European Journal of Medicinal Chemistry 157 (2018) 198e228

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Review article

Medicinal properties of terpenes found in Cannabis sativa and

Humulus lupulus
Tarmo Nuutinen a, b, *
a
Department of Environmental and Biological Sciences, Univerisity of Eastern Finland (UEF), Finland
b
Department of Physics and Mathematics, UEF, Finland

a r t i c l e i n f o a b s t r a c t

Article history: Cannabaceae plants Cannabis sativa L. and Humulus lupulus L. are rich in terpenes e both are typically
Received 9 March 2018 comprised of terpenes as up to 3e5% of the dry-mass of the female inflorescence. Terpenes of cannabis
Received in revised form and hops are typically simple mono- and sesquiterpenes derived from two and three isoprene units,
30 July 2018
respectively. Some terpenes are relatively well known for their potential in biomedicine and have been
Accepted 31 July 2018
Available online 4 August 2018
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,
Keywords:
Monoterpene
analgesic, anticonvulsive, antidepressant, anxiolytic, anticancer, antitumor, neuroprotective, anti-
Sesquiterpene mutagenic, anti-allergic, antibiotic and anti-diabetic attributes, among others. Because of the very low
Cannabis sativa toxicity, these terpenes are already widely used as food additives and in cosmetic products. Thus, they
Humulus lupulus have been proven safe and well-tolerated.
Medicine © 2018 Elsevier Masson SAS. All rights reserved.
Medicinal property

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
2. Myrcene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
3. b-caryophyllene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
4. Caryophyllene oxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
5. Humulene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
6. a-Pinene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
7. b-Pinene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
8. Linalool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
9. Limonene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
10. Perillyl alcohol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
11. Terpinolene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
12. g-Terpinene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
13. a-Terpinene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
14. Terpineols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
15. Geraniol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
16. Nerolidol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
17. Borneol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
18. a-Bisabolol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
19. Bisabolenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
20. b-elemene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
21. Fenchone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
22. Pulegone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215

* Department of Environmental and Biological Sciences, Univerisity of Eastern Finland (UEF), Finland.
E-mail address: tarmon@uef.fi.

https://doi.org/10.1016/j.ejmech.2018.07.076
0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.
 T. Nuutinen / European Journal of Medicinal Chemistry 157 (2018) 198e228 199

23. a-Phellandrene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215

24. b-eudesmol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
25. Other terpenes found in cannabis and hops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
26. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
27. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220

1. Introduction Fig. 1A) is frequently the most abundant terpene encountered in

cannabis and hops. For instance, the total terpenes of the cannabis
Plants produce terpenes for interactions with other organisms drug chemotype 'blueberry' consist of up to 78% MYR [13].
[1]. Terpenes protect plants against pathogens like mold, fungus and Furthermore, some drug chemotypes, possibly due to the founder
bacteria, and can attract pollinating insects or repel herbivores. effect and selective breeding, show a high but stable composition
Thousands of terpenes have been found across the plantae, but only a with respect to this terpene; for instance, the medical cannabis
small percentage of all terpenes have been identified [2]. Despite the chemotype, with low THC and high CBD, also known as “canna-
diversity of the known terpenes, some are concentrated in certain tonic”, mainly expresses myrcene: 63 ± 11.5% of total terpenes
phyla or families such as Cannabaceae. Terpenes found in Cannabis (n ¼ 15 from 10 different sources [13]). In hops, myrcene may be
sativa (cannabis) and Humulus lupulus (hop), or more precisely, in present in a proportion of up to 52% [3]. It is well established as a
their essential oils (EOs), are mainly mono- and sesquiterpenes: up flavor ingredient in the food industry and as a fragrance in soap and
to 99% of all terpenes found in the EO of hops [3] and up to 98% in detergent products. It is also found in lemon grass, bay leaves,
cannabis EO [4]. Cannabis and hop produce and accumulate a ylang-ylang, wild thyme, parsley, cardamom, and basil. It is claimed
terpene-rich resin in glandular trichomes, which are most abundant to have sedative properties, but there is only very weak support for
on the surface of female inflorescences. Some terpene synthases are this to date: in one study [14], it increased barbiturate-induced
specialized to produce strictly one terpene, while others are multi- sleeping time and motor relaxation in mice, but only with doses
substrate enzymes producing more than one terpene [5]. Recently, as high as 200 mg/kg.
a transcriptome analysis of trichomes of the hemp variety “Finola” MYR decreased IL-1b-induced nitric oxide (NO) production,
identified 33 complete terpene synthase (TPS) genes and an addi- nuclear factor kappa-light-chain-enhancer of activated B cells
tional 18 putative TPSs. At the protein level, 40 enzymes involved in protein (NF-kB), c-Jun N-terminal kinase (JNK) and p38 mitogen-
the synthesis of terpenes were identified in hop [6]. activated protein kinase (p38 MAPK) activation and the expres-
Cannabis and hops have been used in traditional medicine for sion of inflammatory, inducible nitric oxide synthase (iNOS) and
millennia around the world. However, all of the active constituents catabolic, matrix metalloproteinases 1 and 13 (MMP-1, MMP-13
and their mechanisms of action have not yet been explored. genes (IC50 ¼ 37.3 mg/ml)) [15]. These anti-inflammatory and anti-
Naturally, the action of cannabis is mostly based on cannabinoids, catabolic effects in the cell model of osteoarthritis together imply
but not all of its medicinal properties. Hops, which are devoid of a potential to slow down the progression of osteoarthritis. In mice,
cannabinoids, have been used as sedative means e.g. for the MYR ameliorated heart tissue damage after global cerebral
treatment of insomnia, depressive symptoms, irritation, nervous ischemia/reperfusion (I/R) by increasing the levels of glutathione
tension, delirium, anxiety and digestive disorders [7]. Cannabis has (GSH) and anti-oxidative enzymes; glutathione peroxidase (GPx),
been used in traditional Chinese medicine for the treatment of pain, catalase (CAT) and superoxide dismutase (SOD) [16]. In a mouse
tetany and convulsions, gout, mania, insomnia, panting and cough, model of cerebral ischemia, MYR (200 mg/kg) was able to suppress
headache, menstrual irregularities, itching and anemia [8], and oxidative stress by restoring levels of GSH, GPx, and SOD after the
traditional Indian medical texts have proposed the use of cannabis cerebral ischemia [17]. It also suppressed the formation of thio-
to stimulate digestion, and act as an analgesic and nervous system barbituric acid reactive substances (TBARS). These mechanisms
stimulant, as well as for its sedative, spasmolytic, diuretic, aphro- resulted in a significant neuroprotection. In another study, MYR
disiac, anti-parasitic and anti-viral actions, in the treatment of orally administered to rats at a dose of 200 mg/kg/day protected
glaucoma and in skin care [9]. against environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-
The medicinal properties of some terpenes were reviewed by dioxin-induced liver damage [18]. In the liver samples, taken at 60
Russo in 2011 [10] and by Russo and McPartland in 2001 [11]. In days, GSH, CAT, GSH-Px and CuZn-SOD were substantially
turn, hop terpenes have not yet been reviewed. More generally, increased and the formation of TBARS was again decreased in
monoterpenes provide various medicinal properties, including comparison to the control. MYR also show anti-ulcer activity [19];
antimicrobial, antioxidant, anticancer, antiarrhythmic, anti- an oral administration of MYR at a dose of 7.50 mg/kg increased the
aggregating, anesthetic, anti-nociceptive, anti-inflammatory, anti- levels of GPx, glutathione reductase (GSR), and total GSH in gastric
histaminic, anti-spasmodic, antitumor and anti-diabetic [12]. These tissue. The protective effects of MYR may in part be mediated by the
can be also attributed to the mono- and sesquiterpenes found in decreased production of an inflammatory prostaglandin E2 (PGE-
hops and cannabis, and the current review extends the list with 2) [20]. At the doses of 5 and 10 mg/kg, MYR was able to prevent
many other medicinal properties evidenced by numerous in vitro, hypernociception in both mechanical and thermal tests on mice
in vivo and clinical trials. It also shows some new data derived from [21]. Another study, with neuropathic mouse models, showed that
publicly available data about the terpene contents of cannabis the anti-hypernociceptive activity of MYR may be mediated by a2-
samples [13] and biological databases. adrenoceptor-stimulated release of endogenous opioids [22]. It is
known that some agonists of the a2-adrenergic receptor are
frequently used in veterinary anesthesia.
2. Myrcene Several studies have shown that MYR is not mutagenic e.g. by
the Ames test [23]. More importantly, studies have found the
Myrcene (MYR, the molecular structure of which is shown in
200 T. Nuutinen / European Journal of Medicinal Chemistry 157 (2018) 198e228
Fig. 1. Molecular structures of A) myrcene, B) b-caryophyllene, C) caryophyllene oxide and D) humulene.
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 pro-
tective 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.
3. b-caryophyllene
b-caryophyllene (BCP, Fig. 1B) is frequently the predominant
terpenoid in cannabis and present in hops. For instance, it com-
prises 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 canna-
binoid receptor 2 (CB2), being a selective, full agonist of the re-
ceptor [28]. This also makes it the only phytocannabinoid found
outside the Cannabis genus to date. The CB2 agonism may be the
most important feature of BCP against inflammation. For instance,
it produces strong anti-inflammatory activity at a dose of 5 mg/kg
in wild type but not CB2 knockout mice [28]. Thus, the specific CB2
agonist holds a great deal of potential for the treatment of various
diseases without any intoxicating effects [29]. The Ki value of BCP is
150 nm while EC50 is 1.9 mM [30].
BCP possesses significant anticancer activities, affecting the
growth and proliferation of numerous cancer cells. The anticancer
qualities are reviewed in detail elsewhere [31]. Briefly, the most
important function of BCP may be the induction of the expression
of proapoptotic and cancer-suppressing genes, encoding proteins
like p53, bcl-2-like protein 4 (bax), Bcl-2 homologous antagonist/
killer (bak), caspase 8, caspase 9 and ATM serine/threonine kinase
(ATM) and suppression the genes encoding anti-apoptotic genes,
such as B-cell lymphoma 2 (bcl-2), E3 ubiquitin-protein ligase
Mdm2 (mdm2), cyclooxygenase 2 (COX-2), and myeloblastosis c-
myb (c-myb). In addition, despite the low cytotoxicity, it can
potentiate the efficacy of classical cancer drugs by augmenting their
concentrations inside the cells [32]. Furthermore, it exhibits a
synergistic anticancer activity with humulene and iso-
caryophyllene, as shown by studies on MCF-7, DLD-1 and L-929 cell
lines [33]. Lastly, 10 mg/ml BCP was able to potentiate the activity of
paclitaxel (PAC) by a 10-fold increase. Possible mechanisms include
the capability to alter levels of mitogen-activated protein kinase
(MAPK) and PI3K/AKT/mTOR/S6K1 (phosphatidylinositol-3-kinase/
protein kinase B/mammalian target of rapamycin/ribosomal pro-
tein S6 kinase b-1) and signal transducer and activator of
transcription 3 (STAT3) pathways [31].
Various other pharmacological activities of BCP include car-
dioprotective, hepatoprotective, gastroprotective, neuroprotective,
nephroprotective, anti-inflammatory and immunomodulatory ac-
tions (reviewed in Ref. [34]). BCP possesses potent therapeutic
promises for neuropathic pain, and neurodegenerative and meta-
bolic diseases; the mechanisms of action are mediated not only
through CB2 agonism but also via the activation of peroxisome
proliferated activator receptors (PPARs), the toll like receptor
complex CD14/TLR4/MD2, synergy with m-opioid receptor-
dependent pathways and antagonism on nicotinic acetylcholine
receptor a7 (a7-nAChRs), among others. The beneficial effects of
BCP (25 mg/kg twice/day) in a multiple sclerosis (MS) model
included the suppression of neuroinflammation by the upregula-
tion of interleukin 10 (IL-10) and a reduction in the production of
interferon g (IFN-g) [35]. The efficacy was comparable to that of a
research chemical JWH-015, which is a CB2 agonist. Furthermore,
the immunomodulatory effect of BCP seems to be related to its
ability to inhibit microglial cells, and CD4þ and CD8þ T lympho-
cytes. Through the activation of CB2, axonal demyelination was
ameliorated and the immune balance of the cells was restored. BCP
displayed marked inhibitory effects in various inflammatory
experimental models in mice and rats [36]. BCP diminished tumor
necrosis factor a (TNFa) release and reduced the production of PGE-
2, iNOS and COX-2 expressions. The effects were comparable to
those of the corticosteroid dexamethasone in animal models.
BCP inhibits hypoxia-induced cytotoxicity and neuro-
inflammation, inhibiting NF-kB activation in microglia [37]. Also, it
inhibits the release of pro-inflammatory cytokines, IL-1b, TNF-a,
and IL-6 and reactive oxygen species (ROS). In a PC12 cell model, by
activating tropomyosin receptor kinase A (TrkA) receptors, BCP
(10 mM and less) evoked neurogenesis independently from neuro-
trophic factor (NGF) or cannabinoid receptors [38]. This was due to
its ability to increase the expression of axonal-plasticity-associated
proteins: growth-associated protein-43 (GAP-43), synapsin and
synaptophysin. BCP also protected astrocytes against Glu-induced
cytotoxicity. It ameliorated the Glu-induced increase in intracel-
lular ROS and mitochondrial dysfunction by the activation and
nuclear translocation of nuclear factor (erythroid-derived 2)-like 2
(Nrf2) [39]. Nrf2 activation was only partly dependent on the
activation of CB2. Another study [40], using a rat model of I/R injury,
showed that BCP was able to activate the PI3K/Akt signaling
pathway and thus exhibited neuroprotection in the hippocampus,
as evidenced by reduced apoptosis. This was seen as the down-
regulation of expression of the pro-apoptotic Bax and p53 pro-
teins and up-regulation of the expression of the anti-apoptotic B-
cell lymphoma 2 (Bcl-2) protein. Moreover, BCP, at mM concentra-
tions, significantly reduced the number of necroptotic neurons,
infarct volumes and neuronal necrosis in a cerebral I/R injury model
[41]. BCP in combination with hydroxypropyl-b-cyclodextrin
diminished cognitive deficits in vascular dementia (VD) rats and
attenuated learning and memory deficits in rats [42]. Moreover,
BCP promoted cerebral blood flow, and increased the expression
levels of CB2 in the hippocampus and white matter tissues and the
 T. Nuutinen / European Journal of Medicinal Chemistry 157 (2018) 198e228
expression of PI3K, which has been linked to the induction of long-
term potentiation (LTP). Protective features were also seen in
experimental autoimmune encephalomyelitis (EAE) models of MS
in the mM range [43]: in vitro (20 mM) and in vivo (25 mg/kg/day),
BCP inhibited the production of H2O2, NO, IFN-g, and TNF-a, while,
in vivo, also IL-17. BCP treatment significantly reduced the number
of inflammatory infiltrates and attenuated the neurological damage
in the central nervous system (CNS).
Recent studies have suggested a protective role of the canna-
binoid signaling system in Parkinson's disease (PD) [29]. The neu-
roprotective effects of BCP were found in a 1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine (MPTP)-induced murine model of PD
[44]. BCP (10 mg/kg, intraperitoneal, [i.p.]) improved motor func-
tion, protected dopaminergic neurons in the substantia nigra and
striatum and diminished glial activation. Additionally, BCP inhibi-
ted the levels of inflammatory cytokines in the nigrostriatal system.
These effects were reversed upon treatment with a CB2 selective
antagonist, AM630, implicating the involvement of CB2 agonism. In
a rat model of PD, the neuroprotective property of BCP (with daily
intake for 4 weeks at a dose of 50 mg/kg) against rotenone-induced
oxidative stress and neuroinflammation was demonstrated [45].
Again, BCP was able to rescue the loss of dopaminergic neurons.
Furthermore, it decreased the activation of microglia and astrocytes
by the reduced expression of ionized calcium-binding adapter
molecule 1 (Iba-1) and glial fibrillary acidic protein (GFAP). In
addition, it restored levels of the antioxidant enzymes and GSH and
down-regulated the pro-inflammatory molecules COX-2 and iNOS.
In another in vivo study, BCP (50 mg/kg) suppressed neutrophil
migration and chemokine (C-X-C motif) ligand 1 (CXCL1), leuko-
triene B4 (LTB4), IL-12, and the NO production induced by Myco-
bacterium bovis [46].
CB2 agonists are also known to possess analgesic properties
[29]. This is also true in the case of BCP; in mouse models of in-
flammatory and neuropathic pains, BCP at a dose of 1 mg/kg exer-
ted analgesic effects, evidenced as reduced thermal hyperalgesia,
mechanical allodynia and spinal neuroinflammation [47]. However,
BCP failed to show these effects in CB2-knockout mice, implicating
the involvement of CB2-dependent mechanisms. BCP alleviated
PAC-induced peripheral neuropathy in a mouse model and reduced
the activation of p38 MAPK and NF-kB and the increased Iba-1 and
IL-1b levels, while treatment with AM630 (a CB2 antagonist)
abrogated these effects [48]. In addition, BCB seems to be an
anxiolytic and anti-depressant, but these effects can be fully
reversed by treatment with AM630 [49]. This indicates that CB2
signaling is also involved in depression. Another study [50] noted
that the anxiolytic effect of BCP was mediated by 5-HT (1 A)
without the involvement of GABA(A) receptors. Finally, BCP
(100 mg/kg) displayed anticonvulsant activity against seizures
induced by pentylenetetrazole (PTZ) [51]. No adverse effects were
observed.
In hypercholesterolemic rats, BCP treatment was able to protect
the cardiac tissue against atherosclerosis by antioxidant mecha-
nisms [52]. It prevented lipid oxidation by decreasing cardiac levels
of ROS and TBARS and increasing GSH levels and GPx activity. BCP
also significantly decreased the atherogenic index (AI) and coro-
nary risk index (CRI). Another study also reported that BCP reduced
the total serum levels of cholesterol and triglycerides while
macrophage infiltration to the aortic surface was also inhibited
[53]. Importantly, it was able to prevent the attachment of leuko-
cyte cells to endothelial cells. Moreover, it inhibited the induction of
vascular cell adhesion molecule-1 (VCAM-1) both in vitro and
in vivo through CB2 activation.
Several recent studies have suggested that the CB2 cannabinoid
receptors in the brain play a major role in feeding behavior,
addiction and alcohol reward [29]. Indeed, the CB2 agonist BCP
201
decreased alcohol consumption in mice in a dose-dependent
manner [54]. This was reversed when the mice were pre-injected
with AM630. On the other hand, BCP is protective against chronic
and binge alcohol intake-induced liver injury and inflammation
[55]. It suppressed the pro-inflammatory phenotypic ‘M1’ switch of
Kupffer cells and neutrophil infiltration by decreasing the expres-
sion of VCAM-1, E-Selectin and P-Selectin. It also beneficially
influenced hepatic metabolic dysregulations (steatosis, protein
hyperacetylation and PPAR-a signaling). These protective effects of
BCP against alcohol-induced liver injury were again dependent on
CB2 agonism. Nonetheless, in Caenorhabditis elegans, the adminis-
tration of 50 mM of BCP increased the lifespan by over 22%
(P  0.0001) and decreased free radical levels in cells [56].
Furthermore, BCP diminished intestinal pro-aging lipofuscin levels.
Moreover, this terpene potentially inhibits spontaneous contrac-
tion in the guinea pig ileum at a concentration of 1.5 mM https://
pubchem.ncbi.nlm.nih.gov/compound/5281515#. Finally, BCP
shows antibacterial properties [34] and is safe and anti-mutagenic
[57].
