**1. Introduction**

Sesquiterpenoids are an extremely wide group of secondary metabolites found in the plant kingdom [1,2]. Their main carbon backbone consists of 15 atoms and several synthases action leads to a grea<sup>t</sup> variety of chemical structures. In particular, bicyclic sesquiterpenoids have raised attention in the bio-pharmacological field, even as essential oils. Among them, β-caryophyllene is one of the major component of essential oils extracted from spice and food plants [3–7], such as black pepper (*Piper nigrum* L.), rosemary (*Rosmarinus o*ffi*cinalis*), cinnamon (*Cinnamomum* spp.) oregano (*Origanum vulgare* L.) basil (*Ocimum* spp.), thyme (*Thymus vulgaris*), sage (*Salvia o*ffi*cinalis*), mint *(Mentha piperita***)**, ginger (*Zingiber o*ffi*cinale*), chinotto (*Citrus Myrtfolia* Raf) [8], and cloves (*Syzygium aromaticum*) [9,10]. It was also found in citronella, (*Cymbopogon*), pine tree (*Pinus*), *Chenopodium ambrosioides*, *Cannabis sativa*, in plants of the genus *Copaifera*, *Artemisia*, *Murraya*, *Cordia*, *Spiranthes*, *Ocimum*, *Croton* [9,10], and in the leaves of *Annona Cherimola* [11]. This plant compound was approved by Food and Drug Administration (FDA) and European Food Safety Authority (EFSA) and it is used as flavour enhancer [9] and in cosmetics [12]. In nature, BCP mainly occurs as *trans*-caryophyllene ((E)-BCP) (**1**) mixed with small amount of the isomers (Z)-β-caryophyllene ((Z)-BCP) (**2**) and α-humulene (α-caryophyllene) (**3**) [12], and its oxidation derivative, β-caryophyllene oxide (BCPO) (**4**) (Figure 1).

**Figure 1.** Structures of sesquiterpenes (**1**–**4**).

BCP belongs to cannabinoid family, which are ligands of the cannabinoid receptors present in the organism. Cannabinoid receptors CB1-R and CB2-R are metabotropic receptors are G protein (protein binding GTP)-coupled receptors, involved in the regulation of neurotransmitters responsible for maintaining an energetic balance, in the metabolism, and in the immune response. The aforementioned receptors are bound and activated by endogenous cannabinoids, derivatives of arachidonic acid, including 2-arachidonoylglycerol and *N*-arachidonoylethanolamine, better known as anandamide. Both receptors are bound by a lot of proteins in various pathways, acting as mediators of cellular responses to biological molecules.

Unlike the main traditional cannabinoids, such as Δ8-tetrahydrocannabinol (**5**), Δ9- tetrahydrocannabinol (**6**) and cannabinol (**7**) (Figure 2), able to activate both receptors, BCP has a very different chemical structure and it is a selective agonist of CB2-R. Furthermore, BCP has no side effects, not activating CB1-Rs (mainly expressed in the central nervous system, but also in the liver, lungs, heart, blood vessels and digestive tract). CB2-Rs are mainly found in peripheral tissues and in immune system cells (B and NK lymphocytes, macrophages, mast cells) and, to a lesser extent, in the central nervous system (brain, neurons, microglia) [13]. The expression of CB2-Rs in the central nervous system is increased in neurodegenerative pathologies, such as Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS) and in some tumours like gliomas [14].

The binding of BCP to CB2-Rs is responsible for many cellular events:


**Figure 2.** Structures of cannabinoids (**5**–**<sup>7</sup>**).

In addition to the involvement in physiological functions, CB2-Rs are implied in numerous pathological processes, so they can represent an interesting target in order to obtain agonist molecules for the treatment of many pathological conditions, including neuropathic pain, inflammation, neuroinflammatory and neurodegenerative pathologies (Parkinson's disease, Alzheimer's disease, multiple sclerosis, and amyotrophic lateral sclerosis), spinal and brain injuries, stroke, ischemia, anxiety disorder, depression, colitis, fibrosis and liver ischemia, atherosclerosis, osteoporosis, osteoarthritis, diabetes, obesity, and some types of cancer [9,12–14,16–21].

