiety and digestive disorders [7]. Cannabis has been used in traditional Chinese medicine for the treatment of pain, tetany and convulsions, gout, Amania,CCEPTED insomnia, MANUSCRIPT panting and cough, headache, menstrual irregularities, itching and anemia [8], and traditional Indian medical texts have proposed the use of cannabis to stimulate digestion, and act as an analgesic and nervous system stimulant, as well as for its sedative, spasmolytic, diuretic, aphrodisiac, anti-parasitic and anti-viral actions, in the treatment of glaucoma and in skin care [9]. The medicinal properties of some terpenes were reviewed by Russo in 2011 [10] and by Russo and McPartland in 2001 [11]. In turn, hop terpenes have not yet been reviewed. More generally, monoterpenes provide various medicinal properties, including antimicrobial, antioxidant, anticancer, antiarrhythmic, anti-aggregating, anesthetic, anti-nociceptive, anti-inflammatory, antihistaminic, anti-spasmodic, antitumor and anti-diabetic [12]. These can be also attributed to the mono- and sesquiterpenes found in hops and cannabis, and the current review extends the list with many other medicinal properties evidenced by numerous in vitro , in vivo and clinical trials. It also shows some new data derived from publicly available data about the terpene contents of cannabis samples [13] and biological databases. Myrcene Myrcene (MYR, the molecular structure of which is shown in Fig. 1A) is frequently the most abundant terpene encountered in cannabis and hops. For instance, the total terpenes of the cannabis drug chemotype 'blueberry' consist of up to 78% MYR [13]. Furthermore, some drug chemotypes, possibly due to the founder effect and selective breeding, show a high but stable composition with respect to this terpene; for instance, the medical cannabis chemotype, with low THC and high CBD, also known as “cannatonic”, mainly expresses myrcene: 63±11.5% of total terpenes (n=15 from 10 different sources [13]). In hops, myrcene may be present in a proportion of up to 52% [3]. It is well established as a flavor ingredient in the food industry and as a fragrance in soap and detergent products. It is also found in lemon grass, bay leaves, ylang-ylang, wild thyme, parsley, cardamom, and basil. It is claimed to have sedative properties, but there is only very weak support for this to date: in one study [14], it increased barbiturate-inducedMANUSCRIPT sleeping time and motor relaxation in mice, but only with doses as high as 200 mg/kg. MYR decreased IL-1β-induced nitric oxide (NO) production, nuclear factor kappa-light-chain- enhancer of activated B cells protein (NF-κB), c-Jun N-terminal kinase (JNK) and p38 mitogen- activated protein kinase (p38 MAPK) activation and the expression of inflammatory, inducible nitric oxide synthase (iNOS) and catabolic, matrix metalloproteinases 1 and 13 (MMP-1, MMP-13 genes (IC 50 =37.3 µg/ml)) [15]. These anti-inflammatory and anti-catabolic effects in the cell model of osteoarthritis together imply a potential to slow down the progression of osteoarthritis. In mice, MYR ameliorated heart tissue damage after global cerebral ischemia/reperfusion (I/R) by increasing the levels of glutathione (GSH) and anti-oxidative enzymes; glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD) [16]. In a mouse model of cerebral ischemia, MYR (200 mg/kg) was able to suppress oxidative stress by restoring levels of GSH, GPx, and SOD after the cerebral ischemia [17]. It also suppressed the formation of thiobarbituric acid reactive substances (TBARS). These mechanisms resulted in a significant neuroprotection. In another study, MYR orally administered to rats at a dose of 200 mg/kg/day protected against environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducedACCEPTED liver damage [18]. In the liver samples, taken at 60 days, GSH, CAT, GSH-Px and CuZn-SOD were substantially increased and the formation of TBARS was again decreased in comparison to the control. MYR also show anti-ulcer activity [19]; an oral administration of MYR at a dose of 7.50 mg/kg increased the levels of GPx, glutathione reductase (GSR), and total GSH in gastric tissue. The protective effects of MYR may in part be mediated by the decreased production of an inflammatory prostaglandin E-2 (PGE-2) [20]. At the doses of 5 and 10 mg/kg, MYR was able to prevent hypernociception in both mechanical and thermal tests on mice [21]. Another study, with neuropathic mouse models, showed that the anti- hypernociceptive activity of MYR may be mediated by α2-adrenoceptor-stimulated release of endogenous opioids [22]. It is known that some agonists of the α2-adrenergic receptor are frequently used in veterinary anesthesia.ACCEPTED MANUSCRIPT Several studies have shown that MYR is not mutagenic e.g. by the Ames test [23]. More importantly, studies have found the opposite: MYR exerts its anti-mutagenic activity by inhibiting certain forms of the cytochrome P-450 isoenzymes, which would otherwise cause the activation of pre-mutagens and pre-carcinogens [24]. Furthermore, MYR was efficient against oxidant-induced genotoxicity, which is predominately mediated by its direct radical scavenging activity [25]. Lastly, MYR had a protective role on UVB-induced human skin photoaging [26]. Thus, less toxic MYR could replace the UV-filter chemicals that are currently being used in sunscreen products. In summary, MYR protects the brain, heart and skin tissues from inflammation and oxidative damage; it also shows anti-nociceptive properties. However, there is only weak support for the alleged sedative effect of MYR. β-caryophyllene β-caryophyllene (BCP, Fig. 1B) is frequently the predominant terpenoid in cannabis and present in hops. For instance, it comprises 64% of terpenes in the cannabis drug chemotype 'gorilla glue' [13], but is almost absent (1.2±0.2%) in some other samples [27]. It is typically less abundant in hops, making up to 15% of the EO of a wild-growing hop [3]. It is also widely present in a large number of plants e.g. clove, rosemary, black pepper and lavender. Unlike any other terpenes, BCP has a notable affinity (150 nM) toward cannabinoid receptor 2 (CB2), being a selective, full agonist of the receptor [28]. This also makes it the only phytocannabinoid found outside the Cannabis genus to date. The CB2 agonism may be the most important feature

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