In conclusion, BCP is a selective, full agonist of CB2 and may
mediate it as part of its actions through mu-opioid, alpha7-nAChR
and 5-HT (1 A) receptors. BCP, along with CB2 agonists, participates
in dopaminergic cell protection, the inhibition of microglia and
astrocyte activation and neuroprotection and modulates noci-
ception, feeding behavior, addiction and alcohol consumption, also
preventing alcohol-induced damage. It also shows anti-
inflammatory and anti-convulsive properties. Taken together, it
shows multi-target potential for the treatment of MS and PD and
other neuroinflammatory conditions.
4. Caryophyllene oxide
The oxidized derivative of BCP, caryophyllene oxide (BCPO,
Fig. 1C), is also found in plants outside of Cannabaceae, such as
guava (Psidium guajava), oregano (Origanum vulgare), cinnamon
(Cinnamomum spp.) clove (Eugenia caryophyllata), black pepper
(Piper nigrum), lemon balm (Melissa officinalis) and eucalyptus
(Melaleuca stypheloides), whose EO contains 44% BCPO [58]. BCPO is
a non-toxic and non-sensitizing agent ([10] and refs), which is often
used as a preservative in foods, drugs and cosmetics, as well as an
insecticidal. Unlike BCP, BCPO does not possess any remarkable
binding affinity towards CB2; however, it seems to be a multi-target
molecule, known for its anticancer and analgesic properties. These
properties were reviewed in Ref. [31]. The potential for its use as a
cancer drug is enhanced by negative genotoxic findings and its
efficient absorption through cell membranes [57,59].
In many cancers, the PI3K/AKT/mTOR/S6K1 pathway is over-
active, thus reducing apoptosis and allowing cells to proliferate.
Despite the various anticancer activities of BCPO, the suppression of
this pathway may be its most important mechanism of action [58],
via suppression of the STAT3 (signal transducer and activator of
transcription 3) pathway by activation of the Src homology region 2
domain-containing phosphatase-1 (SHP-1) protein tyrosine phos-
phatase [60]. In vitro, it also induces gene expression of the pro-
apoptotic bax, bak1, caspase 8, caspase 9, and ATM proteins,
while mRNA levels of anti-apoptotic genes, such as bcl-2, mdm2,
and c-myb were decreased [58]. Furthermore, it activated the pro-
apoptotic ERK, JNKs and p38 MAPK pathways but down-regulated
the expression of gene products that mediate cell proliferation:
cyclin D1, COX-2, vascular endothelial growth factor (VEGF) and
apoptosis inhibitors: bcl-2, bcl-xL (bcl extra-large), IAP-1, IAP-2
(inhibitor of apoptosis 1 and 2) and survivin. BCPO also increases
the expression of the tumor suppressor proteins p53 and p21.
Furthermore, a previous study [61] reported inhibition of the
activation of tumor marker protein Transcription factor p65 (p65).
202 T. Nuutinen / European Journal of Medicinal Chemistry 157 (2018) 198e228
Despite the low cytotoxicity, it potentiates the efficacy of classical
drugs [31]. For instance, it enhanced the cytotoxicity of doxorubicin
(DOX) in various cell lines in a synergistic manner [32,62]. Addi-
tional effects are contributed by its capability to inhibit cytochrome
P4503A (CYP3A) activity in the liver [63]. Thus, this terpene in-
creases the levels of CYP3A-metabolized drugs and prolongs their
action. This action is supported by quantitative high-throughput
screening (qHTS) assays, in which it inhibited CYP2C19 and
CYP2C9 at concentrations of 13 mM and 25 mM, respectively
(https://pubchem.ncbi.nlm.nih.gov/compound/6604672#).
It is known that the 15-lipoxygenase (15-LOX) enzyme reacts
with fatty acids producing active lipid metabolites that are involved
in a number of significant diseases: type 1 and type 2 diabetes,
cardiovascular disease, hypertension, renal diseases, and neuro-
logical conditions such as Alzheimer's disease (AD) and Parkinson's
disease (PD). It was reported that BCPO acts as a potent anti-
inflammatory agent by inhibiting 15-LOX enzyme activity [64]. At
a dose of 12.5 mg/kg, BCPO showed significant central and pe-
ripheral analgesic and anti-inflammatory activity [65]. As a calcium
and potassium current suppressor, it could be used in certain types
of arrhythmias [66]. Finally, BCPO is a strong anti-oxidant, bacte-
ricide [67] and fungicide [68].
Unlike BCP, BCPO does not possess any remarkable binding af-
finity towards CB2; however, it seems to be a multi-target molecule,
known for its anticancer and analgesic properties, and it is an anti-
oxidant and bactericide. Moreover, it may have role in the control of
type 1 and type 2 diabetes, cardiovascular disease, hypertension,
renal diseases, and neurological conditions such as AD and PD via
the inhibition of 15-LOX.
5. Humulene
Humulene (HUM, Fig. 1D) is one of the predominant terpenes in
C. sativa and H. lupulus: it makes up 52% [3] and 19 ± 7.6% [5] of
their total volatile fraction, respectively. It is widely found across
the planta e.g. sage, ginseng and Syzygium zeylanicum (Myrtaceae).
HUM is also called a-caryophyllene, but it does not contain the
cyclobutane ring as b-caryophyllene does, and has not yet been
characterized as a CB2 ligand.
However, HUM possesses anti-inflammatory and anticancer
properties. A study revealed that oral treatment with HUM
inhibited a variety of inflammatory factors in mice and rats [36].
The systemic treatment with HUM (50 mg/kg) reduced TNFa and
IL-1b and reduced the production of PGE2, iNOS and COX-2.
Because of these features, it was effective against edema (5 mg/
kg). The study found that the anti-inflammatory effects of HUM
were similar to those of dexamethasone (a corticoid drug).
Furthermore, in another study [69], HUM (oral, 50 mg/kg for 22
days) significantly reduced the recruitment of eosinophils to the
bronchoalveolar lavage fluid by the recovery of IFN-g and the
reduction of IL-5. These are best known for the stimulation of B cell
growth, increased immunoglobulin secretion and the activation of
eosinophils. It also reduced the levels of eosinophil chemotactic
protein (CCL11) and LTB4. Furthermore, HUM decreased the acti-
vation of NF-kB and activator protein 1 (AP-1), the expression of P-
selectin and the increased mucus secretion in the lungs. In six
Fig. 2. Molecular structures of A) a-Pinene, B) b-Pinene, C) linalool, D) limonene and E) perillyl alcohol.
different cancer cell lines, HUM, however, decreased the cellular
GSH levels and promoted ROS production [70]. This resulted in the
inhibition of cancer cell growth. Moreover, HUM induced apoptosis
in colorectal cancer HT29 cells via death receptor 5 (DR5) and the
caspase-8 and -3-dependent signaling pathways with an IC50 value
of 54 ± 3.0 mM [71]. On the other hand, HUM (100 mg/ml) can sub-
stantially e and specifically e increase the secretion of the pro-
inflammatory cytokine IL-8 in human intestinal epithelial Caco-
2 cells [72]. Of particular note, IL-8 is known to exert proangiogenic,
proliferative and pro-motility functions, which is common to
several cancer types.
Nonetheless, HUM provides a basis for the development of new
drugs exhibiting improved properties. A new HUM derivative, 5-
hydroxyzerumbone (5-hydroxy-2E,6E, 9E-humulatrien-8-one),
and a known HUM derivative, zerumboneoxide, were found to
inhibit lipopolysaccharide (LPS)-induced NO production in murine
macrophage RAW 264.7 cells, with IC50 values of 14.1 and 23.5 mM,
respectively [73]. Accordingly, the 5-hydroxy-derivative inhibited
the expression of both iNOS mRNA and protein in a concentration-
dependent manner. This was likely via the regulation of NF-kB
activity [74]. One of the HUM derivatives from Asteriscuc vogelii was
found to be active against P-338 lymphoma, A-549 lung carcinoma,
HT-29 colon carcinoma and MEL-28 melanoma cells with IC50
values of 1e2 mM [75].
Traditionally, HUM-containing plants have been used for the
treatment of insomnia, depression, nervousness, delirium, anxiety
and digestive disorders. This is not yet recognized by modern sci-
ence, but HUM and its derivatives have shown anti-allergic, anti-
inflammatory and anticancer potential.
6. a-Pinene
a-Pinene (a-PN, Fig. 2A) is the most widely encountered
terpenoid in nature, since it is found in conifers in large amounts. It
is sometimes the dominant terpene in cannabis; for instance, an
inflorescence sample of the drug chemotype 'bubba hash' con-
tained 48% a-PN in the total terpenes [13] and the hemp cultivar
finola contained 23 ± 17% [5]. It can be also found in the essential
oils of salvia, Spanish sage, black plum and lesser galangal, etc.
Interestingly, a-PN is source material for the synthesis of the CB2
ligands SR-144,528 and HU-308 [76]. a-PN itself has no known
affinity towards CB receptors. Pine needle oil from crude extract of
pine needles has been used as an anti-cancer agent in traditional
Chinese medicine. Its anti-cancer action is not devoid of scientific
support today.
Initially, a-PN was found to be very effective in the treatment of
metastatic melanoma by its pro-apoptotic and anti-metastatic ac-
tivity in a mouse model [77]. It was able to reduce the number of
lung tumor nodules, likely via caspase-3-induced apoptosis.
In vitro, a-PN down-regulated M-phase inducer phosphatase 3
(Cdc25C) mRNA and protein expression and reduced cycle depen-
dence on kinase 1 (CDK1) in BEL-7402 cells [78]. This was evi-
denced by cell cycle arrest of the cells at the G2/M phase of the cell
cycle. However, effective concentrations were in the mM range. The
mechanisms have been revealed by in vitro and in vivo studies and
included up-regulation of the checkpoint proteins Chk1 and Chk2
 T. Nuutinen / European Journal of Medicinal Chemistry 157 (2018) 198e228
and down-regulation cyclin B, cell division cycle 25 (Cdc25) and
CDK1 levels [79]. In addition, in a mouse xenograft model, the
proliferation marker Ki67 and proliferation cell nuclear antigen
(PCNA) were down-regulated by the administration of a-PN [80].
Last, a synergistic effect with a broad-spectrum anticancer drug,
Baclitaxel, was observed against non-small-cell lung cancer cells
(NSCLC) [81].
a-PN is an antioxidant and anti-inflammatory terpene. A pre-
vious study [82] demonstrated the antioxidant and protective po-
tential of a-PN against H2O2-induced oxidative stress in PC12 cells.
a-PN restored the expression of antioxidant enzymes including
CAT, SOD, GPx, GSR and heme-oxygenase 1 (HO-1). In addition, the
terpenes were able to decrease apoptosis via reduced caspase-3
activity. Another report [83] showed that pretreatment with a-PN
protected U373-MG cells (astrocytes) against H2O2-induced
oxidative injury in terms of cell viability (with IC50 of 80 mM) and
cell morphology. It also promoted ROS scavenging and decreased
the level of lipid peroxidation by increasing the levels of CAT, SOD,
GSR, GPx, and HO-1, thereby increasing intracellular GSH. In mouse
models, the topical application of a-PN exhibited significant anti-
inflammatory and analgesic effects against nociceptive stimulus-
induced inflammatory infiltrates and COX-2 over-expression [84].
The anti-inflammatory action of a-PN was evidenced as reversal of
the LPS-induced production of IL-6, TNF-a, and iNOS in peritoneal
macrophages [85]. Furthermore, a-PN inhibited LPS-induced COX-2
expression and the activation of MAPKs and NF-kB. In a mouse
model of allergic rhinitis, the level of IL-4 was decreased in the
spleen tissues of a-PN treated mice [86]. In addition to the
decreased activity of TNF-a and NF-kB, the activation of receptor-
interacting serine/threonine-protein kinase 2 (RIP2), inhibitor of
nuclear factor kappa-B kinase subunit b (IKK-b), caspase-1, inter-
cellular adhesion molecule-1 (ICAM-1) and macrophage inflam-
matory protein-2 (MIP-2) was inhibited, which may explain the
observed suppression of the migration of eosinophils and mast cells
into an injury lesion. Lastly, it decreased the levels of nasal
immunoglobulin E. a-PN exhibited anti-inflammatory features in
an in vivo model of acute pancreatitis: a-PN reduced the pancreatic
weight in relation to body weight and the serum levels of amylase
and lipase, indicating reversal of the cerulein-induced tissue
damage [87]. This was likely due to the reduced production of
pancreatic TNF-a, IL-1b, and IL-6 and the deactivation of myelo-
peroxidase (MPO) in the pancreas and lungs in response to the a-PN
pretreatment. a-PN was a potent inhibitor of the IL-1b-induced
inflammatory and catabolic pathways in cartilage-derived cells
substantiated as the inhibition of the NF-kB and JNK activation and
the expression of iNOS and the catabolic MMP-1 and -13 genes [88].
While the (þ)-a-PN was the more active than its ()-enantiomer,
the b-pinene was inactive. Again, in yet another report [89], the
activation of p38 and JNK was attenuated by a-PN, but this time in
IEC-6 cells (normal epithelial cells from rat intestine) and after
aspirin-induced oxidative stress. Cell viability was further
increased by the higher antioxidant enzyme activity and GSH
production.
a-PN shows anxiolytic and hypnotic effects upon inhaled
administration. a-PN evokes its hypnotic action through direct
binding to GABAA as a partial modulator at the benzodiazepine
binding site [90]. Additionally, it was capable of increasing the
duration of non-rapid eye movement sleep (NREM) without
affecting the duration of rapid eye movement sleep (REM) and delta
activity. Another study [91] found an anxiolytic effect in terms of
mice behavior in the elevated plus maze (EPM). The effect remained
several days after the highest concentrations of a-PN were reached
in the brain and liver tissues, indicating that the accumulation of a-
PN in the body keeps the anxiolytic-like action constant. In another
study [92], again employing EPM as the probe for the anxiolytic
203
effect of a-PN, an increased locomotor activity was observed. It also
found that the expression of tyrosine hydroxylase (TH) mRNA in the
midbrain was significantly elevated after 60 min of inhalation of
this terpene. Of particular note, because TH catalyzes the rate
limiting step in the synthesis of catecholamines, e.g. epinephrine
and noradrenaline, it could be hypothesized that a-PN contribute to
the increased locomotor activity in EPM, with this mechanism be-
ing greater than the anxiolytic activity via GABAA. Nevertheless, a-
PN is a bronchodilator in humans at low exposure levels and a
broad-spectrum antibiotic against MRSA ([10] and references).
qHTS data suggests that a-PN inhibits IL-1b-induced iNOS (https://
pubchem.ncbi.nlm.nih.gov/compound/440968#) and TIMP1,
MMP1 and MMP13 mRNA expression at a concentration of 200 mg/
ml (https://pubchem.ncbi.nlm.nih.gov/compound/82227#).
In summary, a-PN, according to the studies referred herein,
show anti-metastatic and anti-tumor activities. Moreover, it seems
to be anti-inflammatory, anti-oxidant and an anti-allergic bron-
chodilator and can produce anxiolytic and hypnotic effects via the
GABAergic system. Finally, it provides a molecular basis for the
development of CB2 ligands.
7. b-Pinene
b-PN (Fig. 2B) is one of the most abundant compounds released
by conifers, but it is regularly found in cannabis (6.1 ± 0.4%) [27] and
hop cultivars in moderate amounts [93]. It is found also in e.g.
Cuminum cyminum and Clausena anisata. In contact with air, it is
oxidized to pinocarveol and myrtenol and other molecules and it is
easily converted to other terpenes.
b-PN (100 mg/kg) showed antidepressant and sedative activities
in mice with several experimental models [94]. Elsewhere, it was
indicated that b-PN (100 mg/kg) produces its antidepressant effect
through the monoaminergic system [95]. b-PN exerted supraspinal
anti-nociceptive actions in a hot-plate test with rats and reversed
the anti-nociceptive effect of morphine in a degree equivalent to
naloxone, indicating that b-PN is a partial agonist of the m-opioid
receptors [96]. b-PN, when complexed with b-cyclodextrin, pro-
voked an antihypertensive effect in rats [97]. Furthermore, the b-
PN/b-cyclodextrin complex induced endothelium-independent
vasorelaxation, possibly caused by the inhibition of Ca2þ influx
through L-type Ca2þ channels. In addition to these properties, it
showed synergistic interactions with PAC against non-small-cell
lung cancer cells (NSCLC) [81]. Moreover, b-PN exhibit antiviral
activity against herpes simplex virus [98]. qHTS data suggests that
it could be an antagonists of peroxisome proliferator-activated re-
ceptor d (PPARd) signaling pathway with activity value of 780 nM
https://pubchem.ncbi.nlm.nih.gov/compound/440967# https://
pubchem.ncbi.nlm.nih.gov/compound/14896# and thus, could be
a player in the development of treatments for several chronic dis-
eases, including diabetes, obesity, atherosclerosis and cancer.
8. Linalool
D-linalool (LNL, Fig. 2C) is rarely found as a predominant
terpene in cannabis; e.g. 25% of all terpenes found in the “Sour OG”
drug chemotype [13]. It is a constituent of hop EO at least in minor
amounts [99]. Lavender (Lavandula angustifolia) EO can contain
around 50% linalyl acetate and 35% LNL; of particular note is that
linalyl acetate is readily converted to LNL in the gastric system.
Lavender (EO) is traditionally used for relaxation, treating parasitic
infections, burns, insect bites and spasms [100]. There is growing
evidence to suggest that lavender EO may be an effective medicine
in the treatment of several neurological disorders. Studies con-
ducted on animals and humans have suggested that LNL is the
active ingredient of lavender EO exhibiting anxiolytic, mood
204 T. Nuutinen / European Journal of Medicinal Chemistry 157 (2018) 198e228
stabilizing, sedative, analgesic, anticonvulsive, anti-inflammatory,
antitumor, antibacterial and neuroprotective properties.
LNL exhibits a variety of anticancer activities with distinct
mechanisms. At a concentration of 25 mM, it has been found to
reduce expression of the anti-apoptotic Bcl-2 and Bcl-xl proteins
and increase the expression of the apoptotic activators Bax and Bak
as well as the activity of caspase 3 and caspase 9, leading to
apoptosis in glioma cells [101]. In addition, the protein expression
of Sirtuin 3 (SIRT3) e which is overactive in several cancers e was
significantly inhibited by LNL (25 mM). Besides, LNL has been found
to inhibit the proliferation of breast, colorectal and liver cancer cells
with IC50 values of 224 mM, 222 mM, and 290 mM, respectively [102].
LNL arrested most of the cells in the G1 phase. The same study
remarked that LNL can also stimulate IFN-g, IL-13, IL-2, IL-21, IL-
21 R, IL-4, IL-6sR and TNF-a secretion, which likely activates the
antitumor immunity. Again, with a variety of cancer cell lines, cell
death was observed with LNL treatment in the mM range [103]. This
was due to the inhibition of complex I and II activities of the res-
piratory chain, a decrease in GSH levels and an increase in the
generation of ROS. In addition, LNL selectively diminished the
growth of melanoma cells in comparison to healthy cells [104]. This
selectivity was e at least partly e caspase-3-dependent, because
the protein was expressed in melanoma cells but almost absent
from normal keratinocytes. Moreover, LNL was found to be a pro-
oxidant in tumor tissues but function as an antioxidant in liver
tissue [105]. This dualism was dependent on the regulation of Nrf-2
and p21 proteins, which is an atypical property for liver cancer
drugs. In comparison to another cancer drug, 5-FU, LNL was more
cytotoxic towards U937 and HeLa cells with the IC50 value of 3 and
11 mM, respectively [106]. Interestingly, U937 cells accumulated to
the G0/G1 phase, whereas HeLa cells accumulated in the G2/M
phase. The cytotoxic effect was likely mediated through the up-
regulation of cyclin-dependent kinase inhibitors (CDKIs) and the
expression of the tumor suppressors p53, p21, p27, p16 and p18. It
has also been found that LNL can reverse doxorubicin resistance
[107] and, in combination with nanoparticles (NPs), significantly
improves the cytotoxicity and apoptotic activity against carcinoma
cells [108]. LNL-NP treatment promoted apoptosis in epithelial
ovarian carcinoma cells by increased ROS generation, and the
decrease in mitochondrial membrane potential, and increase,
again, in caspase-3 levels. Moreover, LIN-NPs were able to decrease
tumor size either alone or in a combination with PAC in a xenograft
model in vivo.