Molecular docking studies showed that BPC interacts with CB2-R on the binding site of CP55,940 (an agonist), precisely in a hydrophobic pocket, engaging with hydrophobic amino acid residues (valine-113, phenylalanine-117, isoleucine-198, tryptophan-258 and methionine-265) [12]. The double bond with conformation E of BCP is fundamental for receptor binding. BCPO lacks a double bond in position C4-C5 and it is not able to bind CB2-R [10].

### **2. Biological properties of** β**-caryophyllene**

In the last few years, BCP has represented an important subject of study [9–15,22–65]. In particular, a lot of researches about the its effects in vitro and in vivo on animals have been conducted, thus experimental data about its biological properties have been verified. However, further studies are needed in order to translate the findings in animal models into promising pre-clinical and clinical trials on humans. Pre-clinical studies have revealed that BCP is a modulator of nervous system and exerts beneficial effects on numerous neurodegenerative and inflammatory pathologies. Moreover, it is able to act on the liver and bones, and has antibiotic properties.

Table 1 shows the principal studies on β-caryophyllene.


### **Table 1.** Preclinical studies on β-caryophyllene.


**Table 1.** *Cont.*


**Table 1.** *Cont.*

### *2.1.* β*-Caryophyllene and Nervous System*

Many studies report the beneficial effects of BCP on central nervous system [22–44], in particular against neuroinflammatory and neurodegenerative pathologies.

Neuroinflammation is a process leading to nervous system degeneration, characterised by the activation of monocytes, macrophages, mast cells, lymphocytes, and the production of inflammation mediators, such as nitric oxide (NO), various cytokines (IL-1β, IL-6 and TNF-α), the protein NF-κB (nuclear factor kappa B) and the prostaglandins.

In detail, BCP, administered intraperitoneally in Wistar rats, at the dose of 50 mg/kg, has reduced the activity of inducible nitric oxide synthase (iNOS) and, consequently, nitric oxide production, thus decreasing brain oxidative stress and leading to the inhibition of lipid peroxidation and the depletion of glutathione stores [22], causing striatal and cortical neurotoxicity [9]. Moreover, nitric oxide is involved in the activation of cyclooxygenase (COX), the enzyme synthetizing prostaglandin H2 (PGH2), which is the precursor of the other prostaglandins, in particular Prostaglandin D2 (PGD2) and Prostaglandin E2 (PGE2), responsible for inflammation and pain. In this contest a frontiers of research for BCP and BCPO will be their influence on microRNA, small molecule able to regulate gene expression, considering that they are involved and proposed as biomarkers, in neuroinflammation and pain [66,67].

The essential oil of *Erymanthos erythropappus*, rich in BCP, has been shown to have anti-inflammatory in Wistar rats [23].

It has also been demonstrated that BCP, at the dose of 10 mg/kg, inhibits the transcription of iNOS, IL-1β, IL-6, and COX-2 in C6 microglia cells [9,24].

Furthermore, BCP, tested on the mouse BV2 cell line at the concentrations of 10, 25 and 50 μM, has inhibited NF-κB activation and reduced the production of nitric oxide and PGE2, thus suppressing hypoxia-induced neuroinflammatory response [25,26].

In the case of central nervous system pathologies, Iba-1, analogous protein of Aif-1, present only in macrophages and microglia, is over expressed (for example, in ischemia) [27], whereas GFAP (glial fibrillary acidic protein), which forms intermediate filaments, is expressed in a lot of central nervous system cells (including astrocytes and ependymal cells). In addition, it is involved in cell communication, in the interaction neuron-astrocytes, in the functioning of the blood–brain barrier (BBB), particularly during mitosis, in which it modulates the filament network, and in the repair following brain injuries [9,28]. It has been demonstrated that BCP administered intraperitoneally at the dosage of 50 mg/kg, reduces the activation of astrocytes and microglia, by decreasing Iba-1 and GFAP expression, thus avoiding the death of dopaminergic neurons [22].

Amyloid plaques, formed by β-amyloid peptides, composed in turn by 36–43 amino acids and derived from amyloid precursor protein (APP), characterize Alzheimer's disease. These peptides are responsible for direct toxicity (death of neurons) and indirect toxicity (production of molecules stimulating inflammation). A considerable decrease of the β-amyloid peptide-induced overexpression of TLR4 (Toll-like receptor 4), which determines the activation of monocytes and microglia, has been noted in BV2 microglia cells treated with BCP, and, consequently, the sesquiterpene leads to a clear reduction of the biosynthesis of IL-6, IL-1β, PGE2, TNF-<sup>α</sup>, NF-κB and nitric oxide. In addition, also a reduced expression of COX-2 and iNOS has been observed [26].

Another common condition affecting central nervous system is Parkinson's disease, a neurodegenerative pathology characterized by neuroinflammation, oxidative stress, mitochondrial dysfunction and cell death, particularly in dopaminergic neurons. An in vitro study on SH-SY5Y cells showed that the treatment with BCP inhibits reactive oxygen species (ROS) production, restoring mitochondrial functionality and the levels of the antioxidant glutathione. BCP prevents apoptosis, by inhibiting the expression of Bax and caspase-3 and increasing Bcl-2 one. It reduces the phosphorylation of JNK (c-Jun N-terminal Kinase), which determines the increase of HO-1 (heme oxygenase-1) expression in this pathological condition. All these effects are related to CB2/Nrf2 pathway [29]. Research on a mouse model of Parkinson's disease conducted by Viveros-Paredes revealed that the treatment with BCP, at the dose of 10 mg/kg, enhances motor coordination in mice and protects the dopaminergic neurons from degeneration, reducing the production of inflammatory cytokines, in particular IL-1β, IL-6, and TNF-α [30].

BCP is also cytoprotective towards the central nervous system due to its modulation of the redox state and inflammation, useful during chemotherapy. The main mechanism of action regarding this aspect is the stimulation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) following the activation of cannabinoid receptors CB2. Nrf2 is a transcription factor, whose activity is stimulated by oxidative stress (in particular, reactive oxygen species or ROS) and oncogenes, such as KRAS, BRAF and MYC. Nrf2 increases the expression of the genes involved in cell survival and in the reduction of the inflammatory process and the oxidative stress in SNC. Moreover, nerve growth factor (NGF) is stimulated through Tropomyosin receptor kinase A (Trk A) pathway, which stimulates in turn PI3K-Akt pathway [68].

BCP, through Nrf2 activation, increases the expression of antiapoptotic genes (B-cell leukemia/lymphoma-2 (BCL-2), Mouse double minute 2 (mdm-2), COX-2, c-Myb) and, at the same time, reduces the one of proapoptotic genes (Bax, Bak-1, caspase-8, caspase-9) [31].

All these cellular events determine cell survival and proliferation and angiogenesis [9].

BCP also diminishes the expression of matrix metallopeptidase (MMP-9) and increases the one of occludin, claudin-5, Tight Junction Protein ZO-1 and Growth Associated Protein 43 (GAP-43) [32].

Furthermore, BCP, at the concentrations of 0.5 and 1 μM, exerts cytoprotective effect on C6 glioma cells, increasing the antioxidant activity, through CB2-R-dependent Nrf2 pathway [33]. In addition, administered orally in Wistar rats, at the dosages of 34, 102 and 306 mg/kg, it reduces oxidative stress and neuronal apoptosis, due to the increased expression of Nrf2 and OH-1.

BCP also inhibits the production of nitric oxide, hydrogen peroxide, TNF-<sup>α</sup>, interferon gamma (IFN-γ), interleukin-17 (IL-17) reducing the extent of macrophage infiltration [9].

BCP can induce neurogenesis with a mechanism independent from the activation of CB2-R, Nrf2 and NGF, stimulating Tropomyosin receptor kinase A (TrkA) in SH-SY5Y (Cell lines Homo sapiens, human, bone marrow) and PC12 (Cell line derived from a pheochromocytoma of the rat adrenal medulla) neuroblastoma cells [34].

There are numerous studies regarding the analgesic effects of the sesquiterpene, isolated or in mixture in essential oils. In fact, the essential oil of *Erymanthos erythropappus*, containing BCP as one of the main components, administered orally at the doses of 200 and 400 mg/kg, induces analgesia in Wistar rats [23]. Further, the essential oil of *Senecio rufinervis*, containing about the 6% of BCP, is responsible for the analgesic effect in mice, when administered orally at the doses of 50 and 75 mg/kg [35].