LNL (100 mg/kg) is an antidepressant, which acts through the
monoaminergic system: most likely via noradrenergic and 5-HT1A
receptors [95]. The antidepressant-like effect was observed in mice
as an altered behavior in the forced swimming test (FST) and the
anxiolytic-like activity in EPM [94]. A microarray analysis on the
gene expression profiles from hypothalamus samples, taken from
stressed rats, revealed that the inhalation of LNL restored the
expression of 560 stress-induced genes in comparison to a normal
status [109]. These genes were associated with synaptic trans-
mission via neurotransmitters, anxiolytic neuropeptides like
oxytocin and neuropeptide Y and several major histocompatibility
complex (MHC) class I proteins, which are known to participate in
neural development and plasticity. In mice, the anxiolytic activity of
LNL was also supported by the light/dark test [110]. In addition, it
reduced aggressive behavior and improved social interactions.
Another study [111] demonstrated that the inhalation of ()-LNL
resulted in reduced blood flow in the anterior cingulate cortex and
insular cortex, causing sedative and anxiolytic effects in healthy
males. Their previous study with ()-LNL had shown the involve-
ment of glutamatergic transmission. In addition to anxiolytic and
anti-depressant properties, LNL (single dose of 5, 10 or 20 mg/kg, i.
p.) shows anti-nociceptive effects, as evidenced by the reversal of
PAC-induced allodynia and hyperalgesia via the involvement of the
peripheral opioid system [112]. Accordingly, LNL (50 mg/kg) (as
well as linalyl acetate) evoked local anesthesia in vivo [113]. With
various models of chronic inflammatory and neuropathic pain, LNL
treatment significantly reduced mechanical hypersensitivity and
edema in mice [114]. Moreover, the administration of ()-LNL
inhibited the biting response induced by IL-1b and TNF-a. LNL ex-
hibits an anti-nociceptive effect through the suppression of COX-2
[84]. Via antagonism at the L-glutamate binding site of N-methyl-D-
aspartate receptor, LNL (200 mg/kg) acted as an anticonvulsant in
quinolinic acid induced seizures in vivo [115]. The terpene allevi-
ated maximal electroshock-induced seizures in mice through the
diazepam binding site of the GABAA receptor, as shown by a
reversal of this effect by the receptor antagonist, PTZ, co-treatment
[116]. Both the (þ) and ()-enantiomers showed similar potency
and mechanism of action. Lastly, a double-blind, placebo-controlled
randomized test with the inhalation of 0.5 ml of 1% linalool (total
dose of 5 ml) was able to reduce blood pressure and pulse rate in
patients with carpal tunnel syndrome [117].
As a result of its radical scavenging activity, LNL has antioxidant
and neuroprotective properties against H2O2-induced oxidative
stress, comparable to that of vitamin E or lipoic acid in terms of lipid
peroxidation and oxidant-induced genotoxicity in the guinea pig
brain [118]. Moreover, ()-LNL and its antioxidant properties were
found to be effective against oxygen-glucose deprivation/re-
oxygenation-induced cortical neuronal injury i.e. in an in vitro
model of ischemic stroke [119]. This was independent from the
inhibition of n-methyl-D-aspartate (NMDA)-induced excitotoxicity
but was mediated through the restoration of SOD and CAT activities.
Additionally, ()-LNL inhibited microglial migration induced by the
chemokine (CeC motif) ligand 2 (CCL2). With regard to CCL2, it is
known that elevated levels in glial cells are found e not only in
brain ischemia and injuries e but also in epilepsy and AD. One
study [120] evaluated the effects of LNL on aged, transgenic mice (a
model of AD) and found that LNL (25 mg/kg every 48 h for 3
months) improved learning and spatial memory of the animals
according to behavioral tests. Importantly, LNL showed a significant
reduction in extracellular b-amyloidosis, tau hyper-
phosphorylation, astrogliosis and microgliosis in the hippocampus
and amygdala. Furthermore, the inflammatory responses related to
p38 MAPK, NOS2, COX-2 and IL-1b activities were reversed by the
LNL treatment. The potential of LNL in the alleviation of AD
symptoms was further highlighted in another study [121] where an
AD like state was induced by Ab1-40 injection into the bilateral
hippocampus. Therein, the induced cognitive deficits were
ameliorated by LNL (100 mg/kg, i. p.) treatments, as measured as
behavior in the Morris water maze and step-through tests. The
decrease in apoptosis was mediated through the suppression of
activated cleaved caspases (caspase-3, caspase-9) and suppression
of the oxidative stress activation via Nrf2/HO-1 signaling. In
another study [122], D-galactose- and aluminum trichloride-
induced cognitive defects in mice, representing those seen in AD,
were alleviated by several mechanisms: LNL (100 mg/kg) restored
levels of SOD, GPX, HO-1 and Nrf2 and protected against the
increased activity of acetylcholinesterase (AChE) and the content of
MDA. The study found that the decreased expression of synapse
plasticity-related proteins, calcium-calmodulin-dependent protein
kinase II (CaMKII), brain-derived neurotrophic factor (BDNF), and
TrkB in the hippocampus, were restored by the LNL treatment. In
addition to neuroprotection, LNL (10, 20 and 40 mg/kg i. p.)
exhibited protective properties against LPS and D-galactosamine-
induced liver injuries in mice, manifested as a decrease in patho-
logical liver damage, MDA content, MPO activity and serum alanine
and aspartate transaminases [123]. Furthermore, LNL caused serum
and hepatic TNF-a and IL-6 levels to decrease, along with hepatic
 T. Nuutinen / European Journal of Medicinal Chemistry 157 (2018) 198e228
iNOS and COX-2 expression through the inhibition of NF-kB activ-
ity. The treatment with LNL increased bcl-2 expression and
inhibited caspase-3 and caspase-8 expression, thus, apoptosis.
Moreover, LNL (10, 20 and 40 mg/kg i. p.) treatment reversed the
increase in inflammatory TNF-a, IL-6, IL-1b, IL-8, MCP-1 and NF-kB
levels induced by cigarette smoke in a dose-dependent manner
[124], while in mouse models of chronic inflammatory and
neuropathic pain, LNL inhibited LPS-induced TNF-a, IL-1b, NO, and
PGE2 production [114]. This was further supported by a reduction
in the inflammation in BV2 microglia cells through activation of the
Nrf2/HO-1 signaling pathway. Besides, anti-inflammatory and
analgesic effects were seen in nociceptive stimulus-induced in-
flammatory infiltrates and COX-2 overexpression [84]. LNL was also
protective against t-butyl hydroperoxide (t-BOOH)-induced geno-
toxicity in a reverse mutation assay with Escherichia coli and oxyR
mutants [25]. In the comet assay, in human hepatoma HepG2 and
human B lymphoid NCeNC cells, LNL was effective against t-BOOH
induced DNA damage in thr 0.01 mg/ml range.
LNL exhibited strong antimicrobial activity against perio-
dontopathic and cariogenic bacteria [125], then against
fluconazole-resistant Trichophyton rubrum with a minimum
inhibitory concentration (MIC) value of 260 mg/mL by causing cell
leakage [126]. The LNL was effective against Microsporum species as
much as ketoconazole [127]. It was also active on conidiogenesis
and conidia germination. Last, this terpene showed antifungal ac-
tivity against Candida spp. isolated from patients with oral candi-
diasis [128].
In summary, various in vitro and in vivo studies have shown that
LNL has anti-tumor, anti-convulsant, anti-nociceptive, sedative,
anti-depressant, anti-inflammatory, anti-oxidative, neuro-
protective, hepatoprotective and anti-microbial properties. The
CNS effects are likely to be mediated by neuropeptides, noradren-
ergic and glutamatergic systems, the 5-HT1A receptor and altered
blood flow in the anterior cingulate cortex and insular cortex.
9. Limonene
Limonene (LIM, Fig. 2D) is occasionally the predominant terpene
in Cannabis; for instance, in the 'girl scout cookie' drug chemotype,
it comprised 56% of all terpenes [13], whereas in hops, it seems to
be less abundant. However, it is also found in lemon rind and in
other citrus (up to 97%), ajwain, Bupleurum gibraltarium (up to 96%),
celery (up to 66%), ebolo (up to 70%), Canadian horseweed (up to
70%), and Bolivian coriander (up to 75%) essential oils. R-limonene
and D-limonene are synthesized by two distinct TPSs in plants, but
the terpene is usually present as a racemic mixture. It is used as a
solvent in cleaning products, food manufacturing, perfumery and
hygiene products and as well as an insecticide. Early studies with
LIM have implicated anxiolytic features in mice by serotonergic
action in the prefrontal cortex and dopaminergic function in the
hippocampus, with a 5-HT1A-dependent mechanism ([10] and
references therein). Moreover, it is an immune stimulator via CD4/8
ratio normalization and has been shown to induce apoptosis in
breast cancer cells in a Phase II clinical trial. It is non-sensitizing and
non-toxic with an estimated human lethal dose of 0.5e5 g/kg. The
anti-cancer properties of it has been known for two decades and
have been evaluated in clinical trials. Of particular note, LIM is
hydroxylated to perillyl alcohol (POH) by cytochrome enzymes,
which can be further metabolized to perillyl aldehyde and perillic
acid. Numerous studies have shown that POH is the most active
compound against cancer. However, the current review deals with
both separately, since routes other than oral administration may
not transform it to perillyl derivatives, so it may work as a pene-
tration enhancer for other molecules due to in vitro studies. The
chemopreventive and chemotherapeutic activity against
205
pancreatic, mammary, and prostatic tumors was reviewed in 1996
[129] and again in 2007, also introducing some other actions and
the safety of LIM [130].
LIM can prevent skin tumorigenesis, as shown with a murine
model [131]. The mechanism included the Ras signaling pathway:
LIM decreased the expression levels of Ras and Raf and the phos-
phorylation of extracellular signal-regulated protein kinases 1 and
2 in tumors. It also decreased the expression of anti-apoptotic Bcl-2
and increased the expression of the pro-apoptotic Bax protein.
However, LIM restored the levels of reduced GSH, GPx, GSR, GSR
and CAT, thus indicating that it may be protective towards healthy
cells and tissues. In LS174T colon cancer cells, LIM suppressed the
viability of the cells in a dose-dependent manner, causing apoptosis
via caspase-3 and -9 activations and PARP cleavage [132].
Furthermore, it decreased the levels of p-Akt (Ser473), p-Akt
(Thr308) and p-GSK-3b (Ser9) phosphorylation. The results sug-
gested that the apoptosis was induced by the mitochondrial
pathway and by suppression of the PI3K/Akt pathway. LIM
exhibited synergistic action with berberine against gastric carci-
noma cells by regulating the expression of the Bcl-2 and caspase-3
proteins [133]. In a high throughput gene expression analysis of
HepG2 cells, LIM was found to regulate genes involved in apoptosis,
signal transduction, cancer, the expression of kinases, inflamma-
tion, DNA damage repair and cell cycle control, thus exhibiting its
anticancer activity through the interplay of these pathways [134].
The oral administration of LIM (2 g of limonene daily for 2e6
weeks) to cancer patients induced significant changes in the serum
levels of several metabolites [135]. The metabolic analysis showed
patterns that were related to the decreased levels of cyclin D1 and
adrenal steroids. LIM also regulated the genes linked to the glucose
metabolism. Lastly, LIM may be a good target for the drug design,
because a thiosemicarbazone derivative of LIM showed a lower
value of growth inhibition (IC50: 0.04e0.05 mM) than PAC in a va-
riety of cancer cells [136]. One derivative showed high selectivity
against prostate cancer cells.
The decreased systemic cytokine IL-6 and TNF-a, neo-
vascularization and inhibition of endothelial P-selectin expres-
sion, all induced by a LIM treatment, were found to be important for
wound-healing in murine models of chemically induced dermatitis
and mechanical skin lesions [137]. Moreover, LIM (10 mg/kg/day, up
to 15 days) exerted anti-hyperalgesic effects against mechanical
hyperalgesia in the spared nerve injury model of neuropathic pain
in rats [138]. In another study, LIM (25 mg/kg) presented anti-
nociceptive activity in chemical nociception models in mice [139].
The study also noted that this action was likely independent of
peripheral analgesia by the stimulation of opioid receptors.
Nevertheless, D-LIM and POH showed anti-stress effects in female
Wistar rats in terms of behavioral and physiologic parameters
[140]. Therein, POH was less potent than LIM, which suggests that
the metabolism of LIM extended the release of its active metabolite
POH over the length of the experiments. When rats, with TNBS
(2,5,6-trinitrobenzene sulfonic acid)-induced colitis were fed with
LIM, it significantly lowered the serum concentrations of TNF-a
[141]. The anti-inflammatory effects of LIM also included the inhi-
bition of TNFa-induced NF-kB translocation in fibroblast cultures.
Moreover, in elderly healthy subjects, to whom orange peel extract
was given for 56 days, there were significantly fewer inflammatory
markers e especially peripheral IL-6. In a cell model of osteoar-
thritis, LIM (85 mg/ml) inhibited IL-1b-induced NO production [15].
LIM also decreased the IL-1b-induced NF-kB, JNK and p38 activa-
tions and the expression of inflammatory iNOS and catabolic MMP-
1 and MMP-13 genes; furthermore, it increased the expression of
the anti-catabolic TIMP metallopeptidase inhibitor 1 (TIMP-1) and
the activation of ERK1/2. Furthermore, D-LIM was protective
against DOX-induced renal damage in rats [142]. The
206 T. Nuutinen / European Journal of Medicinal Chemistry 157 (2018) 198e228
administration of DOX caused renal lipid peroxidation, and the
depletion of GSH and anti-oxidant enzymes. Renal damage was
evident in terms of elevated levels of kidney injury molecule-1
(KIM-1), urea and creatinine in blood. Furthermore, DOX caused
an elevation of NFkB, COX-2, and iNOS and NO expression levels.
However, D-LIM-treatment restored the levels of antioxidant en-
zymes, and significantly decreased the inflammatory response and
the levels of kidney toxicity markers KIM-1 and urea and creatinine
in the blood. Also, it reduced the expression of NF-kB, COX-2, and
iNOS and NO. In another study, LIM was found to be a sedative and
motor relaxant: a significant effect was detected with LIM (5 mg/
kg) in OFT and muscle relaxation at the dose of 50 mg/kg. It can also
increase the duration of sleep [14] and be an antidepressant [138].
Its derivative was found to be an anti-convulsant with GABAergic
action [143]. LIM is also antispasmodic, manifested in the inhibition
of ileum contraction [144], and exerts strong anti-viral activity
against herpes simplex in monkey kidney cells (RC-37) [98]. This
was due to the direct interaction with free virus particles. Finally, a
qHTS assay suggested that LIM agonizes the androgen receptor (AR)
signaling pathway with an activity value of 28 mM in the MDA cell
line (https://pubchem.ncbi.nlm.nih.gov/compound/22311#).
Briefly, LIM can promote wound healing and anabolism, while it
can ameliorate stress, depression, inflammation, oxidative stress,
spasms and viral infections. In addition, it shows a variety of anti-
cancer and anti-tumor mechanisms. However, some of these effects
may be due to its conversion to POH in the gastric system. Finally,
its derivatives can be powerful anti-convulsants via GABAergic
action.
10. Perillyl alcohol
While present in Cannabaceae, Perillyl Alcohol (POH, Fig. 2E) is
an abundant terpene in lavender, sage, and peppermint, and
especially in the EOs of mints, cherries, citrous fruits and lemon
grass. It is common ingredient in cleaning products and cosmetics.
It is also a metabolite of LIM via hydroxylation by cytochrome P450
enzymes. It has no known toxicities. POH and its derivatives hold
strong promises in cancer treatments, especially against brain tu-
mors [145]. These are under many preclinical and phase I and II
clinical trials. While it readily crosses the bloodebrain barrier
(BBB), it can give this property to its covalent derivatives like
NEO212, which is a conjugate with temozolomide e a cancer drug
in use against astrocytoma and glioblastoma multiforme. The novel
drug was effective for the treatment of a broad range of
temozolomide-resistant gliomas. POH, having both pro-oxidant
and antioxidant properties, may be useful against malignant
brain tumors e which often display elevated rates of oxygen con-
sumption e but at the same time, POH could be protective toward
tumor neighboring tissue [146]. In addition, POH is able to induce
apoptosis in cancer cells without affecting the normal cells and can
revert tumor cells back to a differentiated state. Nevertheless, in
oral administrations, it shows intestinal side effects. For this reason,
clinical trials in Brazil exploited the intranasal POH delivery, which
showed that long-term, daily chemotherapy was well tolerated and
effective [147].
In addition to being effective against a variety of gliomas, POH
shows potential against other cancer types as well. In particular,
breast, skin and lung tumors could be treated, because adminis-
tration through the skin or by inhalation is possible. Nonetheless,
POH (250 mM) can induce apoptosis in lung cancer cell lines via
increased caspase-3 activity [148]. Breakpoint cluster region
proteineAbelson murine leukemia viral oncogene homolog 1
(BCReABL1) gene fusion is commonly found in leukemia. The Bcr/
Abl protein is a constitutively activated tyrosine kinase that induces
the expression of c-Myc via its downstream target Jak2. The
constant over-expression of c-Myc leads to the unregulated
expression of many genes, which are involved in G1/S phase tran-
sition, causing uncontrollable cell divisions and the avoidance of
apoptosis. A previous study [149] found that POH activated the
Myc-Ornithine decarboxylase (Myc-ODC) apoptotic pathway which
is not protected by the Bcr/Abl anti-apoptotic mechanism in
BCReABL1-transformed cells. A significant inhibition of ODC ac-
tivity by POH was also found in another study [150]. This time, it
was also found that the Ras/Raf/ERK pathway was suppressed by
POH, which in turn limited the formation and progression of 9,10-
dimethylbenz(a)anthracene (DMBA)-initiated and 12-O-tetrade-
canoylphorbol-13-acetate (TPA)-promoted tumorigenesis in Swiss
albino mice skin. Moreover, POH (250 mM) impaired the regulation
of the mevalonate- and the Ras-Raf-MEK-ERK pathways in U87 and
U343 glioblastoma cells [151]. Furthermore, POH inhibited the
post-translational prenylation of GTPase H-Ras and Ras-related C3
botulinum toxin substrate 1 (Rac1) by inhibiting enzymes down-
stream in the mevalonate pathway. Thus, POH prevented them
from being activated. Of particular note is that H-Ras is over
expressed in many cancer types, while the inhibition of Rac1 may
lead to the reversal of tumor cell phenotypes.
The expression of the translation repressor protein eIF4E-
binding protein 1 (4 E-BP1) and activation of the mTOR/4 E-BP1
transduction pathway are dysregulated in a range of malignant
cancers. 4 E-BP1 is hypophosphorylated in quiescent cells, while
the phosphorylation of 4 E-BP1 causes its release from eIF4E,
allowing cap-dependent translation to proceed. However, POH
(400 mM for 16 h) suppressed 4 E-BP1 phosphorylation at Ser65 in
prostate tumor cell lines, DU145 and PC-3, and in Caco2 adeno-
carcinoma cells [152]. Furthermore, 4 E-BP1 phosphorylation at
Thr37 was reduced by POH in DU145 cells. Thus, POH may promote
cell quiescence. The mTOR/4 E-BP1 pathway participates in the
regulation of hypoxia-inducible factor 1-a (HIF-1a) protein levels.
HIF-1a is known to be involved in angiogenesis, energy meta-
bolism, cell survival and tumor invasion. POH (50 mM) was found to
significantly moderate the increase in HIF-1a protein levels
through the inhibition of the mTOR/4 E-BP1 signaling pathway
[153]. In vivo studies further confirmed the inhibitory effect of POH
(three times a week for 40 days at a dose of 50 mg/kg) on the
expression of HIF-1a proteins, leading to a decrease in the growth
of HCT116 cells in a xenograft tumor model. Furthermore, POH
(300 mM) augmented the protein expression of p53 and p21,
induced cell cycle arrest in the G1 phase and reduced cyclin D1, c-
Myc, and Skp2 protein levels [154]. Another study confirmed the
action of POH on p21 but also found that POH (1.0 mM) attenuated
cell proliferation via up-regulation of the expression of cyclin ki-
nase inhibitor proteins and multiple tumor suppressor 2 (MTS-2)
protein [155]. This resulted in the hypophosphorylation of retino-
blastoma (RB), and thus subsequent G1 arrest.
An in vitro study [156] found that POH (400 mM for 16 h) caused
the rapid dissociation of the captured hTERT-mTOR-RAPTOR com-
plex, which resulted in the decrease of telomerase activity. Poly-
cyclic aromatic hydrocarbon-induced CYP1B1 activity was
suppressed by POH (500 nM) [157]. Of particular note, CYP1B1 is
known to be overexpressed in a wide range of tumors, especially as
high levels of expression are observed in estrogen-mediated dis-
eases. VEGF is up-regulated in many tumors and its contribution to
tumor angiogenesis is well described. POH (100 mM), however,
decreased the release of VEGF from cancer cells, while, on the other
hand, stimulated the expression of angiopoietin 2 (Ang2) by
endothelial cells [158]. The results suggest that POH may suppress
neovascularization and induce vessel regression in tumors. The AP-
1 transcription factor is a dimeric complex that contains members
of the JUN, FOS, ATF and MAF protein families. The AP-1 factor
transduces the growth signals to the nucleus, leading to the
 T. Nuutinen / European Journal of Medicinal Chemistry 157 (2018) 198e228
expression of genes involved in growth and malignant trans-
formation in many cell types. Thus, AP-1 proteins are often
considered as oncogenes, but recent studies have challenged this
view: especially JUNB and c-FOS have been shown to possess
tumor-suppressor activity. In breast cancer cells, POH was able to
induce the expression of c-fos and c-jun genes and the phosphor-
ylation of c-Jun [159]. In the experiments, these changes appeared
concurrently with the transcriptional activation of an AP-1-
dependent reporter gene. The results suggest that POH can alter
the expression of AP-1 target genes, especially those with tumor-
suppression activity. Hepatoma cells (HepG2, SMMC-7721 and
MHCC97H) exhibit higher cell invasiveness and migratory capacity
in comparison to normal liver cells (HL-7702). These properties
were significantly suppressed by POH treatment with an IC50 value
of ~100 mM [160]. This occurred through the Notch signaling
pathway and increased E-cadherin. A decrease of E-cadherin, and
thus cellular adhesion, is known to promote tumor metastasis. The
abnormal activation of Akt is commonly present in tumors; the
dysregulation affects downstream processes, including the control
of cell survival, proliferation, migration and angiogenesis. Akt
activation via Ser473 phosphorylation was increased in DU145 cells
by POH, suggesting that it may take control over the malignant
events [152]. The Na/K-ATPase a1 subunit is commonly overex-
pressed in glioblastoma cells suppressing apoptosis [161]. Previ-
ously, the authors demonstrated by an enzyme kinetic study that
POH is an Na/K-ATPase inhibitor. This time, they found that POH
activated p38 and JNK in human glioblastoma cells, non-tumor
monkey kidney and mouse astrocytes. An inhibitor of Src kinase
abrogated the activation of JNK1/2 in U87 cells, suggesting that the
NKA-Src is involved in this apoptotic mechanism. Furthermore, the
inhibition of JNK1/2 reduced the apoptosis of in POH-treated glio-
blastoma cells, thus suggesting that JNK1/2 is involved in pro-
grammed cell death. Last, POH (500 nM) inhibited the expression
and function of the androgen receptor in human prostate cancer
cells [162]. This was manifested in the inhibition of androgen-
induced cell growth and the androgen-stimulated secretion of
prostate-specific antigen and human glandular kallikrein in the
human prostate cancer cell line LNCaP.
Ethanol use increases serum aspartate aminotransferase,
alanine aminotransferase, lactate dehydrogenase and hepatic MDA.
Also, ethanol administration decreases hepatic reduced GSH con-
tent and the activity of various antioxidant enzymes. In a rat model,
these changes were observed, while the ethanol treatment
increased TNF-a production and NFk-B activation. POH (50 mg/kg)
treatment prior to ethanol treatment, however, effectively dimin-
ished these markers of acute liver injury, normalized endogenous
enzymatic and non-enzymatic defense system, and down-
regulated TNF-a and NFk-B production [163]. Perillic acid, a
metabolite of POH, was also found to exert anti-inflammatory
features by suppressing pro-inflammatory IL-2 production in
mitogen-activated T lymphocytes [164]. This happened via the
ability of perillic acid to interrupt signaling in the Ras/MAP kinase
pathway by depleting farnesylated Ras levels. POH (75 mg/kg)
significantly suppressed the indicators of allergic airway inflam-
mation, such as airway eosinophilia [165]. In addition, cytokine
production in the thoracic lymph nodes of the mice was
Fig. 3. Molecular structure of A) terpinolene, B) g-terpinene, C) a-terpinene, D) a-terpineol and E) terpinen-4-ol.
207
substantially suppressed. The results demonstrated that POH
effectively suppressed antigen-induced immune responses in the
lungs. Thus, POH holds potential for the treatment of immunologic
lung disorders such as asthma. On the other hand, POH can activate
innate immune responses in the pulmonary alveoli of mice, which
manifest as increased splenocyte and cervical lymph node cell
proliferation, IgM production, alveolar macrophages and IgA-
producing lymphocytes [166]. However, negative changes in body
weight or liver, brain and lung morphology were not found.
Moreover, POH (25 mM) can reverse hypoxia/reoxygenation-
induced injury in HK-2 cells by decreasing ROS levels, apoptosis
and ER stress and ameliorating the cell viability via activation of the
PI3K/Akt/eNOS pathway [167]. In a rat model of middle cerebral
artery occlusion, oral POH (25 mg/kg once daily for 7 days)
administration significantly attenuated neurological deficits and
reduced infarct volume in a dose-dependent manner [168]. The
POH treatment also inhibited oxidative stress and lipid peroxida-
tion and increased the levels of GSH and CAT, GPx, and GSR en-
zymes. Moreover, POH suppressed the levels of inflammatory IL-1b,
TNF-a, IL-6, COX-2, NOS-2 and NF-kB. In addition to this, a POH-
dependent reduction of serum concentrations of IL-6 and TNF-a
was observed in mice with chemically-induced skin lesions [137].
Finally, tissue regeneration was improved and neovascularization
decreased. These factors accelerated the wound healing.
POH blocked formalin-, capsaicin-, and glutamate-induced
orofacial nociceptive behavior in mice [169]. The action of POH
was comparable to that of morphine in all tests. When 10 mg/kg
POH was administered to stressed rats, a significant anti-stress
action was evident, as measured by changes in behavioral and
physiologic parameters [140]. Potentially, it could alleviate the
symptoms of AD, since a previous study [170] found that POH
decreased amyloid-b peptide-induced mitochondrial dysfunction
and cytotoxicity in SH-SY5Y neuroblastoma cells. Moreover, POH
exhibited antibiotic effect against Candida albicans [171] and Plas-
modium falciparum in experimental cerebral malaria [172]. In a
qHTS assay, POH was able to inhibit 15-LOX with an activity value of
12.6 mM https://pubchem.ncbi.nlm.nih.gov/compound/10819#
In summary, POH and its derivatives exhibit diverse anticancer
and anti-tumor properties, especially against gliomas. It is an anti-
inflammatory, antioxidant, hepatoprotective, nociceptive, anti-
fungal and anti-parasitic agent. It may be effective against symp-
toms of AD.
11. Terpinolene
Terpinolene (TPL, Fig. 3A) is also known as d-Terpinene due to its
close similarity to other terpinenes. It is sometimes the chief
terpene found in a cannabis sample e.g. in the 'durban poison' drug
chemotype (55%) [13]. It has been found in a variety of plant sources
such as sage, apple, cumin, lilac, tea tree and lemon, but it is pri-
marily isolated from pine and fir trees.
Downstream of PI3K signaling, Akt is a regulatory protein
involved in the metabolism, proliferation, cell survival, growth and
angiogenesis, which is up-regulated in many cancers. In a previous
study [173], 0.05% TPL was found to decrease the expression of Akt
(but not Erk2) in myelogenous leukemia (K562) cells. In
208 T. Nuutinen / European Journal of Medicinal Chemistry 157 (2018) 198e228
comparison, LNL was unable to down-regulate Akt. In another
study [174], the proliferation of primary rat neurons and N2a
neuroblastoma cells was significantly diminished after treatment
with TPL (10 mg/l). However, the TPL treatment increased TAC in
primary rat neurons, but not in N2a cells. Furthermore, TPL was not
genotoxic to the normal cells. This was supported by another report
[175], which revealed that TPL exhibited no genotoxicity against
human primary lymphocytes either. Again, TPL treatment
(10e75 mg/L) increased the cellular TAC levels (but not TOS levels).
Moreover, the inhalation of TPL (0.1 mg) e via nasal absorption into
the body e showed sedative effects in mice [176]. Analysis of the
structure-function relationship of TPL (in the comparison to TPL
analogs) revealed that the double-bond in the side-chain or pi
bonds in the six-membered ring play important roles in the seda-
tive effect. In addition, TPL effectively prevented LDL-oxidation
against copper-induced oxidation in human blood plasma prepa-
rations [177]. This protective effect may be beneficial toward
atherogenesis and coronary heart disease. TPL has been shown to
be a transdermal penetration enhancer for indomethacin, a non-
steroidal anti-inflammatory drug (NSAID) in rats [178,179]. An as-
sociation of ineffective oral doses of TPL (3.125 mg/kg) and diclo-
fenac (1.25 mg/kg) showed a synergistic anti-inflammatory and
anti-nociceptive effect in mouse model of inflammatory hyper-
algesia [179]. The TPL-diclofenac combination reduced neutrophils,
macrophages and lymphocytes in the paw as observed by histo-
logical analysis. A possible mechanism of action of the analgesic
effect might be mediated via 5-HT2A receptors, since ketanserin - an
antagonist of 5-HT2 receptors in rodents - completely reversed the
antinociceptive effect. Importantly, this combination lacks the risk
of gastric injury.
TPL show anticancer, antioxidant and anti-inflammatory prop-
erties along with efficiency against LDL oxidation, and sedative and
penetration enhancing features. Also, it is anti-nociceptive via the
5-HT2A receptor.
12. g-Terpinene
In addition to cannabis and hops, g-Terpinene (g-TPN, Fig. 3B)
has been isolated from a variety of plant sources such as savories
(Satureja) and thyme. Terpene is used as a flavoring agent and is not
acutely toxic at least at 2 g/kg in rats [180]. g-TPN (1.6e50 mg/kg)
showed an anti-nociceptive effect in the formalin, capsaicin, and
glutamate-induced pain models in rats [180]. When the animals
were pretreated with naloxone, glibenclamide, atropine and
mecamylamine in the glutamate test, they reversed the g-TPN
induced anti-nociception suggesting that the anti-nociceptive
mechanism involves cholinergic and opioid systems. The study
excluded the possibility of muscle relaxant activity or central
depressant effects, which could otherwise explain the changes in
the rat behavior. Another study [181] presented the anti-
inflammatory and edema-relieving properties of g-TPN in Swiss
mice with carrageenan-induced paw edemas. Similar actions of g-
TPN were seen towards PGE2, bradykinin and histamine-induced
inflammations. g-TPN reduced pro-inflammatory cytokine IL-1b
and TNF-a production and limited neutrophil migration into the
inflamed site. In addition, g-TPN possessed strong radical scav-
enging activity; it was able to trap approximately 1.2 radicals while
protecting erythrocytes, methyl linoleate and DNA in vitro [182]. In
comparison, this non-conjugated diene was superior to its conju-
gated counterpart a-TPN. Moreover, g-TPN can inhibit LDL oxida-
tion in synergism with Rutin e a common flavonoid glucoside in
foods [183]. In addition, it prevents the lipid peroxidation with
different mechanisms to those of vitamin E [184]. Lastly, g-TPN
suppressed the increase in the serum lipid concentrations in Triton
WR1339-treated rats [185]. In summary, g-TPN exhibited anti-
inflammatory and anti-nociceptive features together with benefi-
cial effects on the oxidation status and blood concentrations of LDL
particles, and thus, possesses potential against cardiovascular dis-
eases. Furthermore, studies have shown that g-TPN has extraordi-
nary ROS scavenging activity along with sedative, anti-nociceptive
and anti-inflammatory properties. Furthermore, it is effective to
lower LDL and lipid oxidation and the serum levels of lipids.
13. a-Terpinene
a-Terpinene (a-TPN, Fig. 3C) is found in cannabis and hops and is
commonly used as a fragrance compound. It is found in allspice and
many EOs e.g. from tea tree and Litsea ceylanica (20%), but it is
usually produced industrially from a-pinene. a-TPN shows no
embryofetotoxicity following the oral administration of 30 mg/kg
to rats [186] and is also not mutagenic according to the Ames tests
[187]. a-TPN shows good ROS scavenging activity, trapping
approximately 0.7 radicals when protecting erythrocytes and 0.5
radicals when protecting methyl linoleate [188]. a-TPN is a strong
antioxidant since it auto-oxidizes rapidly in comparison to many
other compounds, preventing these from degradation. However, it
can also form allergens by auto-oxidation according to a previous
study [189]. a-TPN alone (at a dose of 1.0 ml/kg) increased longevity
in mice infected with Trypanosoma evansi [190]. When combined
with diminazene aceturate, it was able to cure nearly 60% of the
infected animals. However, the daily oral administration of 0.5 ml/
kg for 10 days induced memory deficits in rats accompanied by
alterations in Na (þ), K (þ)-ATPase and NTPDase activity and DNA
damage [191]. Taken together, even though a-TPN is a strong
antioxidant and antibiotic, its use may not be recommended
because of its serious adverse effects on brain health.
14. Terpineols
a- and g-terpineols (TOLs, molecular structure of a-terpineol is
presented in Fig. 3C), and terpinen-4-ol (T4OL, Fig. 3E) isomers are
likely derived from their terpinene counterparts by hydration in
plants. They are present in a variety of plant sources such as tea tree
oil, cajuput oil, pine oil, and petitgrain oils, lilacs, pine trees, lime
blossoms and eucalyptus. T4OL is present at high concentrations
(30e48%) in tea tree EO and at up to 29% in lavender EO [192]. They
can be synthesized from more abundant terpenes limonene and
pinenes.
a-TOL was tested against several cancer cell lines, of which it
was most powerful against small cell lung carcinoma (IC50:
0.26 mM) [193]. In addition, the results showed that a-TOL is an NF-
kB inhibitor, which was seen as NF-kB translocation and its reduced
activity using two NF-kB assays. This led to down-regulation of the
expression of several NF-kB-related genes such as IL-1b and IL1R1.
g-TOL significantly suppressed BEL-7402 cell proliferation in a
dose-dependent manner (smallest dose tested: 40 mg/mL) [194]. It
promoted apoptosis, visualized as morphological changes such as
cell shrinkage, the deformation and vacuolization of mitochondria,
nuclear chromatin condensation and fragmentation and the for-
mation of apoptotic bodies. Furthermore, g-TOL treatment accu-
mulated the cells in the G1 or S phase and blocked cell proliferation.
Similarly, T4OL showed anticancer activity in Hep-G2 hepatocel-
lular carcinoma cells by inducing apoptosis and DNA fragmenta-
tion, and by the inhibition of cell migration and the accumulation of
cells in the sub-G1 cell phase [195]. Anti-tumor effects were also
observed in vivo with a mouse model, in which the administration
of only 10 and 20 mg/kg of T4OL decreased the tumor weight and
tumor volume in a dose-dependent manner. Moreover, in another
study [196] using a xenograft model of ICR-SCID mice implanted
with HCT116 cells, the local injection of 200 mg/kg of T4OL
 T. Nuutinen / European Journal of Medicinal Chemistry 157 (2018) 198e228
increased apoptosis and inhibited tumor growth, likely by caspase-
3/7 pathway and through increased ROS generation by mitochon-
dria. Furthermore, T4OL inhibited the proliferation of colorectal,
pancreatic, prostate and gastric cancer cells (at the lowest con-
centration of 0.005%) either alone or in combination with various
anticancer agents with notable synergism [197]. In a xenograft
model, it (0.2%) reduced tumor volumes alone or in combination
with other drugs. T4OL induced autophagy and apoptosis in human
leukemic HL-60 cells and thus cell deaths with an IC50 of 30 mM via
the activation of microtubule-associated protein 1 A/1 B-light chain
3 (LC3-I/II), Beclin-1 and Autophagy 5 (ATG5) proteins [198].
Finally, T4OL shows anticancer and anti-tumor activities via the
activation of caspases 9 and 3 and the cleavage of poly (ADPribose)
polymerase (PARP), by decreasing the mitochondrial membrane
potential (MMP) and XIAP protein and survivin levels, and by the
elevation of the Bax/Bcl-2 ratio and lastly, by the increase in p53
levels [199].
T4OL (25 mg/kg i. p. or 10 ng intracerebroventricularly) exhibi-
ted anticonvulsant action as it inhibited PTZ-induced seizure via
GABAergic system [200]. However, the anticonvulsant action
exerted by the terpene alcohol was not reversed by flumazenil, a
selective antagonist of the benzodiazepine site of the GABAA re-
ceptor, suggesting that T4OL does not bind to the classical
benzodiazepine-binding site. Furthermore, T4OL decreased the
sodium current through voltage-dependent sodium channels, thus,
its anticonvulsant effect may be related to the changes in neuronal
excitability. This could lead to a depressant effect in the central
nervous system (CNS). Another study [201] showed neuronal
excitability and voltage-dependent Kþ currents in the somatic
sensory system where intact and dissociated neurons of rat dorsal
root ganglia were inhibited. In turn, a-TOL, complexed with b-
cyclodextrin, effectively reversed the effects of mechanical and
TNF-a-induced hypernociceptions without causing any defects in
the force or motor coordination in mice [202]. Similar results were
also observed upon carrageenan-induced local hypernociception,
which was reversed by i. p. injection of 25 mg/kg of a-TOL [203].
The antinociceptive effects were reversed by naloxone or
ondansetron-treatments suggesting an involvement of the opioid
and 5-HT receptors [202]. These findings were supported by mo-
lecular docking studies. By using the acetic acid writhing reflex and
formalin, glutamate, and capsaicin-induced nociception tests, the
anti-nociceptive effects of a-TOL (25 mg/kg) were found in terms of
reduced nociceptive behavior in mice [204]. However, this can also
be, at least partly, because of the sedating properties a-TOL, which
appear with an inhalation of the terpene [205].
a-TOL is a spasmolytic agent as a consequence of its inhibitory
effect on ileum contraction [144], but a-TOL (50 mg/kg) was able to
induce gastric retention independently from the activity on the
smooth muscle cells [206]. The mechanism of the latter rests on the
cholinergic/nitrergic signaling in the vagus nerve. In turn, T4OL
(with IC50 of 170 mM) relaxed rabbit duodenum in vitro in a
myogenic manner, which was mediated through calcium antago-
nism [207]. A similar action was found with isolated rat aortic ring
preparations whose calcium influx through voltage-operated cal-
cium channels was altered in the smooth muscle cells [208]. At mM
concentrations, it reduced the contraction force in the isolated left
atrium of rat hearts [209]. Conversely, at mM concentrations, it
caused a positive inotropic effect and extrasystole. It provoked the
arrhythmia likely by its effects on intracellular Ca2þ handling.