Other studies have reported the same results in mice at the doses of 1 and 5 mg/kg intraperitoneally and at the dosages of 2.6 mg/kg/die per *os*for 2 weeks, alone or in combination with the docosahexaenoic acid (DHA) [9]. The essential oil of *Vitex agnus-castus*(BCP accounts for about 7% of the oil), has exhibited analgesic effect in Wistar rats which were subjected to an immersion test.

BCP, at the dose of 25 mg/kg, decreases the extent of peripheral neuropathyin mice, by the activation of cannabinoid receptors CB2-R and the inhibition of the MAPK p38, resulting in a reduced transcriptional activity of NF-κB, involved in phlogosis [36].

It has been demonstrated in mice that BCP actually exerts its antinociceptive action by activating CB2-Rs, in particular towards primary sensory neurons. In fact, the analgesic effect manifests itself in wild-type mice, but not in the CB2-R knockout mice. The activation of cannabinoid system indirectly leads to the modulation of opioid system. In more detail, it stimulates β-endorphin release, activating opioid receptors μ on primary afferent neurons. BCP has been shown to boost the painkilling effect of morphine, used in the treatment of severe pain, making possible the reduction of the dose of the drug and, consequently, decreasing its side effects [37].

Instead, there was no indication of synergy between BCP and the other components of the essential oils [13].

The derivative BCPO has not elicited any interest as an analgesic [13], since it does not bind to cannabinoid receptors due to the different molecular structure.

BCP could result potentially useful in multiple sclerosis managemen<sup>t</sup> (pathology characterized by axon demyelination and neuroinflammation), as demonstrated by previous study which investigated the therapeutic potential of BCP on experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS).

The research has shown that the treatment with BCP reduces interferon-γ (IFN-γ) production, the main activator of macrophages, and increases the levels of interleukin-10 (IL-10).

Moreover, it inhibits cell T migration. The oral treatment with 50 mg/kg of BCP twice a day has decreased hyperalgesia induced by the EAE and protected from brain damage, inhibiting cytokine biosynthesis and restoring the activity of catalase, superoxide dismutase and glutathione peroxidase, all enzymes involved in the detoxification of oxidant substances [38].

Two essential oils containing BCP, basil one, administered intraperitoneally (at the doses of 100, 150 and 200 mg/kg), and *Baccharis uncinella* one, administered per *os* (at the doses of 50 or 100 mg/kg), exhibit significant sedative effect [39,40].

These early results on the animal model could be attributed to BCP, being a valid alternative to the most common sedatives and anaesthetics.

The latter drugs have contraindications in patients with cardiovascular, neuromuscular and cerebrovascular diseases, and could cause many side effects, such as convulsions, insomnia, agitation, dependence and, in the most extreme cases, lead to death.

Basil essential oil has also exerted anxiolytic effect [39]. The same results have been obtained in mice, when BCP has been orally administered at the dose of 50 mg/kg [41].

Therefore, all these data sugges<sup>t</sup> the possible use of the sesquiterpene as a substitute of the most common anxiolytics, like benzodiazepines and the selective serotonin reuptake inhibitors (SSRI), which cause many adverse effects, such as movement problems, sedation, dependence, muscle relaxation, anterograde amnesia, teratogenic risk, reduction of bone mineral density.

BCP also exhibits antidepressant properties, as revealed by a research conducted on albino Swiss mice receiving 50 mg/kg of the BCP per Os (oral somministration). The mechanism of this e ffect is due to the activation of CB2-R [41].

Various studies have shown muscle relaxant e ffect of BCP in animals. In this regard, the compound, alone or as a constituent of the essential oil from *Pterodon polygalaeflorus*, exerts antispasmodic activity on rat isolated ileum at variable concentrations (1 to 1000 μg/mL) [42].

BCP, orally administered at the dosages of 10, 30, and 100 mg/kg in C57BL/6 mice, exerted a dose-dependent anticonvulsant e ffect [9,43]. Furher research on mice has revealed that the sesquiterpene inhibits tonic-clonic seizures in MES (maximal electro shock seizure) test and reduces kainate-induced neurotoxicity, suggesting a possible use of the sesquiterpene for the treatment of epilepsy [44].