However, in vivo, according to one study [210], intravenous bolus
doses (1e10 mg/kg) of T4OL immediately and dose-dependently
decreased the mean aortic pressure in conscious rats by a direct
myogenic action. Nevertheless, NO plays a primary role in the in-
duction of hypotension and vasorelaxation. Via activation of the
NO-cGMP pathway, and the subsequent NO release from epithelial
209
cells, a-TOL was able to induce these beneficial cardiovascular
events with doses starting form 1 mg/kg in vivo and in mM con-
centrations in vitro [211]. It remains to be determined whether a-
TOL and T4OL share common mechanisms for smooth muscle cell
relaxation.
After cerebral ischemia, induced by transient bilateral common
carotid artery occlusion in male Wistar rats, a-TOL (100 mg/kg) was
able to improve spatial memory and synaptic plasticity in the
hippocampus [212]. This was likely and at least partly due to low-
ered levels of MDA and lipid peroxidation in hippocampus. More-
over, a-TOL (10 mg/kg) presented gastroprotective activity in two
widely used models of gastric ulcers for the evaluation of anti-
ulcerogenic drugs [213]. Furthermore, the data of the study indi-
cated that its gastroprotective activity does not involve gastric acid
secretion inhibition or an increase in prostaglandin synthesis.
However, in LPS-stimulated human macrophages, T4OL and a-TOL
suppressed the production of inflammatory IL-1b, IL-6 and IL-10
[214]. The inhibitions were mediated via interference from the
NF-kB, p38 or ERK/MAPK pathways. In a mouse experimental
model of colitis, T4OL (10 mg/kg) inhibited NF-kB and nucleotide-
binding domain and leucine-rich repeat protein-3 (NLRP3)
inflammasome activation and the subsequent IL-1b secretion [215].
Of particular note, NLRP3 inflammasome is also an important
player in chronic airway diseases such as asthma and chronic
obstructive pulmonary disease. a-TOL and its derivatives were
effective bronchodilators in a model of asthma in rats [216], and
also down-regulated the levels of pro-inflammatory IL-4 and IL-17.
In another study [217], an anti-inflammatory action of a-TOL
(3.1 mg/L) on IL-6 segregation and the IL-6 receptor was found. This
monoterpene alcohol also decreased the levels of pro-
inflammatory IL-1b, IL-6, TNF-a, COX-2, and iNOS, and inhibited
the activation of NF-kB, while it increased the expression of the
anti-inflammatory cytokine IL-10 [218]. In addition, a-TOL was a
strong antibiotic when tested against E. coli (MIC value of
0.78 m$props_value{literPattern}/mL) [219]. Moreover, it effectively
killed periodontopathic and cariogenic bacteria in the mouth,
suggesting that it could be used in toothpaste formulations, for
instance [220]. Furthermore, a-TOL inhibited the growths of
G. vaginalis with MIC value of 0.06% (v/v) and C. albicans with MIC
0.125% (v/v), which was comparable to the action of clotrimazole
[218]. The antimicrobial features were confirmed in vivo (vaginal
cavity of mice). In turn, T4OL was effective against oral candidiasis
with a dose of 50 mL of 40 mg/mL in a murine model [221]. It also
suppressed the infection-induced secretion of the pro-
inflammatory TNF-a and macrophage inflammatory protein 2-a
(MIP-2) from macrophages. Finally, T4OL inhibited the growth of
pathological fungi C. posadasii, mycelial H. capsulatum, and yeast-
like H. capsulatum with MICs of 250 mg/mL, 30 mg/mL, and 10 mg/
mL, respectively, presumably by disturbing the fungal membranes
and reducing the ergosterol content of cells [222].
In conclusion, studies have shown that all g-TOL, a-TOL and
T4OL may exhibit anticancer properties, but that only T4OL has
been shown to be anti-tumorigenic. Both a-TOL and T4OL exhibit
anti-inflammatory, spasmolytic, vasodilatory and antibiotic fea-
tures. Whereas a-TOL shows potential against pain, spasms, asthma
and neurological damages, T4OL is an anticonvulsant that acts via
several different mechanisms.
15. Geraniol
Geraniol (GOH, Fig. 4A) is used in perfumes and as a flavoring
agent, and also as a preservative, insect repellent and attractant. It
is the chief component of palmarosa oil and the second most
abundant in rose oil, as well as being found in citronella oils (lemon
grass) and Geranium. Actually, it is found at least in 160 plant EOs
210 T. Nuutinen / European Journal of Medicinal Chemistry 157 (2018) 198e228
Fig. 4. Molecular structures of A) geraniol, B) nerolidol, C) borneol, D) bisabolol and E) bisabolene.
(references in Ref. [223]) including cannabis and hops. GOH is
important in the biosynthesis of other terpenes. The LD50 value of
GOH is 3.6 g/kg in rats [224]. To date, experimental evidence sup-
ports the therapeutic or preventive effects of GOH towards different
types of cancer (reviewed in Ref. [225]). The anticancer mecha-
nisms include the Ras-ERK and AKT-mTOR signaling, altering the
Bcl-2/Bax ratio, regulation of AKT-mTOR, cyclins A, B, D and E and
CDK1 and CDK4, as well as p21Cip1 and p27Kip1, AMP-activated
protein kinase (AMPK), PCNA, mutant p53 and c-Fos. Further-
more, GOH suppresses angiogenesis via the downregulation of
VEGF [226].
GOH suppressed skin inflammation through a variety of mech-
anisms [225]. It inhibited the expression of COX-2, NF-kB, TNF-a, IL-
6, IL-1b and pp38. Moreover, it restored the levels SOD, GSR, GPx,
GST, CAT, GSH and NO and reduced MDA content. It showed
beneficial attributes towards hyperglycemia and diabetic cardiac
complications alleviating cardiac ischemia and oxidative stress in
streptozotocin-induced diabetic rats [224,227]. Mechanisms of ac-
tion of GOH on the impaired vascular reactivity of aortic rings
isolated from diabetic rats or in rats with metabolic syndrome were
revealed: it was able to block both voltage-dependent and receptor
operated calcium channels in an endothelium-independent
pathway [227]. Another study implicated a possible car-
dioprotective effect using an in vitro model of normal neonatal rat
ventricular cardiomyocytes, in which hypoxia-reoxygenation stress
was introduced [228]. The addition of 5 mM of GOH to the cell
culture was able to decrease the endogenous production of ROS in
the stressed cardiomyocytes. GOH treatment also increased phos-
phorylated AMPK levels implicating the reconstitution of energy
homeostasis. In another study [229], mice were fed for three weeks
with feed supplemented with 25 mmol of GOH per kg, and bene-
ficial effects were found. Plasma lipid levels were reduced, which
was likely due to upregulation of the acetyl-CoA carboxylase 1
(ACACA) enzyme, which is the rate-limiting enzyme in fatty acid
synthesis. In addition, cholesterol synthesis was reduced due to a
decrease in cholesterol 3-hydroxy-3-methyl-glutaryl-coenzyme A
reductase (HMGCR) protein levels and catalytic activity. The mRNA
levels of LDL and VLDL receptors were upregulated, which suggests
the enhanced uptake of these lipoprotein particles from the serum.
Chronic inflammation is a key player in atherosclerosis. A study
[230] found that hamsters fed an atherogenic diet supplemented
with 100 mg/kg of GOH showed reduced remodeling of the heart
and aorta tissues in comparison to the control group, because GOH
was able to enhance the free radical scavenging, and also due to its
anti-inflammatory effects.
In a rat model of colitis, GOH (250 mg/kg) replenished the
depleted antioxidant capacity and showed anti-inflammatory ac-
tion by inhibiting the colon contents of PGE2 and IL-1b and sup-
pressed immunological responses, possibly via Wnt/GSK-3b/b-
catenin, p38 MAPK, NFkB, and PPARg signaling pathways [231]. In
turn, in dextran sulfate sodium-induced colitis mouse model, GOH
(30 mg/kg) strongly improved the histological signs of colitis [232].
Furthermore, this terpene alcohol significantly reduced COX-2
expression in the gut wall as well alleviated colitis-associated
dysbiosis. Moreover, the administration of 120 mg/kg of GOH
reduced systemic inflammation, as evidenced by a significant
decrease in the IL-10, IL-17, TNFa, and IFNg levels. Independently
from that, another study [233], using the same colitis model,
confirmed the effect of GOH on the TNF-a, IL-1b, IL-6 and COX-2
levels. Furthermore, the study found that GOH reduced the activ-
ity of MPO and the amount of iNOS in the colon tissue. Not sur-
prisingly, these effects were dependent on the activity of IkBa and
the degradation of NF-kB. The pre-treatment with GOH also
ameliorated the reduction in the amount of reduced GSH and SOD
activity and the lipid peroxidation and reduced the nitrite levels in
colon tissue. These three studies together strongly suggest that
GOH could be useful in the treatment of inflammatory bowel
disease.
Rats received 250 mg/kg/day of GOH for four weeks after trau-
matic spinal cord injury introduced by a surgical procedure [234].
The observed regeneration at the injury site by the GOH treatment
was mediated through the modulation of NF-kB and p38 MAPK,
which likely involved the actions of TNFa and IL6. Furthermore, the
inflammatory response, oxidative stress, and caspase-9 and -3 ac-
tivities were significantly suppressed. In addition, the regeneration
of liver tissue was evidenced by another study [235], in which, soon
after partial hepatectomy, TNFa and IL6 gene expressions were up-
regulated in the GOH-treated groups. Also, alanine transaminase
levels were diminished. The action of GOH was similar to that of
Silymarin e a drug used for the treatment of cirrhosis. Again,
in vivo, GOH (12.5 mg/kg) exhibited protective effects, this time
against the acute lung injury induced by the administration of LPS
[236]. Acute respiratory distress syndrome, a disease that causes
morbidity and mortality in critically ill patients, is characterized by
chronic inflammation, pneumonia, interstitial edema, sepsis and
increased apoptosis. However, GOH treatment for ill mice amelio-
rated pathological injuries and pulmonary cell apoptosis, and MPO
activity and increased the production of IL-1b, IL-6, and TNF-a.
In vitro, GOH reversed the effects of LPS by reducing the iNOS and
COX-2 levels and the expression of Toll-like receptor 4 (TLR4), thus
preventing NF-kB-dependent actions. The apoptosis was alleviated
by the altered Bcl-2/Bax ratio and the reduced Caspase-3 expres-
sion. In mice, ovalbumin-induced allergic asthma was attenuated
by treatment with 100 mg/kg of GOH [237]. The terpene alcohol
administration reduced the levels of pro-inflammatory IL-4, IL-5
and IL-13, increased the innate immunity, promoting interferon-g
in the bronchoalveolar lavage fluid, and reduced the amounts of
ovalbumin-specific IgE in the serum. Furthermore, GOH increased
the production and activation of Nrf2 and the subsequent expres-
sion of the antioxidant enzymes glutamate-cysteine ligase, SOD
and glutathione S-transferase, while decreasing the formation of
MDA in the lungs.
GOH with both oral and i. p. administration of 12.5 mg/kg
reduced writhing behavior induced by acetic acid in mice [223].
Furthermore, a 12.5 mg/kg i. p. injection reduced paw licking time
in the formalin test, and in the glutamate test with 50 mg/kg
administration, suggesting the involvement of glutamatergic
mechanisms. This was supported by an experiment in which a non-
selective NMDA receptor antagonist, dizocilpine, produced similar
results. The opioid antagonist naloxone failed to reverse the anti-
nociceptive activity of GOH, suggesting a non-opioid pathway for
the mechanism of action. Of particular note, involvement of
 T. Nuutinen / European Journal of Medicinal Chemistry 157 (2018) 198e228
GABAergic mechanisms cannot be excluded. Nevertheless, GOH
potentially ameliorated the symptoms and progression of AD in
mice [238] and Drosophila [239]. In mice, AD symptoms were
induced by the chronic intake of neurotoxin 1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine and probenecid. This led to an increase
in apoptotic cells in the nigrostriatal region, neurodegeneration and
motor impairment in mice. However, GOH (100 mg/kg) treatment
ameliorated these effects, evidenced by improved motor behavior
and altered levels of Bcl2, Bax and cytochrome-C [238]. The loss of
dopaminergic neurons in a transgenic Drosophila model was
reversed by exposure to 10 mM of GOH. Furthermore, the experi-
ments resulted in a significant delay in the loss of climbing ability,
improved overall activity and lowered oxidative stress in the brains
of the flies [239]. Neuropathy, in turn, is a result of several envi-
ronmental factors such as medications, diabetes, traumatic injuries
and chronic alcohol consumption. However, it can be also induced
by acrylamide in an experimental mouse model. A previous study
[240] showed that the adverse changes induced in the model, such
as oxidative stress, can be reversed. This was seen as the ability of
GOH (100 mg/kg) treatment to normalize the levels of antioxidant
enzymes, NO and GSH in the sciatic nerve and cortex and cere-
bellum brain regions, and decreased the levels of MDA and cyto-
solic calcium in the same brain regions. Furthermore, AChE activity
was reduced. GOH was an anti-depressant for chronically stressed
mice [241]. The three-week administration of GOH (20 mg/kg/day)
facilitated depressive behaviors in both the forced swimming and
tail suspension tests. Furthermore, the treatment normalized the
levels of stress induced pro-inflammatory cytokine IL-1b. The
induced neuroinflammation was reduced, probably by inhibition of
the NF-kB pathway and NLRP3 inflammasome expression. Lastly,
according to qHTS assays, GOH agonized the estrogen receptor a
(ER-a) signaling pathway with an activity value of 2.2 mM and
inhibited 15-LOX with a concentration of 7.9 mM (https://pubchem.
ncbi.nlm.nih.gov/compound/637566#) while its cis-isomer
agonized TRPA1 at 11.7 mM (https://pubchem.ncbi.nlm.nih.gov/
compound/643820#).
In conclusion, GOH possesses potential for the treatment of
several diseases and conditions; these include cancer, colitis,
inflammation, diabetes, cardiac dysfunction, atherosclerosis, tissue
injuries, allergic asthma, pain, PD, neuropathy and depression.
16. Nerolidol
Nerolidol (NOH, Fig. 4B) is herbivore and pathogen-induced
volatile in plants, but it is regularly found across the planta,
including Cannabaceae, and makes up 74e95% of the total terpenes
in paper bark tea tree oil. It is found in the EOs of the ginger family
(up to 90%), Siparuna (90%), Myrceugenia (90%), Piper claussenianum
(83%), Melaleuca quinquenervia (87%), New England peppermint
tree (80%), Salvia (72%) and in neroli, from which it got its name.
NOH is commonly used across industries e.g. in cosmetics,
cleansers and as a food flavoring agent. In early studies, it was
found to possess sedative effects (reviewed in Ref. [10] & refer-
ences). As more recently reviewed [242], NOH has also been shown
to possess anti-oxidant, anti-bacterial, anti-fungal, broad-spectrum
anti-parasitic, anti-nociceptive, anti-inflammatory, anti-ulcer and
anticancer properties. In addition, it has shown great promise for
the treatment of neurodegenerative diseases featuring anti-
cholinesterasic, antioxidant, anti-nociceptive, anti-inflammatory
and anxiolytic activities, as reviewed in 2017 [243]. Because of the
low acute toxicity (dermal LD50 in rabbit higher than 2000 mg/kg
and oral LD50 in rats 5 g/kg and mice 10 g/kg) it may have great
clinical potential.
NOH exerts an anxiolytic effect without altering the motor co-
ordination in mice [244]. In comparison to diazepam (1 mg/kg), it
211
produced the same effect in EPM with the administration of 50 mg/
kg, in open field tests (OFT) with 25 mg/kg, and did not affect motor
coordination by the rotarod test with any concentration tested. In
addition, NOH (daily three doses of 12.5 mg/kg) significantly sup-
pressed the sub-convulsive PTZ-induced progression of kindling
[245]. Additionally, NOH ameliorated depression and memory
impairment, as indicated by improved behavior in the tests. These
behavioral observations were associated with up-regulated neu-
rotransmitters and decreased oxidative stress. A previous study
[246] demonstrated the anti-nociceptive activity following the oral
administration of 200 mg/kg of NOH, which was possibly via the
GABAergic system and anti-inflammatory activity due to the sup-
pression of TNF-a and IL-1b. In a rat model of PD, a daily dose of
50 mg of NOH was able to reverse rotenone-induced loss of dopa-
minergic neurons in the subcortical nuclei and striatum [247]. Also,
this sesquiterpene alcohol ameliorated the decrease in the activity
of antioxidant enzymes, decreased glial activation, and expression
of IL-1b, IL-6, and TNF-a in midbrain tissues and COX-2 and iNOS in
the striatum region. In the mouse hippocampus, a single dose of
25 mg/kg of NOH was found to be effective against oxidative stress
in neuronal cells, comparable to ascorbic acid [248]. This was at
least in part due to the increase in the protein levels of SOD and CAT.
Furthermore, the sedative effects in terms of behavior in OFT were
comparable to that of diazepam. NOH-loaded nanospheres were
able to alleviate Trypanosoma evansi infection-induced memory
impairment in terms of mice behavior [249]. In addition, the
treatment was able to prevent oxidative stress and alterations in
Naþ, Kþ-ATPase and AChE activities caused by the infection. Using
the same model for the mouse liver, heart and blood, the authors
demonstrated that the use of NOH and NOH-loaded nanospheres
resulted in similar effects, ameliorating the oxidative stress and
inflammation via the inhibition of TNFa, IL-1b and TLR4 [250].
According to high throughput data, NOH inhibited LPS-induced
production of TNF-a (with an activity value of 2.0 mM) and IL-
12p40 (7.6 mM) (https://pubchem.ncbi.nlm.nih.gov/compound/
5284507#). It also inhibits the activity of 15-LOX (12.6 mM). It
showed antimalarial activity against Plasmodium falciparum
trophozoite (0.12 mM) (https://pubchem.ncbi.nlm.nih.gov/
compound/5356544#). Furthermore, it is an agonist (36 mM)
operating in the AP-1 signaling pathway (https://pubchem.ncbi.
nlm.nih.gov/compound/8888#).
In summary, NOH is a sedative, anti-oxidant, bactericide and
fungicide featuring broad-spectrum anti-parasitic, anti-
nociceptive, anti-inflammatory, antidepressant, anxiolytic and
anticancer properties. It has also shown great promise for the
treatment of neurodegenerative diseases and protection against
environmental stress in the kidney, liver, brain, blood and epithelia.
17. Borneol
Borneol (BOH, Fig. 4C) is found up to 14% in cannabis cultivars
[251] and can be found in several species of Artemisia, Blumea and
Kaempferia and Dryobalanops and EOs of many medicinal herbs,
such as valerian. It has low toxicity: the oral LD50 of BOH is
3000e5800 mg/kg in rodents and 3200 mg/kg in rabbits. BOH has
been and is still used in traditional Chinese medicine formulations
as a drug enhancer and to mitigate the effects of heart disease.
Indeed, it may play a role in the control of heart disease because of
its anticoagulant and fibrinolytic effects [252]. It also ameliorates
ischemic stroke with an ED50 value of 0.36 mg/kg [253]. This occurs
via suppression of the expression of TNF-a, iNOS, IL-1b and COX-2.
BOH is well known for its BBB permeability enhancing effect,
reviewed in Ref. [254]. Mechanisms of BBB permeability involve the
modulation of ABC transporters, including permeability glycopro-
tein P-gp, tight junction proteins, and the enhancement of
212 T. Nuutinen / European Journal of Medicinal Chemistry 157 (2018) 198e228
vasodilatory neurotransmitters. For instance, it enhances the de-
livery of classical cancer drugs cisplatin [255] and PAC, restoring its
efficacy against multidrug-resistant cancers [256].
Like camphor (which is the corresponding ketone of this
alcohol), BOH is a potent TRPM8 agonist, evidenced by the
increased TRPM8 influx Ca2þ currents at mM concentrations in
whole cell assays [257]. Moreover, it antagonized TRPA1 receptor,
as camphor does, but with an IC50 of 0.3 mM in whole cell assays
[258]. The analgesic efficacy of topical BOH was found in a ran-
domized, double-blind, placebo-controlled clinical trial with 122
patients experiencing postoperative pain [259]. Using mouse
models of pain, it was found that the analgesic effect was mediated
independently from TRPA1-or GABAA-receptors. It was found that
the TRPM8 channel was the molecular target of BOH, which evoked
downstream glutamatergic mechanisms in the spinal cord, while
another study [260] found that hyperalgesia was alleviated by the
enhancement of GABAergic transmission in the spinal cord. The
analgesic effects on mechanical hyperalgesia and neuropathic pain
were gained in mice following the oral administration of 125 mg/kg
or intrathecal injection of 15 mg of (þ)eBOH. Indeed, yet another
study [261] found that both (þ)eBOH and ()eBOH were powerful
positive modulators of GABAA receptors, but not at the benzodi-
azepine binding site. It potentiated the action of low concentrations
of GABA by more than 1000%. The efficacy was at least equivalent to
that of the anesthetic etomidate and much greater than that of
diazepam or 5-a-pregnan-3a-ol-20-one [261]. In addition, BOH
inhibited nicotinic acetylcholine receptors and the effect was more
potent than that of lidocaine, suggesting potentially strong local
anesthetic properties [262]. The anti-nociceptive properties of BOH
(5 mg/kg) have been demonstrated by a variety of mouse models of
pain (acetic acid, formalin, hot plate, and grip strength tests) [263].
The rotarod test revealed no defects in motor coordination, impli-
cating that the behavior was truly due to the anti-nociceptive ef-
fects. Finally, the dose of 50 mg/kg was similar to that of 20 mg/kg
of indomethacin on the acetic-acid-induced writhing test and it
was comparable to morphine in a hot plate test [264]. The tests
were controlled with rotarod and grip strength tests.
BOH (1.0 mg/kg) exerted neuroprotection and promoted the
recovery of neurological and sensimotor functions by diminishing
the loss of dendritic spines after permanent ischemic stroke [265].
Furthermore, BOH significantly decreased the infarct volume by a
reduction in the expression levels of iNOS and TNF-a. In a rat model
of global cerebral I/R, BOH improved the ultrastructure of neurons
and intracellular calcium content in both the cortex and hippo-
campus and reduced apoptosis by decreasing the expression of p53
and caspase-3 [266]. BOH increased neuron autophagy in the hip-
pocampus, likely by the regulation of Unc-51 like autophagy acti-
vating kinase (ULK1), a kinase involved particularly in autophagy. In
another study [267], BOH (0.003 mM) reduced neuronal injury,
nuclear condensation, ROS generation, iNOS expression and a loss
of mitochondrial membrane potential, which were first induced by
oxygen-glucose deprivation/reperfusion. Moreover, BOH inhibited
caspase-related apoptotic signaling pathway, release of pro-
inflammatory factors, IkBa degradation and blocked NF-kB p65
nuclear translocation. Moreover, () and (þ) BOH treatment
(100 mM) protected SH-SY5Y cells against Ab-induced toxicity
[268]. It reduced ROS generation by up-regulating the expression of
HO-1 and Nrf2, while it reduced apoptosis by altering Bcl-2/Bax
ratio. In vitro, in RAW 264.7 macrophages, BOH reversed LPS-
induced increase in the levels of inflammatory factors including
NO, TNF-a and IL-6, while in vivo, it lowered the endotoxic fever
induced by LPS in rats [269]. In LPS-induced acute lung injury
mouse model, BOH (20 mg/kg) mitigated pulmonary inflammation
in terms of inflammatory infiltration, histopathological changes,
cytokine production and pulmonary edema [270]. Furthermore,
BOH significantly reduced the phosphorylation of NF-kB/P65, IkBa,
p38, JNK, and ERK, suggesting that BOH suppressed inflammation
through inhibition of the NF-kB and MAPKs signaling pathways. In
a 2,4,6-trinitrobenzene sulfonic acid-induced mouse model of co-
litis, BOH (0.09% in diet) notably decreased IL-1beta and IL-6 mRNA
levels [271]. In a mouse model of peritonitis, BOH (25 mg/kg)
reduced leukocyte migration induced by the inflammatory agent
carrageenan [264]. The efficiency was comparable to that of aspirin
(200 mg/kg).
The oral administration of BOH (25 mg/kg/day) for four weeks
significantly attenuated fasting blood glucose, glycated hemoglo-
bin, urea, alanine aminotransferase, aspartate aminotransferase,
MDA concentration and the atherogenic index in diabetic rats [272].
Furthermore, BOH reversed the increase in body weight and
improved plasma insulin, cellular contents of glycogen and anti-
oxidant enzymes and GSH. In an NO-deficient animal model of
hypertension, BOH (50 mg/kg) reversed increased levels of the
structural modification in proteins and triglycerides and improved
liver health in terms of alterations in proteins, lipids, and glycogen
in the liver [273]. In a cell model of oral submucous fibrosis, BOH
possessed anti-fibrosis activity due to its inhibitory effects on fi-
broblasts' mitosis, collagen and TIMP-1 production [272]. This in-
dicates that BOH could be useful in the treatment of Crohn's
disease. On the other hand, BOH was found to improve wound
healing in rats by reducing MPO activity and increasing the collagen
production [274]. Furthermore, BOH showed synergistic effects
with several drugs independent from its penetration enhancement
feature. These included G2/M arrest by curcumin [275] and bisde-
methoxycurcumin [276]. It fortified the effect of edaravone against
DSS-induced colitis by polarizing macrophages via the JAK2-STAT3
signaling pathway [277]. It induced apoptosis together with cur-
cumin in human melanoma cells [278], and exhibited DNA pro-
tection in HepG2 cells and on plasmid DNA against Fe2þ-induced
damage [279]. In the qHTS assay, BOH targeted H2A histone family
member X (H2AX) with an activity value of 1.6 mM (https://
pubchem.ncbi.nlm.nih.gov/compound/1201518#).
Antipyretic, anti-nociceptive, antioxidant, anti-inflammatory,
neuroprotective and DNA preserving actions can be attributed to
BOH, which also improves the efficiency of other drugs either by its
permeability-enhancing properties or independently from that.
18. a-Bisabolol
Bisabolol (BISA, Fig. 4D) is widely used in the cosmetics industry.
BISA was sometimes the second most abundant terpene (17%) out
of the 200 analyzed samples of cannabis [13] and is present in hops
(at levels of up to 16%) [3]. In addition to cannabis and hops, this
unsaturated monocyclic sesquiterpene alcohol e also known as
levomenol e is found in EOs from the candeia tree, salvia, Plinia,
Eremanthus and cat's claw. It is the main constituent in some Eos;
for instance, more than 80% of the EO from Myoporum crassifolium
(a figwort from the Pacific islands) is BISA. Yet another rich source
of BISA is chamomile oil, which is mainly composed of ()-a-BISA
[280]. BISA is safe at a daily dose of 200 mg/kg for four weeks via
dermal administration on rats without any adverse effects. The
LD50 for acute toxicity for rats and mice is around 15 ml/kg. It re-
sults in notable, in the order of 10e100 times, enhancement in the
permeability of other drugs. Furthermore, it is anti-mutagenic in
bacteria, mammalian cells and drosophila [281,282].
The BH3-interacting-domain death agonist (BID) is a pro-
apoptotic protein belonging to the Bcl-2 family. BISA may induce
apoptosis by direct interaction with BID. Furthermore, BISA also
interacts with the kisspeptin receptor 1, which is associated with
tumor mobility and invasiveness. These actions of BISA, especially
against pancreatic cancer, have been recently reviewed [283]. In
 T. Nuutinen / European Journal of Medicinal Chemistry 157 (2018) 198e228
addition to pancreatic cancer, BISA was effective against breast
cancer in HER-2/neu transgenic mice model [284]. Therein, 10 mg/
mouse of BISA downregulated fibroblast growth factors FGF
(involved in angiogenesis) and anti-apoptotic survivin (Birc5).
Moreover, BISA was effective against leukemia, including Imatinib-
resistant cases, with IC50 of 14 mM in vitro [285]. In another study
[286], glioma cells, which were again treated with BISA, showed
decreased viability, which coincided with the increase of ecto-50 -
NT/CD73 activity. However, this was reversed following pre-
treatment with an A3 antagonist, suggesting that the A3 receptor
is involved in the anti-proliferative effect of BISA. Besides, BISA also
exerted its anticancer feature by inducing pores in mitochondria
and lysosomes [287]. BISA was able to activate the pro-apoptotic
caspases-8,-9,-3 and promoted expression of Fas, as well down-
regulating the expression of the anti-apoptotic protein Bcl-2
[288]. The translocation of Bax, Bak and Bid suggested the
involvement of the mitochondrial pathway. Again, apoptosis of
endothelial cells was induced by BISA, as evidenced by the release
of cytochrome c from the mitochondria, a reduction of the Bcl-2/
Bax ratio and the activation of caspase 3 [289]. In turn, at a non-
apoptotic concentration (0.25 mM), BISA showed a differentiating
effect, resulting in growth inhibition, a reduction of invasiveness
and tubule stabilization. The results also suggested that BISA could
be effective in reducing angiogenesis in a variety of tumors. Novel
derivatives of BISA also showed anticancer properties: they pre-
vented the progression of pancreatic cancer via the inhibition of
AKT in vitro [290]. Finally, it is a potential adjuvant in formulations
containing 5-aminolevulinic acid, indicating effectiveness for the
photodynamic therapy treatment of oral cancers [291].
In contrary to the pro-apoptotic effects, BISA (5 mg/ml) was
found to be anti-apoptotic and anti-amyloidogenic against Ab25-
35-induced neurotoxicity in PC12 cells [292]. The treatment with
BISA caused a reduction in the levels of Ab-induced chromosomal
damage and apoptosis, similar to the standard drug donepezil
(50 mg/ml). Furthermore, in a PD-model of Drosophila, BISA e
administered as a food supplement at a concentration of 5 mM e
was effective against rotenone-induced mortality, locomotor defi-
cits and a high level of oxidative stress [293]. BISA (7.7e31 mg/ml)
suppressed the ROS production induced by either corpusculates of
Candida albicans or N-formyl-methionyl-leucyl-phenylalanine in a
concentration-dependent manner [294]. Moreover, a previous
study [295] showed that BISA was able to decrease oxidative stress
and inflammatory events associated with the gastric lesions
induced by ethanol. Furthermore, at 100 mg/kg it was protective
against indomethacin-induced ulcers by increasing the bioavail-
ability of gastric sulfhydryl groups and altering the activity of MDA,
MPO, SOD and CAT and the nitrite amount in favorable manners
[296]. Likewise, BISA (100 mg/kg) was nephroprotective in an I/R
model of acute kidney injury; BISA was able to reverse I/R-induced
alterations in diuresis, water intake, urinary osmolality, plasmatic
creatinine, urea and uric acid, creatinine clearance, proteinuria and
microalbuminuria [297]. Moreover, it reduced KIM-1 levels and
restored favorable TBARS and GSH levels in kidney tissue. Also, BISA
(30 mg/kg) was able to decrease LPS-induced inflammation in the
lung tissue by altering MAPK signaling and the phosphorylation
levels of ERK1/2, JNK, and p38 [298]. Another study [299] indicated
that BISA, in part, exerts its anti-inflammatory effects by down-
regulating the expression of iNOS and COX-2 genes through the
inhibition of NF-kB and AP-1 (ERK and p38) signaling. In vitro and
in vivo models of skin inflammation, induced by LPS or 12-O-tet-
radecanoyl-phorbol-13-acetate (TPA), demonstrated that BISA can
decrease the production of the pro-inflammatory cytokines TNF-a
and IL-6 [300]. In a recent clinical trial, BISA ameliorated atopic skin
[301]. Another clinical trial found it useful for vascular leg ulcer
treatment [302].
213
BISA exhibits anti-nociceptive effects in vivo with a single dose
of 25 mg/kg; it was especially effective in pain related to inflam-
mation [303]. It also suppressed the inflammation in relation to
leukocyte migration, protein extravasations, the amount of TNF-a
and neutrophil degranulation. Again, a 25 mg/kg (i.p.) dose of this
monocyclic terpene alcohol significantly reduced nociceptive
behavior in various tests, including both phases of the formalin test
[304]. It was more effective than the whole fraction of EO of Stachys
lavandulifolia (56.4% of BISA). Pretreatment with BISA-
nanocapsules (200 mg/mL) was also effective against corneal
nociception, which was induced by topical application of 5 M of
NaCl [305]. On the other hand, a molecular docking study [306]
indicated that BISA is not a TRPV1 agonist (note: both agonist and
antagonist may alleviate pain) but may induce a modulatory in-
fluence on other vanilloid receptors. The study further suggested
that the anti-nociceptive effects are not mediated through binding
at a2-adrenoceptors, since yohimbine did not reverse it. The anti-
nociceptive effects of the oral (100 mg/kg) or topical administra-
tion (50 mg/mL) of BISA were achieved in terms of changes in
behavior in various rodent models of pain, including formalin- or
cinnamaldehyde-induced orofacial pain [307]. With a variety of
tests, the authors found that the effect may be e at least in part e
effective through TRPA1 antagonism. In addition, BISA (0.5 mM)
decreased peripheral nerve excitability in terms of the compound
action potential characteristics [308]. Thus, the effect of BISA might
be caused by an irreversible blockade of voltage-dependent sodium
channels. Of particular note is the fact that this does not exclude the
involvement of TRPV1 currents. Interestingly, BISA also inhibited
ACh-induced a7 receptor-mediated currents (IC50 3.1 mM) [309]. It
is a little bit surprising that the inhibition of the a7-nAChRs ago-
nism would provoke the seen anti-nociceptive and anti-
inflammatory actions. Nevertheless, in EPM, BISA (1 mg/kg)
showed anxiolytic-like and sedative properties with involvement
of GABAergic but not serotoninergic systems [310]. Of particular
note, these sedative properties may party explain the anti-
nociceptive like behavior seen in previously-mentioned behav-
ioral studies. Taken together, the anti-nociceptive effect of BISA is
likely to be mediated by a variety of mechanisms, but the exact
mechanisms still need to be elucidated. Finally, BISA is a vasodilator
[311] and relaxant of smooth muscles in the trachea [312]. This may
be mediated through the inhibition of voltage-dependent Ca2þ
channels.
BISA is a broad-spectrum anti-parasitic drug. It shows
effectiveness towards Acanthamoeba castellani in vitro [313],
Trypanosoma evansi in vitro and in vivo [314] and Leishmania
tropica in a hamster model [315]. Furthermore, it powerfully
inhibited Aspergillus fumigatus Af239 growth via microsomal
D24-sterol methyltransferase e a crucial enzyme in the
ergosterol biosynthetic pathway [316]. This makes it potential
fungicidal agent against other fungi too. Indeed, a-BISA showed
antifungal activity against Microsporum gypseum, Microsporum
canis, Trichophyton violaceum, Nannizzia cajetani, Trichophyton
mentagrophytes, Epidermophyton floccosum, Arthroderma gyp-
seum, Trichophyton rubrum and Trichophyton tonsurans [317].
The results of this study showed that BISA presented a
modulatory synergistic effect for some antibiotics such as
gentamicin. It is also effective against multidrug-resistant
bacteria [318]. In addition, it efficiently inhibited spore
germination. In short, BISA exhibits anticancer and anti-tumor
activities, mainly via pro-apoptotic mechanisms. In contrast, its
anti-apoptotic function may protect healthy cells and be neu-
roprotective. Moreover, it participates in gastro- and neph-
roprotection, mainly via its anti-inflammatory mechanisms,
and is anti-nociceptive, likely via a number of mechanisms.
Finally, it is anxiolytic and a broad-spectrum antibiotic.
214 T. Nuutinen / European Journal of Medicinal Chemistry 157 (2018) 198e228
19. Bisabolenes
a-, b- and g-bisabolene (Fig. 4E) are found e in addition to in
cannabis and hops e in a wide variety of plants including cubeb,
lemon and oregano. Various natural derivatives of bisabolenes can
function as pheromones in a variety of insects e.g. in fruit flies. They
are also produced by several fungi, but their biological role in fungi
remains unknown. b-Bisabolene has a balsamic odor and is
approved in Europe as a food additive. b- and g-bisabolenes have so
far been found to possess anticancer properties. In a previous study
[319], g-bisabolene (10 mM) showed anti-proliferative and
apoptosis-inducing features in neuroblastoma cells. Pathways
included the CK2a-p53 pathway in mitochondria-mediated
apoptosis, and the phosphorylation of ERK1/2, protein phospha-
tases 1 (PP1), and p53. g-Bisabolene also decreased the phos-
phorylation of histone deacetylase 2 (HDAC2), and p53 was found
to be acetylated, which enhances the expression of p53-regulated
apoptotic genes [320]. Besides, b-bisabolene (1.12 g/kg i. p. twice
in week for 2 weeks) showed anti-tumor properties; it was effective
in reducing the growth of transplanted 4T1 mammary tumors
in vivo by inducing apoptosis [321]. In seizure models in zebrafish
(30 min 23 mM) and mouse, bisabolene inhibited PTZ-induced sei-
zures [322]. However, the neuromodulatory action remains un-
known. Moreover, b-bisabolone showed a synergistic bactericidal
activity with ampicillin against resistant Staphylococcus aureus
[323]. In summary, bisabolenes may show some anticancer, anti-
tumor, anti-convulsive and anti-bacterial features.
20. b-elemene
b-elemene (ELE, Fig. 5A) is found in wild hops from Lithuania at
levels of up to 14% [3] and in notable amounts in the medical
cannabis cultivar 'bedropuur' [324]. It contributes to the floral
aromas of many plants and is used as a pheromone by some insects.
It is also found in the Chinese medical herb Rhizoma zedoariae,
which has been used for its alleged properties to improve blood
circulation and alleviate pain.
The efficacy of ELE in cancer treatments was reviewed in 2013
[325] and 2017 [326]. In 2013, a meta-analysis of thirty-eight trials
revealed that chemotherapy supplemented with ELE was signifi-
cantly associated with an improved response in the treatment of
various tumors and leucopenia when compared with chemo-
therapy alone [325]. However, these pooled reports did not show an
improved survival rate of ELE plus chemotherapy in comparison to
chemotherapy alone. A recent (2017) meta-analysis, which
included eleven randomized controlled trials (765 patients), sug-
gested that ELE (injections) might enhance the effectiveness of
radiotherapy in cancer treatments [327]. Recent studies (reviewed
in 2017 in Ref. [326]) have shown that ELE possesses an anti-
proliferative effect on cancer cells by promoting apoptosis, cell
cycle arrest and necrosis. Furthermore, it was also able to induce
protective autophagy in some cancerous cell lines and was less
cytotoxic to normal cells in comparison to other chemotherapeutic
agents. ELE may be effective against cancer cells of multiple origins,
including endocrine, urinary, reproductive, digestive, immune,
respiratory, nervous and integumentary systems. Because ELE is
non-toxic towards normal cells, it is highly promising for the
Fig. 5. Molecular structures of A) b-elemene, B) fenchone, C) pulegone, D) a-phellandrene and E) b-eudesmol.
treatment of cancer [328]. ELE suppresses proliferative signaling,
such as via the MAPK and PI3K/Akt/mTOR pathways, induces cell
death, up-regulates growth suppressors, deactivates invasion and
metastasis, interacts with replicative immortality and attenuates
angiogenesis. ELE is also a potential target for the drug design, for
instance, an isopropanolamine derivate improved its anticancer
profile and aqueous solubility [329], whereas Furoxan-based NO-
donating ELE hybrids were found to improve the anticancer efficacy
and were more anti-proliferative against three cancer cell lines
when compared to the parent compound ELE [330]. IC50 values of
the derivatives were in the nM range. The mechanisms were G2 cell
cycle arrest and the induction of apoptosis by deactivation of the
PI3K/Akt pathway. It also suppressed tumor growth in a xenograft
mouse model at a dose of 60 mg/kg.
Recent studies suggest that ELE is protective against athero-
sclerosis in vivo and in vitro. In the early development of athero-
sclerotic lesions, monocytes are recruited by active endothelial
cells. This makes the inhibition of monocyte-endothelial cell in-
teractions a potential target in the prevention of atherosclerosis.
In vitro, ELE inhibited monocyte adhesion and transendothelial
migration through the suppression of the NF-kB-dependent
expression of cell adhesion molecules and through prevention of
the activation of the MAPK signaling pathway [331]. Also, it pro-
tected the endothelial cells from hydrogen peroxide-induced cell
injury and decreased the generation of ROS. In a rabbit model of
atherosclerosis, the placebo group was fed an atherogenic diet prior
to balloon angioplasty-induced endothelial injury, which showed
an increase in the thickness of the atherosclerosis lesion and
increased levels of TC, TG, and LDL-C [332]. ELE treatment, however,
reversed these effects and reduced the infiltration of macrophages.
In vitro, this aliphatic monocyclic sesquiterpene (0.1 mM) decreased
the levels of TNF-a and IL-6. Furthermore, ELE can increase the
survival rate of human umbilical vein endothelial cells in vitro; this
is likely due to its capability to decrease the MDA content and in-
crease TOC, SOD, CAT and GPx activities [333]. Moreover, in a flow
culture, ELE reduced the migration of vascular smooth muscle cells,
but not endothelial cell migration. In vivo, it inhibited smooth
muscle cell proliferation and migration and thus, neointima for-
mation after vascular injury. In ApoE/ mice, ELE inhibited the
atherosclerotic lesion size and increased the stability of plaques by
alleviating vascular oxidative stress and preventing pro-
inflammatory cytokine production [334]. Last, ELE-coated stents
promoted endothelialization after stent implantation, while it
inhibited the proliferation of vascular smooth muscle cells [335].
A case study [336] demonstrated that the injection of ELE during
bronchoscopy resulted in the inhibition of the proliferation of fi-
broblasts and in lower airway granulation; thus, the results sug-
gested a new way to treat airway stenosis. In addition, in a clinical
trial [337], all patients with diagnosed chylothorax experienced a
resolution of symptoms with 1e5 injections of ELE to the pleural
cavity. In one study [338], which used a model of liver fibrosis in
rats, ELE was administered via i. p. injections into rats for 8 weeks
(0.1 ml/100 g bodyweight per day) and was found to downregulate
the CCl4-induced levels of plasma endotoxins, serum TNF-a, and
hepatic CD14 expression. This alleviated the development of the
hepatic fibrosis. In a model of MS, the treatment of C57 mice with
ELE significantly delayed the onset of autoimmune
 T. Nuutinen / European Journal of Medicinal Chemistry 157 (2018) 198e228
encephalomyelitis by improving Th17 and Treg balance [339]. The
treatment drastically reduced the IL-17, IL-6, IL-23 levels and RAR-
related orphan receptor g t (RORgt) expression and upregulated the
Forkhead box P3 (Foxp3) expression in both the periphery and
inflamed spinal cord. In macrophage cells (RAW264.7), the
expression of IL-6, TNF-a and IL-1b, induced by LPS, was signifi-
cantly suppressed by a treatment with ELE (10 mg/mL) via the
inactivation of b-catenin [340]. Furthermore, ELE fought effectively
against LPS-induced expression of iNOS and IL-10, thus intensifying
the before-mentioned anti-inflammatory effects.
In summary, ELE holds potential for the treatment of cancer,
atherosclerosis, MS, airway and liver fibrosis and other diseases in
which excessive ROS production and inflammation play roles.
21. Fenchone
In addition to cannabis and hops, fenchone (Fig. 4B) is found in
fennel, olive leaves, flowering parts of Lavandula stoechas while its
EO can contain 30% of fenchone. Fenchone is used as a flavoring
agent in foods and in perfumery. Traditionally fennel products are
used for the improvement of food digestion and the prevention of
flatulence. Biological effects of fenchone per se have so far been
limited to two studies. First, fenchone was able to inhibit carcinoma
progression in vivo by inducing cell cycle arrest in the S phase [341].
Furthermore, 60 mg/kg fenchone reduced tumor volume and mass
and increased the survival rate of treated animals. Second, fen-
chone was found to augment wound healing in rats [342]. It also
showed anti-inflammatory and antimicrobial activities and
increased collagen synthesis. Lavender EO, containing fenchone
(30%), a-pinene (23%), Camphor (16%), Camphene (7.8%), signifi-
cantly protected against the increase of blood glucose and
increased the antioxidant enzyme activities, and thus protected
against oxidative stress in alloxan-induced diabetes in rats [343].
Fenchone derivatives exhibit antimicrobial features. Fenchone
and fenchyl alcohol significantly reduced C. albicans biofilm for-
mation [344]. Notably, fenchyl alcohol at a concentration of 0.01%
clearly inhibited hyphal formation by downregulating biofilm-
related genes. Furthermore, fenchyl alcohol reduced C. albicans
virulence in a Caenorhabditis elegans nematode model, whereas the
N-acyl derivative of fenchone was found to be effective against
Mycobacterium tuberculosis H37Rv in vitro [345]. Moreover, de-
rivatives bearing a sulfonamide functional group were found to
have comparable activity to ethambutol and possess lower cyto-
toxicity. Aminoethyl substituted 2-endo-fenchol was studied as a
scaffold for the synthesis of a series of 31 amide structures and
in vitro, some of these presented promising antimicrobial activity
with concentrations of 0.2 mg/ml with low cytotoxicity [346].
22. Pulegone
In addition to Cannabaceae, pulegone (PUL, Fig. 5C) is widely
present in the Mentha genus e.g. in Mentha pulegium, from which
PUL gets its name, and in Calamintha nepeta (lesser calamint) oil,
which contains PUL up to 85% [347]. It is also found in Agastache
formosanum oil and rosemary for instance. This monoterpene ke-
tone is used as a flavoring agent and in perfumery. It is claimed to
possess antispasmodic, emmenagogue, diaphoretic, diuretic and
CNS strengthening properties. The biological activities of the oil
includes but is not limited to antimicrobial, antioxidant and anti-
inflammatory, anti-ulcer and insecticidal properties, while PUL
per se shows antimicrobial, anti-parasitic, anti-inflammatory,
spasmolytic, antidiarrheal, central depressant, anti-nociceptive,
antipyretic and antihistaminic features. Indeed, PUL is a potent
histamine receptor 1 antagonist with similar action to mepyramine
and dexchlorpheniramine [348]. This could also explain the
215
spasmolytic activity.
Ambulation in ICR mice was promoted by PUL intake [349].
When dopamine receptor antagonists were co-administered with
PUL, the effect of PUL on ambulation was diminished. A pretreat-
ment with the dopamine depletor reserpine produced no subse-
quent sensitivity to the effect of PUL. These results suggest that the
dopaminergic system is involved in the ambulation. Conversely, in
another study [350], PUL was found to cause a notable decrease in
the ambulation of mice. Besides, it increased pentobarbital-induced
sleeping time, suggesting that it is a depressant in CNS. Interest-
ingly, it also augmented the latency of PTZ-induced convulsion. The
same study yet showed that PUL is an anti-nociceptive molecule
and its action was not blocked by naloxone e a non-selective opioid
receptor antagonist. In another study [351], employing mouse
behavioral tests, PUL was found anxiolytic and psychostimulant.
Only Verapamil, a drug that blocks voltage-dependent calcium
channels, decreased the PUL-induced psychostimulation. Its
anxiolytic-like actions were not mediated through the benzodiaz-
epine site of the GABAA receptor. However, PUL may have adverse
effects on mammalian heart function since it may have an effect on
Ca2þ homeostasis and causes negative inotropism in the mamma-
lian myocardium [352].
Moreover, the main constituents of the C. nepeta oil (limonene,
menthone, pulegone, menthol) were tested against variety of mi-
croorganisms; while PUL showed antimicrobial activity, particu-
larly against all of the Salmonella species, limonene and menthone
were inactive in this respect [353]. It also inhibited the growth of
Aspergillus flavus at 0.8 mg/mL (https://pubchem.ncbi.nlm.nih.gov/
compound/442495#). The sub-chronic toxicity of PUL was inves-
tigated in rats for 28 days [354]. The daily exposure to the dose of
80 mg/kg of PUL induced atonia, decreased blood creatinine con-
tent and terminal body weight, and caused histopathological
changes in the liver and in the white matter of cerebellum. With
higher doses, in long-term studies with rodents, PUL showed no
carcinogenic properties, but increased the incidence of various
tumors and nephropathy in males as well other non-neoplastic
lesions in the liver [355]. Taken together, even though PUL ex-
hibits a variety of beneficial properties, it also exhibits adverse ef-
fects for heart, renal and liver functions, and may therefore not be
suitable as a pharmaceutical in high doses for longer term use.
23. a-Phellandrene
a-Phellandrene (a-PA, Fig. 5D), in addition to C. sativa and
H. lupulus, is found also in Eucalyptus phellandra, from which it got
its name. a-PA has also been isolated from the oil of water fennel
and Canada balsam oil and Schinus terebinthifolius (rose pepper)
fruits. The phellandrenes are used in fragrances and are approved
as flavoring agents in the EU.
It has been reported that a-PA modulates immune responses in
mice. A previous study [356] demonstrated that a-PA (25 mg/kg)
increased the phagocytosis of macrophages from blood samples,
promoted the natural killer cell activity of splenocytes and
increased Be and T-cell proliferation. In another report [357], a-PA
induced autophagy in human liver tumor cells (J5) by regulating
mTOR and LC3-II expression, p53 signaling and NF-kB activation. In
addition, a-PA (50 mg/kg) was able to prevent carrageenan-
induced neutrophil accumulation, inhibit leukocyte rolling adhe-
sion and the production of the pro-inflammatory cytokines TNF-a
and IL-6 in vivo [358]. These results point out that a-PA is an anti-
inflammatory agent acting via the modulation of neutrophil
migration and mast cell stabilization. In mouse leukemia cells
(WEHI-3), a-PA induced the production of ROS, diminished mito-
chondrial membrane potential and released cytochrome c,
apoptosis-inducing factor (AIF) and endo G from the mitochondria
216 T. Nuutinen / European Journal of Medicinal Chemistry 157 (2018) 198e228
[359]. These together led to G0/G1 arrest and apoptosis of these
cancer cells.
a-PA (3.1 mg/kg) exhibited anti-hyperalgesic effects against
mechanical and cold hyperalgesia, and was anti-depressive in SNI
rats [360]. Pretreatments with naloxone, glibenclamide, L-arginine,
atropine or yohimbine reversed the anti-nociceptive effect of a-PA,
implicating that the mechanisms of action involve the gluta-
matergic, opioid, nitrergic, cholinergic and adrenergic systems. Oral
administration for up to 15 days of a-PA (10 mg/kg) significantly
obtunded hyperalgesia in a spared nerve injury model of neuro-
pathic pain and inhibited sensitivity to a cold stimulus [138]. In
addition, a-PA is a potential fungicide. It was effective against (plant
pathogen) Penicillium cyclopium by disrupting the integrity of the
fungal cell membrane [361]. Thus, it may also be effective against
some human pathogens. In addition, its metabolite 5-p-menthene-
1,2-diol exhibited antibacterial and anti-candidal properties, com-
parable to standard antimicrobial agents [362]. Thus, it may be
useful in antibiotic drug design in the future. Lastly, qHTS assays
suggest that a-PA could be an antagonist (~50 mM) of the farnesoid-
X-receptor (FXR) and an agonist (~50 mM) of the antioxidant
response element (ARE) signaling pathway (https://pubchem.ncbi.
nlm.nih.gov/compound/442482#).
Taken together, a-PA is a pro-apoptotic, immunomodulatory,
anti-inflammatory, anti-nociceptive, anti-depressive and anti-
microbial terpene.
24. b-eudesmol
In addition to cannabis and hops [363], b-eudesmol (b-EOH,
Fig. 5E) is present in Atractylodes lancea and Zingiber (gingers), for
example. A. lancea is used in traditional Chinese medicine to ease
gastrointestinal problems, eliminate pathogens and treat head-
aches, body aches, fever and blocked nasal passages. b-EOH exhibits
pro-apoptotic, anti-proliferative and antitumor properties. b-EOH
inhibited the proliferation of human lung (A549) and colon (HT29
and Caco-2) cancer cells in vitro [364]. It also inhibited the cell
adhesion and migration of A549 and HT29 cells. In a human chol-
angiocarcinoma xenograft mouse model, b-EOH (100 mg/kg for 30
days) reduced tumor size by 92% and lung metastasis by 95% [365].
The survival time of the xenograft mice was prolonged by 64%
compared with untreated controls. In HL60 cells, b-EOH induced
apoptosis by the cleavage of caspase-3, caspase-9 and poly (ADP-
ribose) polymerase, and by the downregulation of Bcl-2 expression
[366]. Involvement of the mitochondria was evidenced by the
release of cytochrome c and decreased membrane potential and the
activation c-Jun N-terminal kinases (JNK) in b-EOH treated cells.
Moreover, b-EOH inhibited the growth of several cancer cell lines
with IC50 values ranging from 17 to 25 mg/ml [367]. Again, increased
caspase-3 activation promoting mitochondria mediated apoptosis
was found.
b-EOH showed an anti-inflammatory action via suppression of
the calcium ionophore A23187-induced caspase-1 activation in
mast cells [368]. In addition, it inhibited the production of IL-6 and
suppressed the activation of p38 MAPK, NF-kB and caspase-1, and
the expression of receptor-interacting protein-2. It is known that
Fig. 6. Molecular structures of A) isopulegol, B) isoborneol, C) sabinene, D) 3-carene and D-cadinene.
stem cell factor (SCF) participates in allergic reactions through the
differentiation and migration of mast cells. The treatment of rat
peritoneal mast cells (RPMCs) with b-EOH (2 mM) markedly sup-
pressed SCF-induced mast cell migration and morphological alter-
ations in a concentration-dependent manner [369]. b-EOH also
reduced SCF induced F-actin formation, the activation of Fyn ki-
nase, Rac1 GTPase and p38 MAPK, and the expression of TNF-a and
intercellular adhesion molecule-1. Also, in PC-12 cells, it induced
neurite outgrowth in 100 mM concentrations with the involvement
of phosphoinositide-specific phospholipase C (PI-PLC) and MAPK
[370]. Taken together, b-EOH may have a regulatory role via the
MAPKs in mast cell-mediated inflammatory and allergic diseases
and neuronal differentiation. It also inhibited superoxide produc-
tion in A549 cells [364].
b-EOH (50 mg/kg) prevented convulsions and lethality induced
by maximal electroshock or an organophosphate [371]. When the
CA1 pyramidal layer of hippocampal slices was studied, b-EOH
reduced the high potassium (8.5 mM)-induced electrographic
seizure activity. It was also able to reverse the neuromuscular
failure, which was confirmed by another study [372] in which b-
EOH completely blocked the neuromuscular junction at 200 mM
concentration. With the patch-clamp technique, it was shown that
b-EOH blocked the nACh channels in both the open and closed
conformations, and accelerated the desensitization of nAChR. Thus,
b-EOH could be useful as a muscle relaxant in anesthesia. A pre-
vious report [373] demonstrated that the stimulation of TRPA1 by
b-EOH (0.14 ppb in drinking water) increased appetite and weight
gain in vivo. b-EOH significantly elevated plasma levels of ghrelin e
a peptide hormone, which is known to control appetite. Also,
gastric vagal nerve activity (GVNA) was increased, which was
eliminated by treatment with a TRPA1 inhibitor (HC-030031).
In vitro, it activated human TRPA1 with an EC50 value of
33 ± 0.38 mM. Besides, b-EOH has been reported to stimulate gastric
emptying and small intestinal motility by inhibition of the dopa-
mine D2 and 5HT3 receptors [374].
In conclusion, b-EOH is a molecule with anticancer, anti-
inflammatory and anticonvulsive properties, and can stimulate
neurite outgrowth, appetite and gastric emptying at low
concentrations.
25. Other terpenes found in cannabis and hops
Isopulegol (Fig. 6A), which differs from menthol only by a single
extra double bond, presented depressant- and anxiolytic-like ef-
fects when it was administered to male mice with the i. p. dose of
25 mg/kg [375]. The effect was similar to that of diazepam (1 mg/
kg), but without the loss in general motor activity in the OFT.
However, the overall sedative effect was likely due to a similar
mechanism of action to that of benzodiazepine i.e. the positive
modulation on GABAergic receptors. This was supported by another
study [376]. In the study, isopulegol (100 mg/kg) significantly pro-
tected animals against PTZ-induced convulsions and related mor-
tality. It also significantly prevented the increase in lipid
peroxidation and reversed the PTZ-induced loss of CAT activity and
GSH in the hippocampus of mice. Moreover, isopulegol presents
 T. Nuutinen / European Journal of Medicinal Chemistry 157 (2018) 198e228
significant gastroprotective effects in both ethanol- and
indomethacin-induced ulcer models [377]. The results showed that
the effects appear to be mediated, at least in part, by endogenous
prostaglandins and K (ATP) channel opening. Furthermore, iso-
pulegol restored GSH levels. Lastly, isopulegol derivatives have
been created with different properties, especially to enhance the
transdermal penetration [378].
Isoborneol (Fig. 6B), a geometric isomer of borneol, protected
SH-SY5Y neuroblasts against oxidopamine-induced apoptosis at
low mM concentrations [379]. Oxidopamine is neurotoxic research
chemical, which selectively destroys dopaminergic and noradren-
ergic neurons in the brain. Thus, isoborneol could be an ameliora-
tive supplement for the treatment of PD. It is also a potent inhibitor
of herpes simplex virus type 1 [380]. Isoborneol and its acetate
ester were effective inhibitors of the motility of mice after inhala-
tion; this sedative effect was mediated by positive modulation of
the GABAA receptor [261]. However, borneol produced a stronger
effect.
The efficacy of sabinene (Fig. 6C), a major constituent of carrot
seed oil, was studied in silico against the drug target L-asparaginase
[381]. This bicyclic monoterpene yielded the lowest the docking
score in comparison to ciprofloxacin, eucalyptol and cinnamalde-
hyde, suggesting that sabinene could be a safe alternative in the
treatments of infections e.g. caused by multidrug-resistant Salmo-
nella. Moreover, sabinene (0.6 m$props_value{literPattern}/mL)
demonstrated a strong anti-inflammatory activity through the
iNOS inhibition [382]. Another bicyclic sesquiterpene that includes
a rare cyclopropane ring, 3-carene (Fig. 6D), and its pyrazole de-
rivatives, may act in cell differentiation and maturation, and exhibit
beneficial effects on bone health. First, it was supported by an in
silico study [383], in which Sphingosine-1-phosphate receptor 1
(S1P1) agonism was found. Secondly, at a low concentration such as
5 mM, it significantly stimulated the activity and expression of
alkaline phosphatase, an early phase marker of osteoblastic dif-
ferentiation, in mouse osteoblastic MC3T3-E1 subclone 4 cells
[384]. The stimulatory effect of 3-carene on the mineralization
might be associated with its potential to induce the protein
expression/activation of the MAPKs, osteopontin and type I
collagen. If the effects are mediated through S1PR1 signaling, as
previously suggested in an in silico study, it may also have a role in
endothelial cell migration and vascular maturation. S1PR1 is
known to be involved in tumor angiogenesis and typical lesions in
MS. These facts together make 3-carene and its derivatives attrac-
tive S1PR1 agonist/antagonist. Furthermore, along with a-pinene,
3-carene was the most potent inhibitor of AChE out of 17 screened
bicyclic monoterpenoids [385]. Of particular note, AChE inhibitors
are used to treat neurodegenerative conditions like AD, Lewy Body
Dementia, PD and schizophrenia. Last, it showed antispasmodic
activity against oxytocin-induced contraction in the Wistar rat
uterus at concentration of 2.2 mg/ml (https://pubchem.ncbi.nlm.
nih.gov/compound/26049#).
D-cadinene (Fig. 6E) is a potent larvacide against common ma-
laria, dengue and filiriasis vectors [386]. In addition, it and its de-
rivatives were found to be effective fungicides against various
infectious fungi [387]. In addition, it exhibited high antimicrobial
activity against Streptococcus pneumonia [388]. Finally, it inhibited
the growth of ovarian cancer cells via the induction of caspase-
Fig. 7. Molecular structures of A) selinene, B) valencene, C) copaene, D) farnesol and E) myrcenol.
217
dependent apoptosis and cell cycle arrest in the G0/G1 phase in a
dose-dependent manner [389]. Selinene (Fig. 7A), which is present
in Callicarpa macrophylla (57%) and in cannabis in moderate
amounts (up to 9%) [390], exhibited strong anti-inflammatory,
analgesic, and antipyretic activity, comparable to those of the
standard drugs: ibuprofen, paracetamol and indomethacin [391].
Valencene (Fig. 7B), a close molecular relative to cadinene and
selinene, possesses various biological effects such as anti-allergic
and anti-melanogenetic activity. In addition, it had desirable ef-
fects on the skin lesions in a mice model of atopic dermatitis [392].
Valencene significantly ameliorated the symptoms and restored the
decreased expression of filaggrin (filament aggregating protein).
Furthermore, it reduced the serum levels of IgE, IL-1b, IL-6, and IL-
13. In vitro, it reversed the TNF-a and IFN-g-induced expression of
many pro-inflammatory chemokines including CCL17, CCL22,
CXCL8, GM-CSF, and I-CAM through the blockade of the NF-kB
pathway. On the other hand, it increased the expression of the skin
barrier protein, involucrin e which was likely the reason for the
observed reduction on itching behavior in mice. Copaene (Fig. 7C),
which contains two 6-member rings fused together with cyclo-
butane ring, significantly reduced the cell proliferation of several
cell lines at the concentration of 200 mg/l [393]. In addition, it was
anti-mutagenic and antioxidant by increasing TAC levels at con-
centration of 50 mg/l. Copaene is also known for its actions in plant-
insect interactions.
Farnesol (Fig. 7D), an acyclic sesquiterpene and the simplest
alcohol that can be directly made from farnesyl-pyrophosphate
(FPP), is also a structural isomer of nerolidol (Fig. 4B). It partici-
pates in the quorum sensing of several species and is used for its
anti-allergic and antibiotic properties. It plays multiple roles in both
cell proliferation and apoptosis, and thus, in cancer [394].
Furthermore, it has roles in embryonic development, prenylation of
proteins e and thus the protein trans-localization and fate, endo-
plasmic reticulum stress and the subsequent expression of the
related genes and levels of some hormones. Moreover, farnesol was
neuroprotective and anti-nociceptive in acrylamide-induced neu-
ropathy in mice by the suppression of reactive gliosis and related
inflammatory events [395]. In detail, farnesol supplementation
(100 mg/kg) showed a remarkable improvement in gait perfor-
mance, neuromuscular function and fine motor coordination,
reversed the adverse changes in the GSH, lipid peroxidation, pro-
tein carbonyl, hydroxide, hydroperoxide and nitrite levels, and
alleviated the histological aberrations and reactive gliosis. The last
one occurred by the downregulation of Glial fibrillary acidic protein
(GFAP) and ionized calcium-binding adapter molecule-1 (Iba-1) in
the cortex, hippocampus and striatum. Lastly, farnesol, at least
partly, reversed the acrylamide-induced increase in the levels of
TNF-a, IL-1b and iNOS. Furthermore, in obese mice, farnesol limited
weight gain by the inhibition of adipogenesis through the up-
regulation of AMP-activated protein kinase, an increase in the
expression of uncoupling protein 1 and PPARg coactivator 1 a, and
the browning of white adipose tissue [396]. In addition, farnesol
was cardioprotective in rats; a dose of 1 mg/kg/day significantly
decreased infarct sizes [397]. The effect was mediated through the
increased protein geranylgeranylation but independent from the
antioxidant effect of farnesol. Farnesene, an aliphatic hydrocarbon
derived from FPP (lacks the eOH group in comparison to farnesol
218 T. Nuutinen / European Journal of Medicinal Chemistry 157 (2018) 198e228
(Fig. 7D), is present in both cannabis and hops in low to moderate
amounts and probably functions as a herbivore repellent since it is
also an insect alarm pheromone. It is neuroprotective against H2O2-
induced neurotoxicity in vitro [398]. It suppressed the H2O2-
induced cytotoxicity, genotoxicity and oxidative stress in the
newborn rat cerebral cortex cell cultures. Myrcenol (Fig. 7E), an
alcohol derivative of myrcene, which is present at least in hops, is
an insect pheromone and a sedative by the allosteric modulation on
GABAA receptor expressed in Xenopus oocytes [399]. The same
study found, interestingly, that beer itself induced the same effect
on GABAA receptors, and furthermore, the pentane extract of the
beer, hop oil and myrcenol potentiated the action of GABA on the
receptor. When myrcenol was injected into mice, it increased the
sleeping time induced by pentobarbital. b-Ocimene (Fig. 8A), which
differs from MYR only by the positions of double-bonds, is found in
taget EO (up to 70%) and thyme EO (up to 44%). b-ocimene and
myrcene synthases shares 92% amino acid identity. b-ocimene is
also commonly found in cannabis at moderate concentrations (up
to 23% in “white super skunk” drug chemotype [13] and is often the
second most abundant terpene in 'mostly sativa' phenotypes
[400]). It contributes to the distinct sweet herbal scent of cannabis
plants. However, little is known about ocimene, apart from the fact
that it is common pheromone of honeybees and moths and par-
ticipates to insect-plant and even plant-plant interactions [401].
These, common properties of closely related terpenes may indicate
similar actions and potentials in biomedicine; however, these are
yet to be discovered.
Linalyl acetate (Fig. 8B), the acetylation product of LNL, coexists
usually with LNL; however, the enzyme responsible for the acety-
lation reaction has not yet been identified. Linalyl acetate decreased
locomotor activity, and increased sedation and anti-nociceptive
activity in rodents [402]. In addition, it, with the administration
of 100 mg/kg, it was a mitigating agent for acute cardiovascular
events induced by tobacco, while 1 mg/kg was able to decrease
lactate dehydrogenase activity, vascular contractility and NO levels
in mice [403]. Lastly, when added together with limonene, it
exhibited toxicity effect against neuroblastoma cells [404]. Since
linalyl acetate is converted to LNL with oral administration [405], its
effects may be actually due to LNL. However, other routes of
administration could have different effects. Bergamotene (Fig. 8C)
was present as the predominant terpene with 30% proportion in
floral EO of Eugenia klotzschiana [406]. Several studies with EOs
from different plant sources have shown antioxidant, anti-
inflammatory, broad-spectrum antibacterial and anti-parasitic ac-
tivities (not referred herein). However, these data are limited to EOs
in which bergamotene is only one of the terpene constituents; thus,
Fig. 8. Molecular structures of A) b-ocimene, B) linalyl acetate, C) Bergamotene, D) ()-guaiol and b-elemol.
Fig. 9. Molecular structures of A) bulnesol, B) guaiene, C) p-cymene, D) fenchol and E) humulene-1,2-epoxide.
more studies with purified bergamotene are needed.
A bicyclic sesquiterpene alcohol ()-Guaiol (Fig. 8D) is known to
possess antibacterial activity. It is present in 'liz lemon' drug che-
motype (11%) [13] and can be found in volatile fractions of many
medicinal plants e.g. in Ferula ferulaeoides (37%) and Guaiacwood
Oil (40e72%) [407]. Guaiol also shows inhibition against aphids at
low concentrations, but, maybe more importantly, guaiol inhibited
non-small cell lung cancer in vitro and in vivo in terms of tumor
volume [408]. The study indicated its involvement in the cell
autophagy and apoptosis triggered by the regulation of the
expression of RAD51 and subsequent double-strand breaks via
H2AX phosphorylation. It shows low toxicity and no sensitization,
which is indicated by its use in perfumery. Yet three bicyclic ter-
penes co-exist with Guaiol, namely, b-elemol (Fig. 8E), bulnesol
(Fig. 9A) and the precursor molecule guaiene (Fig. 9B). Indeed,
guaiol and guaiene co-exist in notable amounts in 'bedropuur' drug
cultivar, thus, in a cannabis based medicine: Bedrocan [324]. g-
elemene is sometimes present in hop EOs in relatively high pro-
portions (up to 14.0%) [3]. However, the literature is limited to EOs,
which contains g-elemene, and thus nothing can really be said
about the medicinal potential; however, hop EO is likely the richest
source of g-elemene.
p-Cymene (Fig. 9C) is sometimes a significant component of
terpene fraction of C. sativa and H. lupulus. Cymene exhibit a range
of biological activity including antioxidant, anti-inflammatory,
anti-nociceptive, anxiolytic, anticancer and antimicrobial effects
[409]. Of particular note is that p-cymene is readily formed during
prolonged storage of EOs or upon heating from other mono-
terpenes such as myrcene and terpinenes. Fenchol (Fig. 9D), an
isomer of borneol, is present in medical cannabis cultivars up to a
proportion of 5.2% [251]. Nothing is known about the physiological
effects of humulene-1,2-epoxide (Humulene epoxide II [Fig. 9E])
either, even if it was found (up to 7.9%) in the aqueous extracts of 10
cultivars of the “aroma”-type H. lupulus [93]. The same study found
an unidentified terpene (up to 9.7%); thus, these two, along with
other terpenes, can be present in beer in notable concentrations
and contribute to the health effects of moderate beer consumption
[410].
26. Discussion
The terpenes reviewed herein show very low acute toxicity:
typically, acute oral LD50 values are around 5000 mg/kg or higher
e.g. for b-caryophyllene, myrcene, limonene, terpinolene, pinenes,
nerolidol, ocimenes and fenchone [411]. This means that a good
therapeutic index (LD50 of 1%) is achieved with the administration
 T. Nuutinen / European Journal of Medicinal Chemistry 157 (2018) 198e228
of 50 mg/kg of these terpenes e a typical amount for the biological
activity for terpenes reviewed herein. Even lower effective doses
were regularly met in vivo, for instance caryophyllene oxide with
the dose of 12.5 mg/kg showed analgesic and anti-inflammatory
activity [65]. Limonene (10 mg/kg/day) exerted anti-hyperalgesic
effect on neuropathic pain [138] and it was also a sedative at the
dose of 5 mg/kg [14]. 10 mg/kg perillyl alcohol was able relieve
stress [140]. 1 mg/kg of terpenen-4-ol decreased blood pressure
[210], while a-terpineol induced hypotension and vasorelaxation at
1 mg/kg [211]. Furthermore, geraniol was anti-nociceptive at
12.5 mg/kg [223], while nerolidol, at the same dose, ameliorated
depression and memory impairment in mice [245]. Moreover,
borneol ameliorated ischemic stroke with ED50 value of 0.36 mg/kg
[253], and was neuroprotective and reduced infarct volumes
(1.0 mg/kg) [265], while a-bisabolol showed antitumor effect at 10
mg/mouse [284], and anti-nociceptive and anti-inflammatory ef-
fect with a single dose of 25 mg/kg [303,304]. Lastly, a-phellan-
drene (3.1 mg/kg) was effective against mechanical and cold
hyperalgesia, depression [360], and 10 mg/kg neuropathic pain
(pared nerve injury model) [138]. Nonetheless, terpenes are not
mutagenic; actually, some are even anti-mutagenic such as bisa-
bolol [281,282], myrcene [24] and copaene [393]. These facts
together make them safe for biomedical use.
In early animal studies [205], mice exposed to the inhalation of
terpene odors in ambient air for 1 h experienced profound effects
on activity levels; linalool caused a decrease in motility of 73% at a
plasma concentration of 27 nM, while pinene showed a 14% in-
crease at trace concentrations and terpineol a 45% reduction at
34 nM. A double-blind, placebo-controlled and randomized tests
with the inhalation of 0.5 ml of 1% linalool (total dose of 5 ml) was
able to reduce blood pressure and pulse rate in patients with carpal
tunnel syndrome [117]. Moreover, in a double-blind, randomized
and controlled therapeutic set-up, inhalation of EO had significant
effects on physiologic craving of inhalant-drug-addicted persons
[412]. In vitro, b-eudesmol (b-EOH) activated human TRPA1 with
EC50 value of 33 ± 0.38 mM, but more importantly, it increased the
ghrelin concentration in rodents with supplementation of 0.14 ppb
in drinking water [373]. That is comparable to the concentrations
found in beer. This high activity in vivo increasing appetite was not
only due to the previously mentioned mechanism, but b-EOH has
also been reported to stimulate gastric emptying and small intes-
tinal motility by the inhibition of the D2 and 5HT3 receptors. Thus,
one terpene can have notable effects through the cumulative effect
of multiple pathways. Then, multiple constituents of Cannabis and
Humulus preparations can have additive or synergistic effects. For
instance, experiments with different combinations of six to eight
major terpenes, showed both synergistic and antagonistic effects
on each other in cholinergic system [413]. Aromatherapy has been
suggested to provide a potentially effective treatment for a range of
psychiatric disorders by double blind, controlled and randomized
studies [414]. Sedative, antidepressant, anxiolytic and analgesic
effects are most commonly reported. In turn, in mice, the inhalation
of 27 mg linalool or 23 mg linalyl acetate decreased the motility of
normal mice and reversed caffeine-induced over-agitation. I. p.
administration of 0.050e0.1 ml/kg of EO of Eugenia caryophyllata
(clove), comprised of 77% eugenol and 10% b-caryophyllene, sup-
pressed tonic electroshock-induced convulsions and mortality in
mice. In rats, 0.3 mg/kg of Tagetes minuta (Asteraceae) EO (con-
taining mostly ocimene) displayed anxiolytic and antidepressant
effects, while in chickens, administration of 0.04e0.45 mg/kg of the
EO exhibited anxiogenic effects.
It is claimed that terpenes, together with THC or CBD, evoke a
so-called ‘entourage effect’, which means that the terpenes could
have synergistic actions with these cannabinoids [10]. Especially,
myrcene is claimed to induce strong synergistic sedative/
219
immobilizing action with THC e or ‘cough lock’ as recreational
cannabis users refer to it. However, this review did not found any
support for this or for the ‘entourage hypothesis’ in general. Thus, it
may also possible that minor cannabinoids explains the most of
different subjective effects. Especially, CBD is known to antagonize
psychotomimetic action of THC among its other properties in its
own right [415]. In addition, there are several other cannabinoids
with distinct physiological effects. Cannabinoids; cannabinol (CBN),
cannabigerol (CBG), cannabichromene (CBC), D9-tetrahy-
drocannabivarin (D9-THCV), cannabivarin (CBV) and cannabidivarin
(CBDV) among other less abundant cannabinoids has been shown
to act e not only on the classical cannabinoid receptors, CB1 and
CB2 e but also on other receptors like PPARg, 5HT3A, A1A adeno-
sine receptor, a2 adrenergic and on a variety of TRP channels and
the non-classical cannabinoid receptors G protein-coupled re-
ceptors GPR55 and GPR18 [416]. Thus, it is yet unknown whether
different chemotypes of cannabis show their different effects
through this complex interplay or by the support of terpenes.
However, if the terpenes contribute to the sedative effects of
cannabis, then limonene [14], terpinolene [176], nerolidol [249],
bisabolol [310], isopulegol [375], borneol [261], linalool, linalyl ac-
etate and ocimene [414] may be the more probable candidates. Of
particular note is also that myrcene is readily converted (e.g. via
oxidation and upon heating) to citral, nerol, polymeric products,
terpinenes, linalool, geraniol, limonene and especially to p-cymene
[417]. It is not known which terpenes (or other molecules) are
responsible for the sedative effects of hops, and there is no evidence
that humulene would have sedative properties either. Thus, the
sedative mechanisms of these plants are yet to be elucidated.
However, the case of b-caryophyllene (BCP) is especially inter-
esting. The Ki value of BCP is 150 nm, while it agonizes CB2 with an
EC50 value of 1.9 mM [30]. Because it is fat-soluble, the EC50 would
be achieved by a consumption of 5 mg of BCP (by a human with
70 kg body weight and 20% fat). Further, this would correspond to
the consumption of 200 mg dry C. sativa inflorescence (assuming
that 4% of the dry weight is terpenes and that 64% is BCP) [13].
Interestingly, BCP does not seem to share any obvious structural
similarities with other CB2 agonists such as JWH-015 JWH-133 or
AM-1241, indicating a distinct molecular mechanism for the ago-
nism. Thus, synergistic or at least additive effect may be possible
when combined with other CB2 agonists e like THC. CB2 agonists
are known to suppress neuroinflammation and especially micro-
glial activation. Thus, BCP alone or in combination with cannabi-
noids or other drugs has potential for the treatment of neurological
disorders in which microglial over-activation plays a role such as
MS, AD, PD, Huntington's disease, bipolar disorder, depression and
schizophrenia. Indeed, BCP (25 mg/kg/day) attenuated the neuro-
logical damage in CNS in a mouse model of MS [43] and in a rat
model of PD, BCP (50 mg/kg/day) reduced oxidative stress, neuro-
inflammation, glial activation and the loss of dopaminergic neurons
[45].
The production of EOs may be viable with cannabis and hop
cultivars. By preventing pollination field-cultivated hemp yields
18 l/ha of EO ([11] refs therein). In comparison, lavender can pro-
duce 17 l/ha of EO on its third year of cultivation [418]. Because the
synthesis of both cannabinoids and terpenes share common
enzymatic pathways, the drug and landrace chemotypes produces
a higher proportion of terpenes too, in comparison to hemp culti-
vars. If we assume that a female cannabis drug chemotype pro-
duces 500 g/m2 inflorescence and contains 3% terpenes, this would
yield 150 kg/ha. In turn, hops can produce up to 137 l/ha of EO [419].
In comparison, rosemary produces 60 kg/ha and thyme 20e100 kg/
ha of EO [418]. Thus, we can conclude that cannabis and hop can
produce EO in comparable or even higher amounts with other high
yielding plant sources. Terpene profiles vary greatly between the
220 T. Nuutinen / European Journal of Medicinal Chemistry 157 (2018) 198e228
chemotypes and could be further optimized by selective breeding.
Thus, especially, the production of humulene, humulene epoxide II,
myrcene, b-caryophyllene, limonene, ocimene, bisabolol, elemene,
farnesene and terpinolene could be worthwhile.
27. Conclusions
Terpenes are widely used in industry, perfumery, as food addi-
tives and in traditional medicines. They show low toxicity and high
bioavailability and readily cross the skin and BBB. They have a good
therapeutic index i.e. they are well tolerated without side effects
and the therapeutic effects are gained far before the lethal dose.
Many terpenes exhibit high selectivity over receptors such as TRP
channels, and dopaminergic and GABAergic receptors. Only b-car-
yophyllene show high affinity to cannabinoid receptors and the CB2
agonism holds great potential to treat a variety of neuro-
inflammatory diseases. The additive and synergistic action of ter-
penes with other drug molecules, and the improved penetration of
other drugs by either adjuvant or covalent fusion have been already
gained, but little is known about how they affect together with
cannabinoids and the resinous part of hops. Per se they exhibit,
especially, antibiotic, anti-inflammatory, anti-antioxidant, anti-
cancer and anti-tumor activities. They show anticancer activity
often by pro-apoptotic action, but are non-toxic for healthy cells or
tissues e they are rather neuro-, hepato-, and nephroprotective.
However, the pharmacokinetics and pharmacodynamics of many
terpenes are yet to be studied, as well more placebo-controlled
double-blind human trials with pure terpenes are needed.
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